WO2023230439A1

WO2023230439A1 - Fc-engineered hepatitis b virus neutralizing antibodies and uses thereof

Info

Publication number: WO2023230439A1
Application number: PCT/US2023/067296
Authority: WO
Inventors: Davide Corti; Nadine CZUDNOCHOWSKI; Michael Alexander SCHMID; Gyorgy Snell
Original assignee: Vir Biotechnology, Inc.
Priority date: 2022-05-23
Filing date: 2023-05-22
Publication date: 2023-11-30

Abstract

The present disclosure relates, in part, to antibodies, and antigen-binding fragments thereof, that can bind to the antigenic loop region of hepatitis B surface antigen (HBsAg) and, optionally, can neutralize infection hepatitis B virus (HBV), and further optionally, of hepatitis delta virus (HDV). Presently disclosed antibodies and antigen-binding fragments have advantageous production characteristics, such as reduced formation of aggregates and/or improved production titer in transformed host cells, as compared to a reference antibody or antigen-binding fragment. Presently disclosed antibodies and antigen-binding fragments include engineered polypeptides (e.g., Fc polypeptides, Fc polypeptide fragments, Fc fusion proteins) that comprise a variant of an IgG Fc polypeptide (or a portion or fragment thereof), which variants (and the polypeptides that comprise these variants) have one or more improved characteristics over known Fc polypeptides.

Description

FC-ENGINEERED HEPATITIS B VIRUS NEUTRALIZING ANTIBODIES AND USES THEREOF

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The ccoonntteennttss of the electronic sequence listing (930485_443WO_SEQUENCE_LISTING.xml; Size: 156468 bytes; and Date of Creation: May 12, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

Hepatitis B virus causes potentially life-threatening acute and chronic liver infections. Acute hepatitis B is characterized by viremia, with or without symptoms, with the risk of fulminant hepatitis occurrence (Liang TJ, Block TM, McMahon BJ, Ghany MG, Urban S, Guo IT, Locarnini 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]). Despite an efficacious vaccine against hepatitis B being available since 1982, WHO reports that 240 million people are chronically infected with hepatitis B and more than 780,000 people die every year due to hepatitis B complications. Approximately one third of chronic hepatitis B (CHB) patients 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, Locarnini 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]).

For patients infected with HBV, severe complications can develop as a result of coinfection or superinfection with HDV. According to the WHO, hepatitis D infects about 15 million people worldwide. HDV is considered a subviral satellite because it can propagate only in the presence of HBV. HDV is one of the smallest known animal viruses (40 nm), whereby its genome is only 1.6 kb and encodes for S and L HDAg. All other proteins needed for genome replication of HDV, including the RNA polymerase, are provided by the host cell, and the HDV envelope is provided by HBV. When introduced into permissive cells, the HDV RNA genome replicates and associates with multiple copies of the HDV-encoded proteins to assemble a ribonucleoprotein (RNP) complex. The RNP is exported from the cell by the HBV envelope proteins, which are able to assemble lipoprotein vesicles that bud into the lumen of a pre-Golgi compartment before being secreted. Moreover, the HBV envelope proteins also provide a mechanism for the targeting of HDV to an uninfected cell, thereby ensuring the spread of HDV.

Complications caused by HDV include a greater likelihood of experiencing liver failure in acute infections and a rapid progression to liver cirrhosis, with an increased chance of developing liver cancer in chronic infections. In combination with hepatitis B virus, hepatitis D has the highest fatality rate of all the 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 inefficient and is not well-tolerated. Treatment with interferon-alpha results in sustained virological response six months post-treatment in one-fourth of the patients. Also, nucleos(t)ide analogs (NAs) have been widely tested in hepatitis delta, but they appear to be ineffective. Combination treatment using NAs with interferon also proved to be disappointing (Zaigham Abbas, Minaam Abbas Management of hepatitis delta: Need for novel therapeutic Options. World J Gastroenterol 2015 August 28; 21(32): 9461-9465).

Accordingly, new therapeutic options, such as new therapies with neutralizing activity against Hepatitis B and/or D infection are needed.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures provided herein are intended to illustrate subject matter included in the present disclosure in more detail. The figures are not intended to limit the disclosure in any way. Figure 1 shows a diagram summarizing a process by which immune complexes of Fc-engineered anti-HBsAg monoclonal antibodies and HBsAg can have a vaccinal effect when interacting with FcyR of anitgen presenting cells, e.g., dendritic cells.

Figures 2A-2B summarize FcyR binding and other characteristics of Fc variants, relative to wildtype Fc. Bars and values indicate fold-change in binding as compared with wild-type Fc. Fc variants shown were not treated with 2FF. Figure 2A shows binding to FcyRIIA-H (high affinity), FcyRIIA-R (low affinity), FcyRIIB, FcyRIIIA-V (high affinity), FcyRIIIA-F (low affinity), and FcRn (at pH 6). Figure 2B further shows the ratio of FcyRIIA-H/FcyRIIB binding, as well as Clq binding and complement-dependent cytotoxicity (CDC), with the WT "baseline" value indicated by a dashed vertical red line. Binding was measured by a meso scale discovery-based assay (MSD; employing electrochemiluminescence)).

Figure 3 shows binding of certain Fc variants to FcyRIIA-H (high affinity) and FcyRIIB. Plots connected by a line represent the same variant. Variants shown were not treated with 2FF.

Figures 4A-4B shows FcyR signaling through different FcyRs as measured using a reporter cell assay (Promega; tested cells expressed one type/allele FcyR, as indicated). Fc variants shown are fucosylated ("fuc"; 4A/4B) or afucosylated ("afuc"; 4B) as indicated in the figure. Values are calculated from an average of three experiments and indicate fold-change (expressed linear) in area-under-the-curve (plotted in log) as compared to wildtype Fc.

Figures 5A-5C summarize characteristics of certain variant Fes. Antibodies comprising the indicated Fc were expressed as recombinant human IgGl . Variants shown in Figures 5B-1-5B-4 are afucosylated. Binding was measured by a meso scale discovery-based assay (MSD; employing electrochemiluminescence)). Values represent fold-change compared to the antibody comprising wild-type fucosylated human IgGl Fc. Figures 5A-3, 5A-4, 5B-3, and 5B-4 also show fold-change in FcyR signaling, as measured using a reporter cell assay.

Figures 6A-6B summarize SPR assays. Figure 6A is a schematic showing setup for SPR assay to study binding of HBC34-v40-rIgGlm3 Fc variants to FcyR (CAP chip was used for capture of biotinylated FcyR proteins by Streptavidin; HBC34-v40- rIgGlm3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed successively with no regeneration between different concentrations of the same sample; injection: 600 seconds; dissociation 100 seconds each injection). Figure 6B shows an example SPR curves showing binding to FcyRIIIA.

Figure 7 shows a schematic of a cell reporter assay for measuring FcyR- mediated cell signaling induced by an Fc variant antibody

Figures 8A-8B show FcyR signaling through FcyRIIA-H (high affinity, Figure 8A) and FcyRIIB (Figure 8B) by the Fc variant "G236A R292P Y300L" as measured using a reporter cell assay.

Figures 9A-9B show FcyR binding versus signaling through FcyRIIA-H (high affinity, Figure 9A) and FcyRIIB (Figure 9B) by Fc variants. FcyR binding was measured using a Meso Scale Discovery binding assay and FcyR signaling was measured using a reporter cell assay (Promega).

Figures 10A-10B show activation, by anti-HBsAg antibody HBC34v35 containing variant Fc, of lurkat cells expressing human FcyRIIA (H131) (Figure 10A) or FcyRIIIA (F158) (Figure 10B) with target cells line stably expressing HBsAg.

Figures 11A-11D show results from repeat experiments of those in Figures 10A and 10B.

Figures 12A-12B show activation, by anti-HBsAg antibody HBC34v40 containing variant Fc, of urkat cells expressing human FcyRIIIA (Fl 58) (Figure 12A) or FcyRIIA (H131) (Figure 12B) with target cells line stably expressing HBsAg.

Figures 13A-13D show activation, by anti-HBsAg antibody HBC34v40 containing variant Fc, of urkat cells expressing human FcyRIIA. Luminescensce levels are shown for HBC34v40-LS (Figure 13A), HBC34v40-LS-GARPYL (Figure 13B), HBC34v40-LS-GAALIE (Figure 13C), and HBC34v40-LS-GAYL (Figure 13D).

Figures 14A-14F show activation, by anti-HBsAg antibody HBC34v40 containing variant Fc, of urkat cells expressing human FcyRIIIA. Luminescensce levels are shown for HBC34v40-LS (Figure 14A), HBC34v40-LS-GARPYL (Figure 14B), HBC34v40-LS-GAALIE (Figure 14C), HBC34v40-LS-GAYL (Figure 14D), HBC34v40-LS-GARPYL-afuc (Figure 14E), and HBC34v40-LS-GAYL-afuc (Figure 14F).

Figure 15 shows a diagram of monocyte-derived dendritic cells (moDCs) interaction with and activation by immune complexes of anti-HBsAg antibodies and HBsAg antigens.

Figure 16 shows flow cytometry analysis of CD83 expression on moDCs (expressing the indicated FcyR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 pg/mL) and 30 lU/mL HBsAg from HBV+ patient serum.

Figure 17 shows flow cytometry analysis CD83 expression on moDCs in the presence of HBC34-v40 Fc variant antibody (50 pg/mL) and HBsAg from HBV+ patient serum (BioIVT) at the indicated concentration. Left graphs are from an experiment using a first method of pipetting/generating immune complexes of antibody:HBsAg; right graphs are from an experiment using a second method of pipetting/generating immune complexes of antibody: HBsAg.

Figures 18A and 18B show moDC activation by HBC34-v40 Fc variant antibodies. Figure 18A shows moDC activation via immune complexes 30-1,000 lU/mL HBsAg (HBV+ patient sera) and 50 pg/mL HBC34-v40-LS-GRLR, -LS, and - LS-GAALIE variants. Figure 18B shows moDC activation via immune complexes 100, 10, and 1 lU/mL HBsAg (HBV+ patient sera) and 8 pg/mL of the indicated HBC34- v40 Fc variants.

Figure 19 shows a diagram of antigen presentation and activation of T cells by immune complex-activated moDCs.

Figure 20 shows results for NF AT activity in transgenic Jurkat cells. CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated with antigen and HBC34-v40 Fc variant antibody at 0.01, 0.03, 0.06, 0.125, 0.25, 0.5, and 1 pg/mL to form immune complexes, then co-cultured with an HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was GFP-NFAT reporter of Jurkat cells.

Figures 21A-21B show a summary of the results shown in Figure 20. Figures 22A-22C show a comparison of Jurkat TCR reporter assay for three independent experimental repeats at 0.125 pg/mL antibody.

Figure 23 shows a diagram summarizing a process by which moDCs and HBsAg-specific CD4+ memory T cells for a human vaccinee are collected and assayed for T cell proliferation and activation dependent from antigen specificyt and regulated via activation of moDCs by immune complexes.

Figures 24A-24B show percent of CFSE low CD25+ human CD4+ memory T cells from HBV vaccinees with the indicated Fc variant antibody and HBV + patient sera. Figure 21 A and Figure 21B show results for assays performed with 30 lU/mL and 100 lU/mL HBsAg (HBV+ patient sera), respectively. Vertically dispersed data points clustered from left to right as follows: HBC34-v40-GRLR; HBC34-v40-rIgGl-LS; HBC34-v40-rIgGl-LS-GAALIE; HBC34-v40rIgGl-LS-GA; HBC34-v40rIgGl-LS- GAYL; HBC34-v40rIgGl-LS-GARPYL, HBC34-v40rIgGl-LS-GAYL-afuc; and HBC34-v40rIgGl-LS-GARPYL-afuc.

Figures 25A-25B show percent of CD4+ memory T cells that are CFSE low and CD25+, IL-2+, and/or FNF-y+. Figure 22A and Figure 22B show results for assays performed with 30 lU/mL and 100 lU/mL HBsAg (HBV+ patient sera), respectively. Data points for each marker on the X-axis are shown from left to right as follows: HBC34-v40-GRLR; HBC34-v40-rIgGl-LS; HBC34-v40-rIgGl-LS-GAALIE; HBC34-v40rIgGl-LS-GA; HBC34-v40rIgGl-LS-GAYL; HBC34-v40rIgGl-LS- GARPYL, HBC34-v40rIgGl-LS-GAYL-afuc; and HBC34-v40rIgGl-LS-GARPYL- afuc.

Figures 26A-26C show a summary of data from different assays.

Figure 27 shows a diagram summarizing a process by which CD8+ T cells are restimulated within total PBMC from patients with chronic hepatitis B with immune complexes (HBC34 Fc variants + HBsAg).

Figures 28A-28B show proliferation of CD8+ T cells in CFSE-labeled PBMCs from patients with CHB. The CFSE-labelled PBMCs were co-cultured with immune complexes (HBC34 Fc variants + HBsAg) and APCs. Figure 29 shows a diagram summarizing an experimental setup for assessing ex vivo proliferation of T cells from FcyR-expressing mice immunized and boosted with an HBsAg vaccine; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, cocultured with immune complex (antibody :HBsAg antigen)-pulsed BMDCs, and assessed for proliferation on day 6. SEB = Staphylococcal enterotoxin B from S. Aureus.

Figure 30 shows CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in Figure 29, wherein the BMDCs (50,000) were stimulated using immune complexes comprising the indicated HBC34-v40 Fc variant antibody (20 pg/mL) and HBsAg (1000 lU/mL). SEB = 1 pg/mL; Mann- Whitney test.

Figures 31A-31B shows CD4 expression and CFSE staining as in Figure 30. Figure 31A shows data representative of seven independent experiments using HuFcyR mice as T cells donors (n=4 independent experiments) and C57B1/6 mice as T cell donors (n=3 independent experiments). Figure 3 IB shows results of CD4+ T cells stimulated using immune complexes comprising the indicated HBC34-v40 Fc variant antibody (titrated from 60 pg/mL to 0.25 pg/mL) and HBsAg (1000 lU/mL).

Figure 32 shows further analysis of the results of experiments of Figures 31A- 3 IB. (Left) Frequency of CFSE low CD4+ CD44+ T cells following incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells following incubation with moDCs pre-treated with HBsAg (1,000 lU/mL) and the indicated HBC34-v40 Fc variant antibody (20 pg/mL). 50,000 moDCs + 500,000 T cells were tested. SEB = 1 pg/mL; Mann-Whitney test.

Figures 33A-33C show results of experiments to compare activation of CD4+ memory T cells with immune complexes formed with HBsAg and HBC34-v40-Fc variants lacking the LS modification using the methods of Figure 29. Figure 33A shows mean % CFSE of CD44+ CD4+ memory T cells co-cultured with BMDCs stimulated with immune complexes of 1,000 lU/mL HBsAg and the indicated HBC34- v40-Fc variants (titrated from 60 pg/mL to 0.25 pg/mL). Figures 33B and 33C show graphs of the data of Figure 33 A. Figures 34A-34B show results of experiments to compare activation of CD4+ memory T cells with immune complexes formed with HBsAg and the indicated HBC34-v40-Fc variants and afucosylated variants thereof using the methods of Figure 29. Figure 34A shows mean % CFSE of CD44+ CD4+ memory T cells co-cultured with BMDCs stimulated with immune complexes of 1,000 lU/mL HBsAg and the indicated HBC34-v40-Fc variants (titrated from 60 pg/mL to 0.25 pg/mL). Figure 34B shows graphs of the data of Figure 34A.

Figure 35 shows a diagram summarizing the generation of transgenic mice expresing transgenes for a reactive TCR specific for HBsAg residues 126-128.

Figure 36 shows a diagram summarizing an experimental setup for assessing proliferation of transgenic CD4+ T cells expresing transgenes for a reactive TCR specific for HBsAg residues 126-128 in HuFcyR transgenic mice.

Figure 37 shows expansion of HBs-specific Tg CD4+ T cells in mice immunized i.p. with ICs of HBC34-v40 Fc variants (5 pg/mL) complexed with 5000 lU/mL HBsAg.

Figure 38 shows neutralization of HBV genotype D by HBC34-v35 and certain antibodies of the present disclosure, with HBeAg as the viral readout. Calculated EC50 values of each mAb are shown at right. HBC34-v35 (purified IgG and supernatant) and HBC34-v36 (purified IgG) were used as controls.

Figure 39 shows neutralization of HDV HBC34-v35 and certain antibodies of the present disclosure, with HBsAg (genotype A) as the viral readout. Calculated EC50 values of each mAb are shown at right.

DETAILED DESCRIPTION

The present disclosure relates to the field of immunotherapy for hepatitis B virus (HBV) and hepatitis delta virus (HDV). Disclosed antibodies are capable of binding to an epitope located in the antigenic loop region of the S domain of the HBV envelope protein (HBsAg), are capable of neutralizing a HBV infection and, in some embodiments, a HDV infection. Presently disclosed antibodies possess advantageous production properties (e.g., reduced formation of antibody dimers and/or increased production in a host cell) and include an engineered IgG Fc polypeptide (or a portion or fragment thereof), which have one or more improved characteristics over known Fc polypeptides (such as, for example, a reference wild-type Fc polypeptide and/or to known variant Fc polypeptide). The VH and VL sequences of "HBC34-v40", "HBC34-v44", "HBC34-v45", and "HBC34-v50" of the instant disclosure have mutations in L-CDR2 and/or in framework sequence relative to HBC34-v35 (PCT Publication No. WO 2021/262840, incorporated by reference herein in its entirety).

Briefly, HBC34-v35 antibody has favorable binding and neutralization properties, but can form antibody dimers through inter-light chain interactions during antibody product! on/purification. HBC34-v35 dimers have reduced ability to bind to HBsAg as compared to HBC34-v35 antibody monomers. Reducing dimer formation may improve, e.g., efficiency of antibody (or antigen-binding fragment) production and potency of a dose of the antibody (or antigen-binding fragment).

Further, as noted above, the presently disclosed antibodies possess improved characteristics over antibodies comprising known Fc polypeptides. For example, the presently disclosed antibodies may demonstrate increased binding to one or more human FcyRA (e.g., a FcyRIIA and optionally a FcyRIII); decreased/reduced binding to a human FcyRIIB; increased binding to one or more human FcyRA as compared to binding to a human FcyRIIB; increased thermostability as compared to known Fc polypeptides; improved binding to human Clq; increased human FcyRIIIA signaling in a host cell expressing the FcyRIIIA, increased human FcyRIIIA signaling in a host cell expressing the FcyRIIA, decreased human FcyRIIB signaling in a host cell expressing the FcyRIIB, a relative increase in binding to FcyRA as compared to FcyRIIB, improved qualities for production as compared to known Fc polypeptides; and combinations of such features.

In certain embodiments, presently disclosed antibodies can bind to any or all of the known HBsAg genotypes, as well as HBsAg variants, and can neutralize HBV infection, as well as HDV infection. In certain embodiments, a presently disclosed antibodies can bind to and/or can neutralize HBV and/or HDV with similar or even increased potency as compared to HBC34-v35.

In certain embodiments, antibodies comprising a variant Fc polypeptide of the present disclosure provide surprising advantages, such as any one or more of the following: increased binding affinity (e.g. as determined by surface plasmon resonance, e.g. using a Biacore instrument and/or as determined by a electrochemiluminescence assay, such as a meso scale discovery (MSD) assay) for and/or inducing increased signaling (e.g. as determined using (1) an Fc variant antibody (2) antigen-expressing target cells and (3) reporter cells expressing one or more human FcyRA, optionally driving expression of a reporter gene such as, for example, GFP or luciferase) by one or more human FcyRA, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state; decreased binding affinity for and/or inducing decreased signaling of human FcyRIIB, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state; a unique and optionally improved binding profile across human FcyRIIA-H, human FcyRIIA-R, human FcyRIIB, human FcyRIIIA-F, and human FcyRIIIA-V, wherein improved binding comprises an overall increase in binding to and/or activation of FcyRA signaling relative to binding to and/or activation of inhibitory FcyR signaling, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state; increased binding affinity for human Clq , as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state; no detrimental effect or no substantial detrimental effect on thermal stability, a reduced negative effect on thermal stability as compared to a variant Fc polypeptide or fragment thereof not comprising the mutation(s) and/or fucosylation state (e.g., a human IgGl Fc comprising the mutations G236A, A330L, and I332E (e.g. having a smaller decreasing effect, or no decreasing effect, on melting temperature as compared to the antibody comprising a human IgGl Fc comprising the mutations G236A, A330L, and I332E), or having a higher melting temperature than the antibody comprising a human IgGl Fc comprising the mutations G236A, A330L, and I332E)); increasing specific lysis (e.g. via ADCC) by natural killer cells and/or PBMCs (e.g. expressing Fl 58/VI 58 or VI 58/VI 58 FcyRIIIA) against antigen-expressing target cells, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state (e.g. the antibody comprising a human IgGl Fc comprising the mutations G236A, A330L, and I332E); increasing ADCP by monocytes (e.g. CD14+ monocytes, optionally expressing Fl 58/VI 58 FcyRIIA and R131/H131 FcyRIIA or F158/F158 FcyRIIA and R131/H131 FcyRIIA) against antigen-expressing target cells, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state; increasing the percentage of CD83+ cells (e.g. moDCs) and/or increasing expression of CD83 by moDCs in a sample when provided in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided in combination with the antigen; increasing production of one or more cytokine (optionally selected from the group consisting of IL-ip, IFN-y, IL-6, and TNF-a) by moDCs in a sample when provided in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided in combination with the antigen; and/or increasing the ability of moDCs to stimulate antigen-specific CD4+ T cells when provided to the moDCs in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided to the moDCs in combination with the antigen, wherein, optionally, (1) the moDCs and the CD4+ T cells are from the same (optionally antigen-vaccinated) subject and/or (2) stimulation of antigen-specific CD4+ T cells is determined by an increase in CD25 expression and/or an increase in proliferation (e.g. as determined by a reduction in CFSE staining over time) and/or an increase in expression of CD69 and/or an increase in expression of NF AT and/or an increase in expression of CD44, by the antigen-specific CD4+ T cells.

In some embodiments, presently disclosed antibodies possess characteristics such as effector functions, ability to bind human Clq, ability to induce FcyRA-mediated cell signaling, ability to bind to human FcRn, and the like. In certain embodiments, presently disclosed antibodies have one or more altered characteristics (e.g., increased binding to a human FcyRa, decreased binding to a human FcyRIIb, binding to a human FcyRa that is increased relative to the binding to a FcyRIIb, increased binding to a human Clq, increased binding to a human FcRn, an increased Tm, increased binding to a FcyRIIIa, or any combination thereof), as compared to a reference polypeptide or antibody that comprises a variant Fc containing the following mutation(s): G236A; G236S; G236A/A330L/I332E; G236A/A330L/I332E/M428L/N434S; G236A/A330L/I332E/M428L/N434A; G236A/S239D/A330L/I332E; or A330L/I332E.

It will be understood that herein, “FcyRIIA” may be expressed as “FcyRIIa”, “FcyRIIIA” may be expressed as “FcyRIIIa”, “FcyRIIB” may be expressed as “FcyRIIb”, and “FcyRIIIB” may be expressed as “FcyRIIIb”.

Nucleic acids that encode, and host cells that express, such antibodies are also provided herein. In addition, the present disclosure provides methods of using the antibodies described herein in the diagnosis, prophylaxis, and treatment of diseases, as well as in methods of screening.

For example, embodiments of the antibodies according to the present description may be used in methods of preventing, treating, or attenuating, or diagnosing HBV and HDV. In particular embodiments, the antibodies described herein bind to two or more different genotypes of hepatitis B virus surface antigen and to two or more different infectious mutants of hepatitis B virus surface antigen. In specific embodiments, the antibodies described herein bind to all known genotypes of hepatitis B virus surface antigen and to all known infectious mutants of hepatitis B virus surface antigen.

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Throughout this disclosure, unless the context requires otherwise, the term "comprise," and variations thereof, such as "comprises," and "comprising," is used synonymously with, e.g. "having," "has," "including," "includes," or the like, and will be understood to imply the inclusion of a stated member, ratio, integer (including, where appropriate, a fraction thereof; e.g., one tenth and one hundredth of an integer), concentration, or step but not the exclusion of any other non-stated member, ratio, integer, concentration, or step. Any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.

The term "consisting essentially of is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, a Fc, a CH2, a CH3, a CH2-CH3, or a CH1- CH3) or a protein "consists essentially of' a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy -terminus or between domains) 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 a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of an antibody).

In some embodiments, an antibody includes a variant of a CH2, CH3, CH1- CH3, or Fc polypeptide that comprises one or more amino acid substitution relative to a wild-type or parent CH2, CH3, CH1-CH3, or Fc polypeptide, respectively, wherein the one or more amino acid substitution consists essentially of the specified one or more amino acid substitution. In certain further embodiments, a variant of a CH2, CH3, CH1-CH3, or Fc polypeptide comprises one or more additional amino acid substitution (e.g. one or more conservative amino acid substitution and/or one or more amino acid substitution that is remote from the specified one or more amino acid substitutions), provided that one or more characteristics of the claimed subject-matter is retained or substantially retained and is not materially changed, e.g. binding to and/or activation of one or more FcyR, binding to FcRn, melting temperature, binding to Clq, promotion of ADCC, promotion of ADCP, promotion of CDC, formation of an immune complex, activation of dendritic cells (e.g. monocyte-derived dendritic cells) when provided in an immune complex with antigen, or the like. In some embodiments, a claimed subjectmatter comprising one or more amino acid substitution(s) that consist(s) essentially of the specified amino acid substitution(s) is a functional variant of a claimed subjectmatter wherein the amino acid substitution(s) consist(s) of the specified amino acid substitution(s).

In addition, it should be understood that the individual compounds, or groups of compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure.

The terms "a" and "an" and "the" and similar reference used in the context of describing the disclosure (including in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. Recitation of ranges of values herein is intended to serve as a shorthand method of referring individually to each separate 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 nonclaimed element as essential to the practice of the subject matter disclosed herein. The word "substantially" does not exclude "completely"; e.g., a composition which is "substantially free" from Y may be completely free from Y. In certain embodiments, "substantially" refers to a given amount, effect, or activity of a composition, method, or use of the present disclosure as compared to that of a reference composition, method, or use, and describes a reduction in the amount, effect, or activity of no more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%, or less, of the amount, effect, or activity of the reference composition, method, or use.

As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. In certain embodiments, "about" includes ± 15%, ± 10%, or ± 5%.

"Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.

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 a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

As used herein, the terms "peptide," "polypeptide," and "protein," and variations of these terms, refer to a molecule that comprises at least two amino acids joined to each other by a (normal or modified) peptide bond. Accordingly, a protein or polypeptide comprises a polymer of amino acid residues. For example, a peptide, polypeptide or protein may comprise or be composed of a plurality of amino acids selected from the 20 amino acids defined by the genetic code or an amino acid analog or mimetic, each being linked to at least one other by a peptide bond. A peptide, polypeptide or protein can comprise or be composed of L-amino acids and/or D-amino acids (or analogs or mimetics thereof). The terms "peptide", "polypeptide," "protein" also include "peptidomimetics" which are defined as peptide analogs containing non- peptidic structural elements, which peptides are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. In certain embodiments, a peptidomimetic lacks characteristics such as enzymatically scissile peptide bonds.

A peptide, polypeptide or protein may comprise amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids, or it can be composed of amino acids other than the 20 amino acids defined by the genetic code. In certain embodiments, a peptide, polypeptide or protein in the context of the present disclosure can comprise amino acids that are modified by natural processes, such as post-translational maturation processes, or by chemical processes (e.g., synthetic processes), which are known in the art and include those described herein. Such modifications can appear anywhere in the polypeptide; e.g., in the peptide skeleton; in the amino acid chain; or at the carboxy- or amino-terminal ends. A peptide or polypeptide can be branched, such as following an 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 can include acetylation, acylation, ADP- ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation or ubiquitination. Such modifications have been described in the literature (see 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). Accordingly, the terms "peptide", "polypeptide", "protein" can include for example lipopeptides, lipoproteins, glycopeptides, glycoproteins and the like.

Variants of proteins, peptides, and polypeptides of this 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 an amino acid sequence of a defined or reference amino acid sequence as described herein.

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

"Nucleic acid molecule" or "polynucleotide" or "nucleic acid" refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, or the like.

Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, any of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). Also contemplated are microRNA, siRNA, viral genomic RNA, and synthetic RNA. Polynucleotides (including oligonucleotides), and fragments thereof may be generated, for example, by polymerase chain reaction (PCR) or by in vitro translation, or generated by any of ligation, scission, endonuclease action, or exonuclease action.

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 sequences may also include intron(s) to the extent that the intron(s) may be removed through co- or post-transcriptional mechanisms. Different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing, or both.

Variants of nucleic acid molecules of this 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 a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the capacity to encode a fusion protein or a binding domain thereof having a functionality described herein, such as specifically binding a target molecule.

As used herein, the term "sequence variant" refers to any sequence having one or more alterations in comparison to a reference sequence, whereby a reference sequence is any published sequence and/or of the sequences listed in the "Table of Sequences and SEQ ID Numbers" (sequence listing) herein. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. In certain embodiments, a sequence variant in the context of a nucleotide sequence, the reference sequence is also a nucleotide sequence, whereas in certain embodiments for a sequence variant in the context of an amino acid sequence, the reference sequence is also an amino acid sequence. A "sequence variant" as used herein can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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.

"Percent sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. Methods to determine sequence identity can be designed to give the best match between the sequences being compared. For example, the sequences may be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters which originally load with the software when first initialized.

A "sequence variant" in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted, or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one- letter designation (A, C, G, or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence can either result in a change in the respective reference amino acid sequence, i.e. in an amino acid "sequence variant" or not. 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 one or more "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. Nucleotide and amino sequences as disclosed herein refer also to codon-optimized versions of a reference or wild-type nucleotide or amino acid sequence. In any of the embodiments described herein, a polynucleotide of the present disclosure may be codon-optimized for a host cell containing the polynucleotide. Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGene™ tool, or the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include sequences that are partially codon-optimized (z.e., at least one codon is optimized for expression in the host cell) and those that are fully codon-optimized.

A "sequence variant" in the context of an amino acid sequence has an altered sequence in which one or more of the amino acids is deleted, substituted, or inserted in comparison to a reference amino acid sequence. As a result of the alterations, such a sequence variant has an amino acid sequence which 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, per 100 amino acids of the reference sequence a variant sequence that has no more than 10 alterations, i.e. any combination of deletions, insertions or substitutions, is "at least 90% identical" to the reference sequence.

A "conservative substitution" refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution 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 (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

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

In general, alterations in the sequence variants do not abolish or significantly reduce a desired functionality of the respective reference sequence. For example, it is preferred that a variant sequence of the present disclosure does not significantly reduce or abrogate the functionality of a sequence of an antibody, or antigen-binding fragment thereof, to bind to the same epitope, to sufficiently neutralize infection of HBV and HDV, and/or does not cause or increase formation of antibody dimer, and/or is not produced at a lower titer in a host cell, as compared to antibody or antigen binding fragment having (or encoded by) the reference sequence.

As used herein, a nucleic acid sequence or an 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 which is derived from a particular sequence may have an amino acid sequence that is essentially identical to that sequence or a portion thereof, from which it is derived, whereby "essentially identical" includes sequence variants as defined above. A nucleic acid sequence or amino acid sequence which is derived from a particular peptide or protein may be derived from the corresponding domain in the particular peptide or protein. In this context, "corresponding" refers to possession of a same functionality or characteristic of interest. 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" parts of peptides, proteins and nucleic acids are thus easily identifiable to one of ordinary skill in the art. Likewise, a sequence "derived from" another (e.g., "source") sequence can be identified by one of ordinary skill in the art as having its origin in the source sequence.

A nucleic acid sequence or an 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 an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), in particular a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a functional sequence variant as described above of the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). 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.

As used herein, the term "mutation" relates to a change in a nucleic acid sequence and/or in an amino acid sequence in comparison to a reference sequence, e.g. a corresponding genomic, wild type, or reference sequence. A mutation, e.g. in comparison to a reference genomic sequence, 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 (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and/or in the amino acid sequence). Thus, the terms "mutation" or "mutating" shall be understood to also include physically making or inducing a mutation, e.g. in a nucleic acid sequence or in an amino acid sequence. A mutation includes substitution, deletion and/or insertion of one or more nucleotides or amino acids, as well as inversion of several successive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved, for example, by altering (e.g., by site-directed mutagenesis) a codon (e.g., by altering one, two, or three nucleotide bases therein) of a nucleic acid molecule encoding one amino acid to provide a codon that encodes a different amino acid, or that encodes a same amino acid, or by synthesizing a sequence variant.

A "functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding," "similar affinity" or "similar activity" when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant).

As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of this disclosure has "similar binding" or "similar activity" when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity).

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. "Isolated" can, in some embodiments, describe an antibody, antigen-binding fragment, fusion protein, polynucleotide, vector, host cell, or composition that is outside of a human body.

The term "gene" means the segment of DNA or RNA involved in producing a polypeptide chain; in certain contexts, it includes regions preceding and following the coding region (e.g., 5’ untranslated region (UTR) and 3’ UTR) as well as intervening sequences (introns) between individual coding segments (exons).

The term "introduced" 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 wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

The term "recombinant", as used herein (e.g. a recombinant antibody, a recombinant protein, a recombinant nucleic acid, or the like), refers to any molecule (antibody, protein, nucleic acid, or the like) which is prepared, expressed, created or isolated by recombinant means, and which is not naturally occurring. "Recombinant" can be used synonymously with "engineered" or "non-natural" and can refer to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (z.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.

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 a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other selfreplicating 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 encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof.

As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.

The term "expression", as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post- translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a protein (e.g., a heavy chain of an antibody), or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.

As used herein, the terms "cell," "cell line, " and "cell culture" are used interchangeably and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" and "host cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant 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 distinct designations are intended, it will be clear from the context.

The term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule (or, if the context clearly indicates, a fusion protein of the present disclosure).

In certain embodiments, polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (z.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

Antibodies

Presently disclosed embodiments include antibodies that are capable of binding to the antigenic loop region of HBsAg (HBsAg and the antigenic loop region are described in further detail here) and, optionally, neutralizing infection by a hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof; i.e., any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, or all ten of these genotypes. In addition, the antibodies disclosed herein comprise engineered variants of immunoglobulin G (IgG) Fc polypeptides or fragments or portions thereof. As discussed further herein, presently disclosed antibodies possess other advantages, including, for example and not limited to, characteristics that favor production in a host cell, and reduced propensity to form undesirable aggregates, such as dimers.

As used herein, and unless the context clearly indicates otherwise, "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (though it will be understood that heavy chain antibodies, which lack light chains, are still generally encompassed by the term "antibody", though preferred embodiments of the present disclosure comprise both of a VH and a VL, and in some embodiments, both of a heavy chain and a light chain), as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as, for example, a scFv, Fab, or F(ab')2 fragment. Thus, the term "antibody" herein is used 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 (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (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, and heteroconjugate antibodies, multi specific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv, and other antibody formats known in the art. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term "antibody" also encompasses intact or full-length antibodies, including antibodies of any class or subclass thereof, including IgG and sub-classes thereof (IgGl, IgG2, IgG2, IgG4), IgM, IgE, IgA, and IgD.

As used herein, in the context of an antibody, the terms "antigen-binding fragment," "fragment," and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the disclosure that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, a single chain antibody, Fab, Fab’, F(ab')2, Fv or scFv.

Human antibodies are known (e.g., van Dijk, M. A., and van de Winkel, J. G., 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 or a selection of human antibodies 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 antigen 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 the preparation of 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 by 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. The term "human antibody" as used herein also comprises such antibodies which are modified, e.g, in the variable region or the constant region, to generate properties according to the antibodies and antibody fragments of the present disclosure. Antibodies according to the present disclosure can be of any isotype (e.g., IgA, IgG, IgM, IgE, IgD; i.e., comprising an a, y, p, e, or 5 heavy chain). Within the IgG isotype, for example, antibodies may be IgGl, IgG2, IgG3 or IgG4 subclass. In specific embodiments, an antibody of the present disclosure is an IgGl antibody. Antibodies or antigen binding fragments provided herein may include a K or a light chain. Preferably, an antibody or antigen-binding fragment can comprise a X light chain. In certain embodiments, HBsAg-specific antibodies described herein are of the IgG isotype e.g., IgGlM,17 1 allotype and IgGIM, 3 allotype) and may block the release of HBV and HBsAg from infected cells. Accordingly, in certain embodiments, an antibody according to the present description can bind intracellularly and thereby block the release of HBV virions and HBsAg.

The terms "VL" or "VL" and "VH" or "VH" refer to the variable region (also called variable domain) from an antibody light chain and an antibody heavy chain, respectively; typically, these regions are involved directly in the binding of an antibody or antigen-binding fragment to an antigen. A VL (as well as a CL or a light chain) can be a kappa (K) class (also "VK" herein) or a lambda (X) class. The variable binding regions comprise discrete sub-regions 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 refer to sequences of amino acids within antibody variable regions, which, in general, together confer the antigen specificity and/or binding affinity of the antibody, wherein consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from one another in primary amino acid sequence by a framework region. There are three CDRs in each variable region (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3; also referred to as HCDRs and LCDRs, respectively). In certain embodiments, an antibody VH comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3- HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen-binding site through their respective CDRs, though it will be understood that in some cases, a binding site can be formed by or can comprise one, two, three, four, or five of the CDRs which CDR(s) may disposed in the VH, in the VL, or in both.

In certain embodiments, antibody CDRs and amino acid numbering of variable regions are according to the system developed by the Chemical Computing Group ("CCG"); e.g., using Molecular Operating Environment (MOE) software (www.chemcomp.com).

In certain embodiments, antibody CDRs and amino acid numbering of variable regions are according to the IMGT numbering scheme (see, e.g., Lefranc et al., Dev. Comp. Immunol. 27:55, 2003).

Equivalent residue positions can be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).

As used herein, a "variant" of a CDR refers to a functional variant (as provided herein) of a CDR sequence having up to 1-3 amino acid substitutions, deletions, or combinations thereof.

Table 1 provides the CDR amino acid SEQ ID NOs. of certain antibodies, wherein CDRs are defined according to CCG.

Table 1. CDR (CCG numbering) amino acid SEQ ID NOs. of Certain Antibodies

Further, the antibodies of the present disclosure comprise engineered variants of immunoglobulin G (IgG) Fc polypeptides and fragments or portions thereof. By way of background, the Fc region (also called the "Fc domain") of an antibody can interact with Fc receptors and other binding partners such a complement Clq to, for example, initiate, participate, and/or and mediate an immune response against a pathogen or antigen. Presently disclosed Fc variants possess various advantages over native (i.e. wild-type) Fc and/or known Fc variants, such as, but not limited to, increased binding to one or more activation or activating Fc receptor (e.g. FcyRIIa), decreased binding to an inhibitory Fc receptor e.g. FcyRIIb), providing a relative increase in binding to an activating Fc receptor versus to an inhibitory Fc receptor, binding to complement Clq, facilitating or increasing antibody-dependent cellular phagocytosis (ADCP), facilitating or increasing antibody-dependent cell cytotoxicity (ADCC), facilitating or increasing complement, facilitating or increasing intracellular signaling that occurs via an activating Fc receptor, reducing intracellular signaling that occurs via an inhibitory Fc receptor, providing a relative increase in signaling via an activating Fc receptor versus signaling via an inhibitory Fc receptor, facilitating or increasing activation of dendritic cells (e.g. monocyte-derived dendritic cells) when provided thereto in a (variant Fc- bearing) antibody: antigen complex, or the like. In certain embodiments, presently disclosed Fc variants possess improved thermal stability (e.g., a higher Tm, or a Tm that is closer to the Tm of a wild-type Fc polypeptide), similar or improved capacity for production and/or purification, and/or favorable binding to FcRn, e.g. as compared to a wild-type reference Fc polypeptide or a variant Fc polypeptide that does not comprise the specified mutation(s) and/or fucosylation state.

In certain embodiments, the antibody or antigen-binding fragment is capable of eliciting continued protection in vivo in a subject even once no detectable levels of the antibody or antigen-binding fragment can be found in the subject (i.e., when the antibody or antigen-binding fragment has been cleared from the subject following administration). Without wishing to be bound by theory, it is believed that dendritic cells can internalize complexes of antibody and antigen and thereafter induce or contribute to an endogenous immune response against antigen. In certain embodiments, an antibody or antigen-binding fragment comprises one or more modifications, such as, for example, mutations in the Fc listed in Table 2 below, that are capable of activating dendritic cells that may induce, e.g., T cell immunity to the antigen.

An immune response can comprise, e.g., activation of dendritic cells (e.g. as assessed by increased expression of CD80, CD86, CD83, HLA-DR, IL- 12, CD40L, CD40, BAFF, April, or any combination thereof), activation of host T cells (e.g. CD4+ T cells and/or CD8+ T cells, e.g. as assessed by increased expression of CD25, CD137, ICOS, CD4, CD3, CD28, CD8, CD69, or any combination thereof, and/or by increased proliferation), proliferation of HBV-antigen-specific T cells, activation and/or proliferation of host B cells, polyclonal humoral immune response (e.g., production of IgM and/or IgG antibodies), production of one or more cytokine, fever, or the like. In some embodiments, an immune response attenuates an intensity of infection or protects against an infection.

Certain embodiments, the antibody or antigen-binding fragment thereof increases an intensity and/or a duration of an immune response against an antigen or immunogen, as compared to the intensity and/or duration of the immune response elicited by the antigen or immunogen alone. In the presently disclosed methods and uses, boosting is effected by combining an anti-HBV antibody, or an antigen-binding fragment thereof, with an HBV immunogen that comprises or encodes an HBV antigen recognized by the anti-HBV antibody or antigen-binding fragment.

In some embodiments, increasing an intensity of an immune response comprises eliciting an immune action that was not previously observed in the subject’s immune response to the antigen or immunogen without the antibody or antigen-binding fragment (e.g., activation of dendritic cells (e.g. as assessed by increased expression of CD80, CD86, CD83, HLA-DR, IL- 12, CD40L, CD40, BAFF, April, or any combination thereof), activation of host T cells (e.g. CD4+ T cells and/or CD8+ T cells, e.g. as assessed by increased expression of CD25, CD137, ICOS, CD4, CD3, CD28, CD8, CD69, or any combination thereof, and/or by increased proliferation), proliferation of HBV antigen-specific T cells, activation and/or proliferation of host B cells, polyclonal humoral immune response (e.g., production of IgM and/or IgG antibodies), production of one or more cytokine, fever, or the like), increasing the intensity of one or more immune action, or both. A boosted immune response includes a measurable immune response (including, but not limited to, protection from infection) of a longer duration than that of the immune response in a reference subject to the antigen or immunogen without the antibody or antigen-binding fragment. In some embodiments, a boosted immune response includes an increase in one or more of the following: activation of dendritic cells (e.g. as assessed by increased expression of CD80, CD86, CD83, HLA-DR, IL- 12, CD40L, CD40, BAFF, April, or any combination thereof), activation of host T cells (e.g. CD4+ T cells and/or CD8+ T cells, e.g. as assessed by increased expression of CD25, CD137, ICOS, CD4, CD3, CD28, CD8, CD69, or any combination thereof, and/or by increased proliferation), proliferation of HBV antigen-specific T cells, activation and/or proliferation of host B cells, polyclonal humoral immune response (e.g., production of IgM and/or IgG antibodies), production of one or more cytokine. In some embodiments, the increase is detectable in a sample from the subject for up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, up to eight weeks, up to three months, up to four months, up to five months, up to six months, up to seven months, up to eight months, or more. In some embodiments, a boosted immune response includes protection from infection and/or protection from progression of infection.

As discussed further herein, antibodies typically include two heavy chain polypeptides. As discussed herein, an immunoglobulin heavy chain typically includes a variable region (also called a variable domain) and a constant region (also called a constant domain). In that case of, for example, the IgG isotype, the constant region typically comprises a CHI region, a hinge, CH2, and CH3. Heavy chain polypeptide monomers can associate and be held together by shared disulfide bonds to form a dimer; the CH2-CH3 portions of an immunoglobulin heavy chain dimer comprise the Fc (fragment crystallizable) portion or domain of an immunoglobulin, for example, an IgGl antibody. An example of a wild-type human IgGl CH1-CH3 amino acid sequence is provided in SEQ ID NO.:56. An example of a wild-type human IgGl hinge-CH2-CH3 is provided in SEQ ID NO.:57. An example of a wild-type human IgGl CH2 is provided in SEQ ID NO.:58. An example of a wild-type human IgGl CH3 amino acid sequence is provided in SEQ ID NO.: 59. An example of a wild-type human IgGl hinge-CH2 amino acid sequence is provided in SEQ ID NO.:60. It will be understood that a hinge of a hinge-CH2 polypeptide or a hinge-Fc polypeptide can comprise one or more modifications (e.g. mutations) relative to a wild-type hinge sequence, which one or more modification can be further to, e.g., a P230A or S219Y mutation as disclosed herein.

As used herein, unless the context provides otherwise, an "Fc polypeptide" refers to a CH2-CH3 polypeptide. A fragment of an Fc polypeptide can comprise a CH2, a portion of a CH2, a CH3, and/or a portion of a CH3, while not comprising a complete, full-length CH2-CH3. In certain embodiments, Fc polypeptide fragments are provided that comprise a portion of a CH2 and/or a CH3 of sufficient length to comprise the specified amino acid position(s) and variations and, in some embodiments, to possess the recited function or functions.

Presently disclosed antibodies include those that comprise a variant of an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises one or more modifications as compared to the IgG Fc polypeptide or fragment thereof. It will be understood that, unless stated otherwise, a "reference" polypeptide or antibody (e.g., reference IgG Fc polypeptide or fragment thereof, reference antibody, reference CH2 polypeptide, reference IgG hinge-CH2, reference IgG hinge-Fc polypeptide, reference CH3 polypeptide) is preferably identical to the recited molecule (e.g., antibody, variant of an Fc polypeptide or fragment thereof; polypeptide comprising such a variant; antibody comprising a variant of an Fc polypeptide) except for the recited difference or differences (e.g., differences in CDR and/or variable region framework sequence(s) and/or Fc polypeptide).

For example, it will be understood that for a variant IgGl Fc polypeptide that comprises an alanine (A) amino acid at EU position 236, a reference Fc polypeptide includes an IgGl Fc polypeptide that is otherwise identical to the variant except that a native glycine (G) amino acid is found at EU position 236. As another example, for a variant of an Fc polypeptide fragment (e.g., containing a CH2 and a portion of a CH3), a reference Fc polypeptide fragment is preferably of an identical length to the variant and preferably differs from the variant only by the recited features (e.g., amino acid mutation or mutations present in the variant). In some embodiments, a reference Fc polypeptide, Fc polypeptide fragment, or antibody comprises a wild-type amino acid sequence (e.g., wild-type human IgGl). Excepting the recited differences present in the variant, a reference Fc polypeptide, Fc polypeptide fragment, or antibody will be of the same isotype, and, preferably, of the same allotype, as the variant. In the case of a reference antibody, the Fabs or other antigen-binding domains will preferably be identical to those present in the specified antibody comprising a variant Fc polypeptide or fragment thereof.

It will further be understood that when comparing a presently disclosed antibodies with a reference antibody under certain conditions, the conditions (e.g., amount of starting material, temperature, buffer, identity of host cell line, culture conditions, duration of a relevant time period, codon-optimization of an encoding polynucleotide, or the like) will, unless explicitly stated otherwise, be identical as between the presently disclosed molecule(s) and the reference molecule(s), or as close to identical as the conditions permit (e.g., two antibodies may differ in their amino acid sequences by one or a number of amino acids, but will be otherwise identical, and will be encoded by a comparable polynucleotide (e.g., each antibody can be encoded by a respective codon-optimized polynucleotide).

In some embodiments, variants of IgG Fc polypeptides or fragments thereof include one or more amino acid substitution as compared to a reference (e.g. wild-type) IgG Fc polypeptide or fragment thereof. Herein, the position of an amino acid in a variant IgG Fc polypeptide or fragment may be described by referencing the "EU position"; it will be understood that "the EU position" follows the EU numbering system as set forth in Kabat. By way of illustration, it will be understood that in the example of a human IgGl CH1-CH3 amino acid sequence provided in SEQ ID NO.:56, the first amino acid (A) corresponds to EU position 118, and the last amino acid (K) corresponds to EU position 447:

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY

SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

(SEQ ID NO : 56)

Accordingly, it will be understood that unless otherwise indicated, the position of a recited amino acid(s) follows EU numbering for human IgGl even if a complete antibody heavy chain, complete CH1-CH3, complete CH2-CH3, or the like is not present or is not explicitly recited. In other words, for example, if only a hinge-CH2 is described and a CH3 and/or CHI may not be present, the position of the amino acids in the hinge-CH2 is described with reference to EU numbering, unless stated otherwise. Correspondence between EU numbering, Kabat numbering, IMGT exon numbering, and IMGT unique numbering for immunoglobulin G heavy chain constant domain is known in the art and is shown, for example, in the IMGT Scientific chart (www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html; created May 17, 2001, accessed May 23, 2021, last updated January 20, 2020).

In the present Examples, certain Fc variants were generated (expressed in fucosylated and afucosyated human IgGl antibodies of various allotypes) and tested for various properties. Certain embodiments of Fc variants of the present disclosure (fucosylated, unless otherwise indicated) and non-limiting properties of the same are summarized in Table 2; see also Table 7 and Figures 12A-1-12C.

Table 2. Certain Fc variants and properties thereof

Additional features of disclosed Fc variant-containing antibodies are shown in the present Examples and Figures, and described herein.

It will be understood that two or more amino acid substitutions present in a variant can be expressed in a variety of ways, for example, as G236A_Y300L, or as G236A/Y300L. Moreover, a mutation or combination mutation may be referenced using a short form including the original amino acid(s) and the amino acid(s) resulting from the substitution(s). For example, G236A may be described as “GA” or “236A”; G236A_Y300L may be described as “GAYL”; G236A_L328V_Q295E may be described as “GALVQE”; G236A R292P Y300L may be described as “GARPYL”, G236A R292P I377N may be described as “GARPIN”, or the like.

In any of the presently disclosed embodiments, a variant of an Fc polypeptide or fragment thereof can be derived from or comprise a human Fc polypeptide or fragment thereof, and/or can be derived from or comprise a human IgGl, a human IgG2, a human IgG3, or a human IgG4 isotype. In this context, the expression "derived from" means that the variant is the same as the referenced polypeptide or isotype, with the exception of the specified modification(s) (e.g., amino acid substitution(s)). By way of example, a variant Fc polypeptide which comprises a wild-type human IgGl Fc amino acid sequence with the exception of the amino acid substitution mutations

G236A L328V Q295E (and, optionally, other amino acid substitutions) can be said to be "derived from" wild-type human IgGl Fc. In any of the presently disclosed embodiments, a polypeptide, CH2, Fc, Fc fragment, or antibody may comprise human Ig sequence, such as human IgGl sequence. In some embodiments, the polypeptide, CH2, Fc, Fc fragment, or antibody can comprise a native or wild-type human Ig sequence with the exception of the described mutation(s), or can comprise a human Ig (e.g. IgG) sequence that contains one or more additional mutations.

In certain embodiments, a variant Fc polypeptide comprises only the specified or recited amino acid mutations (e.g. substitutions), and does not comprise any further amino acid substitutions or mutations; e.g, relative to the reference polypeptide (e.g., a wild-type Fc polypeptide or fragment thereof). For example, in some embodiments, a variant Fc polypeptide comprising the amino acid substitutions G236A Y300L does not comprise any other amino acid substitutions; i.e., comprises an amino acid sequence that is wild-type except for G236A and Y300L.

In some embodiments, a variant Fc polypeptide may comprise one or more additional amino acid mutations (e.g. substitutions), which can be specified (e.g., M428L_N434S; M428L_N434A). In some embodiments, a further amino acid mutation or mutations is physically remote to the recited amino acid positions in tertiary structure, and/or is of such nature (e.g. is a conservative substitution), so that one or more function of the recited Fc variant or fragment thereof is not reduced or is reduced by no more than 50%, no more than 40%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10% or no more than 5%, or by no more than 10-fold, no more than 9-fold, no more than 8-fold, no more than 7-fold, no more than 6-fold, no more than 5-fold, no more than 4-fold, no more than 3-fold, no more than 2-fold, or no more than 1.5-fold. In some embodiments, a polypeptide comprises the mutations M428L and N434S or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, including those described herein. In certain embodiments, the present disclosure provides an antibody comprising (a) a heavy chain variable region (VH) that comprises a CDRH1 amino acid sequence according to SEQ ID NO.:34, a CDRH2 amino acid sequence according to SEQ ID NO.:35 or SEQ ID NO.:36, and a CDRH3 amino acid sequence according to SEQ ID NO.:37, wherein the CDRs are defined according to the CCG numbering system ; (b) a light chain variable region (VL) that comprises a CDRL1 amino acid sequence according to SEQ ID NOs.:40, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:43, 42, and 45, and a CDRL3 amino acid sequence according to SEQ ID NO.:46, wherein CDRs are defined according to the CCG numbering system, wherein, optionally, the VL comprises a R60N substitution mutation or a R60K substitution mutation relative to SEQ ID NO.:47, and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:47, and further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ ID NO.:47; and (c) a variant of an IgG Fc polypeptide or a fragment thereof comprising at least a portion of a(n e.g. human) IgGl CH2-CH3 or hinge-CH2-CH3 or CH1-CH3 comprising the amino acid mutation(s) set forth in any one of (i)-(xviii): (i) G236A, L328V, and Q295E; (ii) G236A, P230A, and Q295E; (iii) G236A, R292P, and I377N; (iv) G236A, K334A, and Q295E; (v) G236S, R292P, and Y300L; (vi) G236A and Y300L; (vii) G236A, R292P, and Y300L; (viii) G236S, G420V, G446E, and L309T; (ix) G236A and R292P; (x) R292P and Y300L; (xi) G236A and R292P; (xii) Y300L; (xiii) E345K, G236S, L235Y, and S267E; (xiv) E272R, L309T, S219Y, and S267E; (xv) G236Y; (xvi) G236W; (xvii) F243L, G446E, P396L, and S267E; (xviii) G236A, S239D, and H268E, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. In certain embodiments, the antibody is afucosylated. In some embodiments, the polypeptide or antibody further comprises one or more mutation that enhances binding to a human FcRn, such as M428L and N434S mutations or M428L and N434A mutations (EU numbering) or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the polypeptide is afucosylated. In some embodiments, the IgGl heavy chain comprises a CH1-CH3 or a CH2-CH3 or a hinge-CH2-CH3, wherein the CH1-CH3 or CH2-CH3 or hinge-CH2- CH3 has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identity to a wild-type human IgGl CH1- CH3 or CH2-CH3 or hinge-CH2-CH3, respectively.

In some embodiments, the antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs.:61-79, or a variant thereof, e.g. that further comprises one or more mutation that enhances binding to a human FcRn, such as M428L and N434S mutations or M428L and N434A mutations (EU numbering) or any other mutation(s) that enhance binding to a human FcRn, including those described herein. In some embodiments, the polypeptide or antibody comprises an amino acid sequence that differs from the amino acid sequence set forth in any one of SEQ ID NOs.: 61-79 only by one or more IgGl allotype-specific mutations and/or by the presence of M428L and N434S mutations or M428L and N434A mutations or other mutation(s) that enhance binding to a human FcRn.

An antibody of the present disclosure can be fucosylated (e.g., comprising one or more fucosyl moiety, and typically comprising a native (wild-type) fucosylation pattern or a fucosylation pattern that includes one or more additional, or fewer, fucosyl moieties as compared to native), or can be afucosylated. In particular, native IgGl antibodies carry a glycan site at N297, and this is typically the only site where a core fucose moiety may be found in the antibody, though some glycan sites may arise through mutation e.g. in the variable domains) during antibody development. Fucosylation of an Fc polypeptide or fragment thereof, or of an antibody, can be effected by introducing amino acid mutations to introduce or disrupt a fucosylation site (e.g. a mutation at N297, such as N297Q or N297A, to disrupt formation of a glycan that can include a core fucose moiety), though typically it is preferred to maintain N297 and the glycan thereof, such as by expressing the antibody in a host cell which has been genetically engineered to lack the ability (or have an inhibited or compromised ability) to fucosylate the antibody; by expressing the antibody under conditions in which a host cell is impaired in its ability to fucosylate the antibody (e.g., in the presence of 2-fluoro- L-fucose (2FF)), or the like. An afucosylated antibody can comprise no fucose moieties, or substantially no fucose moieties, and/or can be expressed by a host cell that is genetically engineered to lack the ability (or have an inhibited or compromised ability) to fucosylate the antibody and/or can be expressed under conditions in which a host cell is impaired in its ability to fucosylate the antibody (e.g., in the presence of 2- fluoro-L-fucose (2FF)). In some embodiments, a antibody does not comprise a core fucose moiety at Asn297. In some embodiments, afucosylated antibodies have increased binding to FcyRIIIA. In some contexts, addition of 2FF to a culture media comprising host cells expressing an antibody results in about 85% or more of the antibodies not carrying a fucose moiety. Accordingly, a plurality of antibodies may be described as “afucosylated” when the plurality was produced in the presence of 2FF or like reagent. In some contexts, a plurality of antibodies may be described as, for example, afucosylated, meaning that about 85% or more of the single molecules of the plurality do not comprise a fucose moiety. In certain preferred embodiments, an afucosylated antibody or a population or a plurality thereof comprises an asparagine (N) at EU position 297. Fucosylation or lack thereof can be assessed using, for example, mass spectrometry (e.g. Electrospray mass spectrometry (ESI-MS)). In some embodiments, compositions are provided that comprise a plurality of any one or more of the presently disclosed antibodies, wherein the composition comprises afucosylated antibodies.

Also in the present Examples, variant Fes including those comprising the mutations shown in Table 2 above were expressed in afucosylated human IgGl antibodies and tested for various properties, including by comparison to fucosylated wild-type human IgGl antibody. In some contexts, afucosylated polypeptides bearing Fc variants have similar or even improved properties as when fucosylated.

In certain embodiments, presently disclosed antibodies that comprise variants of IgG Fc polypeptides or fragments thereof possess one or more function that is distinct from (e.g. improved as compared to) the corresponding function of a reference Fc polypeptide that comprises the following mutation or mutations: G236A; G236S; G236A_A330L_I332E; G236A_A330L_I332E_M428L_N434S; A330LJ332E; or G236A_S239D_A330L_I332E. For example, in certain embodiments, a presently disclosed variant of an IgG Fc polypeptide or fragment thereof possesses one or more of the following properties, as compared to a reference Fc polypeptide that comprises the following mutation or mutations: G236A; G236S; G236A_A330L_I332E;

G236A_A330L_I332E_M428L_N434S; A330LJ332E; or G236A_S239D_A330L_I332E: increased binding (e.g. affinity) to and/or signaling via a human FcyRIIa H131; increased binding (e.g. affinity) to and/or signaling via a human FcyRIIa R131; decreased binding to (e.g. affinity) and/or signaling via human FcyRIIb; an increased ratio of binding to (e.g. affinity) and/or signaling via a human FcyRIIa (H131, R131, or both) versus the ratio of binding to or signaling via (respectively) a human FcyRIIb; increased binding (e.g. affinity) to and/or signaling via a human FcyRIIIa (VI 58, Fl 58, or both); increased binding (e.g. affinity) to a human Clq; a higher Tm; an improved production titer; an improved signaling in a host cell via a FcyRIIa (H131, R131, or both); increased facilitation of ADCP and/or ADCC by human NK cells and/or human PBMCs when in the presence of antigen-presenting cells; and an improved ability to stimulate moDCs when in an immune complex with antigen.

In the present disclosure, binding of an antibody comprising a variant Fc polypeptide or fragment may be described as increased (or "greater than", or the like) or decreased (or "reduced" or "less than", or the like) as compared to the binding of a comparator (e.g., to a reference wild-type IgGl Fc, or to a reference IgGl Fc that is wild-type except for M428L and N434S mutations or except for M428L and N434A mutations or to a variant IgGl Fc comprising G236A A330L I332E mutations) to a same binding partner. Binding interactions between a variant Fc polypeptide or fragment (or an antibody or polypeptide comprising the same) and a binding partner (e.g. a human FcyR, FcRn, or Clq) can preferably be determined using an electrochemiluminescence assay, more preferably using the Meso Scale Discovery ("MSD"; mesoscale.com) platform. MSD binding assay is similar to ELISA though MSD uses electrochemiluminescence, as opposed to colorimetry, as a detection technique. Other techniques for measuring binding interactions are known and include, for example, ELISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), and the like.

In some embodiments, binding includes affinity, avidity, or both. Affinity refers to the strength of a bond between a binding molecule and its binding partner. In some contexts, binding can include affinity and/or avidity. Unless otherwise indicated, avidity refers to the total binding strength of a molecule to a binding partner, and reflects binding affinity, valency of binding sites (e.g., whether an Fc polypeptide comprises one, two, or more binding sites), and, for example, whether another agent is present that can affect the binding (e.g., a non-competitive inhibitor of the Fc polypeptide).

A binding interaction between a variant Fc polypeptide of the present disclosure and a binding partner can be expressed in terms of fold-change relative to the binding interaction between a reference molecule and the binding partner. For example, binding of a presently disclosed antibody comprising a variant Fc to a human FcyRIIa may be stronger than the binding of the antibody comprising a wild-type Fc to the human FcyRIIa, and the relative increased strength of the variant can be expressed in terms of fold-change e.g., linear scale of area-under-the-curve) relative to the reference molecule binding using the same assay. For example, a variant Fc polypeptide or fragment may bind to a FcyRIIa with a 2-fold, 3-fold, 4-fold, or 5-fold greater binding strength than a reference Fc polypeptide or fragment binds to the FcyRIIa. As another example, a variant Fc polypeptide or fragment thereof may bind less strongly to a FcyRIIb as compared to a reference Fc or fragment thereof; e.g., may have a 0.9-fold binding, 0.8-fold binding, 0.7-fold binding, 0.6-fold binding binding, or the like, as compared to the reference Fc polypeptide or fragment thereof. It will be understood that, for example, the expression "2-fold greater binding as compared to the binding of a reference" means a 2-fold increase in binding as compared to the reference.

Moreover, binding of a variant molecule of the present disclosure to two different partner molecules can be described in terms of a ratio, and this ratio can be compared to a like ratio obtained using a reference molecule with the same assay. For example, a variant Fc polypeptide may bind to a human FcyRIIa H131 five times more strongly than it binds to a human FcyRIIb, while a reference wild-type Fc polypeptide binds to FcyRIIa H131 as strongly as it binds to a human FcyRIIb. In this example, the variant Fc polypeptide can be said to have a 5: 1 (binding FcyRIIIa H131 :binding FcyRIIb) binding ratio, which can be compared to the 1 :1 (binding FcyRIIIa H131 :binding FcyRIIb) binding ratio of the reference wild-type Fc polypeptide.

Variant Fc polypeptides of the present disclosure may also be described in terms of ability to induce signaling in a host cell, wherein the host cell expresses or overexpresses one or more FcyR (e.g., FcyRIIa H131, FcyRIIa R131, FcyRIIb, FcyRIIIa Fl 58, or FcyRIIIa VI 58) and the signaling is induced by binding of the variant Fc polypeptide to the FcyR. Reporter cells useful for determining signaling include, for example, cells in which NF AT drives expression of a luciferase reporter (e.g., available from Promega®).

Unless stated otherwise, FcyRs, FcRn, and Clq as described herein are human.

In some embodiments, an antibody comprising a variant Fc polypeptide or fragment is preferably capable of inducing one or more of: antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP); and complement-dependent cytotoxicity. Assays for measuring these functions are known.

In some embodiments, an antibody comprising a variant Fc polypeptide or fragment preferably has comparable binding to a human FcRn (e.g., at pH 6.0) and/or a comparable in vivo half-life in a mammal as compared to a reference antibody.

In some embodiments, an antibody comprising a variant Fc polypeptide or fragment preferably has increased binding to a human FcRn (e.g., at pH 6.0) and/or increased in vivo half-life in a mammal as compared to a reference antibody.

In some embodiments, an antibody comprising a variant Fc polypeptide or fragment preferably has a melting temperature (Tm) that is less than 12°C, less than 11°C, less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5 °C, less than 4°C, less than 3 °C, less than 2°C, or less than 1°C below the Tm of a reference antibody, or has a Tm that is higher than the Tm of the reference antibody. In some embodiments, the reference antibody comprises a wild-type human Fc polypeptide. In some embodiments, an antibody comprising a variant Fc polypeptide or fragment has a melting temperature that is higher than the melting temperature of a reference antibody that comprises the mutations G236A, A330L, I332E, and, optionally, M428L and N434S.

In some embodiments, an antibody comprising a variant Fc polypeptide or fragment is preferably capable of being produced in a host cell line (e.g., a CHO cell line) at least about as efficiently (e.g., produces at least about the same titer and/or within less than O.l-fold, less than 0.09-fold, less than 0.08-fold, less than 0.07-fold, less than 0.06-fold, less than 0.05-fold, less than 0.04-fold, less than 0.03-fold, less than 0.02-fold, or less than 0.02-fold less) as compared to a reference antibody.

In certain embodiments, an antibody is provided that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GALVQE”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody is provided that comprises a variant of: (i) an IgG hinge-CH2 polypeptide; or (ii) an IgG hinge-Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 230, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAPAQE”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody is provided that comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPIN”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody is provided that comprises a a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 334, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAKAQE”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody is provided that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a serine (S) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSRPYL”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody is provided that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPYL”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody is provided that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAYL”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody is provided that comprises a variant of: (i) an IgG CH2 polypeptide; or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, and a glutamic acid (E) at EU position 268. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GASDHE”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In some embodiments, the antibody comprising the variant Fc polypeptide has increased binding to a human FcyRIIa and/or has decreased binding to a human FcyRIIb, as compared to the binding of a reference antibody to the human FcyRIIa or the human FcyRIIb, respectively, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.

In certain embodiments, the increased binding to a human FcyRIIa comprises more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa as compared to the binding of a reference antibody (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa.

In some embodiments, the human FcyRIIa comprises H131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa H131 as compared to the binding of a reference antibody (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa H131.

In some embodiments, the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa R131 comprises more than 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference antibody (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa R131.

In some embodiments, the decreased binding to a human FcyRIIb comprises less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, or between 0.5- fold and 0.9-fold, of the binding of a reference antibody (optionally comprising a wildtype human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIb.

In any of the presently disclosed embodiments, (1) a ratio of (i) the binding of the variant or polypeptide to a human FcyRIIa to (ii) the binding of the variant or polypeptide, respectively, to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a reference polypeptide to the human FcyRIIa to (iv) the binding of the reference polypeptide to the human FcyRIIb, wherein the reference polypeptide optionally comprises a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the human FcyRIIa comprises H131, R131, or both. In some embodiments, the ratio in (1) is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, or at least 14-fold greater than the ratio in (2).

Also provided is an antibody comprising a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAYL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

In some embodiments, the antibody comprising the variant Fc polypeptide has increased binding to a human FcyRIIa as compared to the binding of a reference antibody to the human FcyRIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.

In some embodiments, the increased binding to a human FcyRIIa comprises at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, or at least 18-fold greater binding to the human FcyRIIa as compared to the binding of a reference antibody (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa.

In some embodiments, the human FcyRIIa comprises H131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16- fold, at least 17-fold, or at least 18-fold greater binding to the human FcyRIIa H131 as compared to the binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa H131.

In some embodiments, the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa R131 comprises at least 4-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g. IgGl) Fc polypeptide or a fragment thereof) to the human FcyRIIa R131.

In certain embodiments, (1) a ratio of (i) the binding of the variant Fc polypeptide to a human FcyRIIa to (ii) the binding of the variant Fc polypeptide, respectively, to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a reference polypeptide to the human FcyRIIa to (iv) the binding of the reference polypeptide to the human FcyRIIb, wherein the reference polypeptide comprises a wildtype human IgG Fc polypeptide or a fragment thereof. In certain embodiments, the human FcyRIIa comprises H131, R131, or both. In further embodiments, the ratio in (1) is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, at least 14-fold, at least 15-fold, at least 16-fold, or at least 17-fold greater than the ratio in (2).

Also provided is an antibody comprising a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPYL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

In certain embodiments, the variant Fc polypeptide has increased binding to a human FcyRIIIa, as compared to the binding of a reference polypeptide to the human FcyRIIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the increased binding to a human FcyRIIa comprises at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, or at least 14-fold greater binding to the human FcyRIIa as compared to the binding of a reference polypeptide optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.

In some embodiments, the human FcyRIIa comprises H131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, or at least 14-fold greater binding to the human FcyRIIa Hl 31 as compared to the binding of a reference polypeptide optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa H131.

In some embodiments, the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa H131 comprises at least 2-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference polypeptide optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa R131.

In certain embodiments, (1) a ratio of (i) the binding of the variant Fc polypeptide to a human FcyRIIa to (ii) the binding of the variant Fc polypeptide, respectively, to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a reference polypeptide to the human FcyRIIa to (iv) the binding of the reference polypeptide to the human FcyRIIb, wherein the reference polypeptide optionally comprises a wild-type human IgG Fc polypeptide or a fragment thereof, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the human FcyRIIa comprises H131, R131, or both. In some embodiments, the ratio in (1) is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, at least 14-fold, or at least 15-fold greater than the ratio in (2). In certain embodiments, the Fc variant polypeptide has increased binding to a human FcyRIIIa, as compared to the binding of a reference polypeptide to the human FcyRIIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the human FcyRIII comprises V158, F158, or both. In certain further embodiments, the increased binding to a human FcyRIIIa comprises greater than 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.0 fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, or at least 3.7-fold greater binding to the human FcyRIIIa as compared to the binding of a reference polypeptide optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIIa.

In certain embodiments, the variant, and optionally the polypeptide, is capable of binding to a human complement component Iq (Clq), wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.

Also provided is an antibodythat comprises a variant of an IgG Fc polypeptide, wherein the variant comprises a serine (S) at EU position 236, a valine (V) at EU position 420, a glutamic acid (E) at EU position 446, and a threonine (T) at EU position 309. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSGVGELT”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

Also provided is an antibody that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide, wherein the variant comprises an alanine (A) at EU position 236 and a proline (P) at EU position 292. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARP”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

In certan embodiments, the variant Fc polypeptide has decreased binding to a human FcyRIIb as compared to the binding of a reference polypeptide to the human FcyRIIb, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the decreased binding to a human FcyRIIb comprises less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, less than 0.5-fold, or less than 0.4-fold as compared to the binding of a reference polypeptide optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIb.

In further embodiments, the variant Fc polypeptide has increased binding to a human FcyRIIa as compared to the binding of a reference polypeptide to the human FcyRIIa, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.

In some embodiments, the increased binding to the human FcyRIIa comprises greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold greater binding to the human FcyRIIa as compared to the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.

In certain embodiments, the human FcyRIIa comprises H131, R131, or both.

In some embodiments, (1) a ratio of (i) the binding of the variant Fc polypeptide to a human FcyRIIa to (ii) the binding of the variant Fc polypeptide to a human FcyRIIb is greater than (2) a ratio of (iii) the binding of a reference polypeptide to the human FcyRIIa to (iv) the binding of the reference polypeptide to the human FcyRIIb, wherein the reference polypeptide optionally comprises a wild-type human IgG Fc polypeptide or a fragment thereof, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embedments, the human FcyRIIa comprises H131, R131, or both. In certain embodiments, the ratio in (1) is at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at least 11-fold, or at least 12-fold greater than the ratio in (2).

Also provided is an antibody that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide, wherein the variant comprises a proline (P) at EU position 292 and a leucine (L) at EU position 300, and wherein, optionally, variant and, further optionally, the polypeptide has increased binding to a human FcyRIIIa with as compared to the binding of a reference polypeptide to the human FcyRIIIa, wherein, optionally, the binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the IgG CH2 polypeptide or IgG Fc polypeptide comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or IgG Fc polypeptide (“RPYL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

In certain embodiments, the human FcyRIIIa comprises V158, F158, or both, and wherein the increased binding to the human FcyRIIIa comprises at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.1 -fold, or at least 5.2-fold greater binding as compared to the binding of a reference polypeptide optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.

Also provided is an antibody that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a leucine (L) at EU position 300. In some embodiments, the IgG CH2 polypeptide or IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“YL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

Also provided is an antibody that comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a lysine (K) at EU position 345, a serine (S) at EU position 236, tyrosine (Y) at EU position 235, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSEKLYSE”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoy slated.

Also provided is an antibody that comprises a variant of: (i) an IgG hinge-CH2 polypeptide or (ii) an IgG hinge-Fc polypeptide or a fragment thereof, wherein the variant comprises an arginine (R) at EU position 272, a threonine (T) at EU position 309, a tyrosine (Y) at EU position 219, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG hinge-CH2 polypeptide or an IgG hinge-Fc polypeptide or a fragment thereof comprises an (e.g. otherwise wild-type) IgGl hinge-CH2 polypeptide or IgG hinge-Fc polypeptide or a fragment thereof (“SYSEERLT”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

Also provided is an antibody that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a tyrosine (Y) at EU position 236. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GY”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

Also provided is an antibody that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a tryptophan (W) at EU position 236. In some embodiments, the IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or Fc polypeptide or fragment thereof (“GW”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoy slated.

Also provided is an antibody comprising a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, wherein the IgG Fc polypeptide or fragment thereof, and optionally the polypeptide, is afucosylated, and wherein, further optionally, the variant comprises a leucine (L) at EU position 330 and a glutamic acid (E) at EU postion 332, wherein, still further optionally, the variant does not comprise an aspartic acid (D) at EU position 239, and, even further optionally, comprises a serine (S) at EU position 239. In some embodiments, the IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or Fc polypeptide or fragment thereof (“GA-afuc” or “GAALIE- afuc”, respectively). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.

Also provided is an antibody that comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a leucine (L) at EU position 243, a glutamic acid (E) at EU position 446, a leucine (L) at EU position 396, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“FLSEPLGE”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody is afucoyslated.

Also provided is an antibody comprising a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, a glutamic acid (E) and EU position 332, a leucine (L) at EU position 428, and a serine (S) or an alanine (A) at EU position 434. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GASDIEMLNS” or “GASDIEMLNA”). In certain embodiments, the antibody has increased binding to a human Clq as compared to the binding of a reference antibody to the human Clq, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the increased binding to a human Clq comprises more than 1-fold, at least 1.5-fold, at least 1.75-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.0 fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, at least 3.7-fold, at least 3.8-fold, at least 3.9-fold, at least 4.0-fold, at least 4.1 -fold, or at least 4.15-fold greater binding to the human Clq as compared to the binding of a reference antibody comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human Clq.

In certain of the presently disclosed embodiments, the antibody: (i) is capable of binding to a human FcyRIIIa, wherein the human FcyRIIIa comprises a VI 58, a F158, or both; (ii) is capable of binding to a human FcyRIIIb; (iii) is capable of binding to a human FcRn, optionally at pH 6; (iv) is capable of binding to a human complement component Iq (Clq); (v) has a higher Tm and/or can be produced at a higher titer as compared to (1) a reference antibody comprising a human IgGl Fc polypeptide comprising the amino acid substitutions G236A, S239D, A330L, and I330E (EU numbering), and optionally not comprising any other amino acid substitutions relative to a wild-type human IgGl Fc polypeptide, (2) a reference antibody comprising a human IgGl Fc polypeptide comprising the amino acid substitutions G236A, A330L, and I330E (EU numbering), and optionally further comprising M428L and N434S mutations and/or M428L and N434A mutations and/or not comprising any other amino acid substitutions and/or not comprising S239D, relative to a wild-type human IgGl Fc polypeptide, (3) a reference antibody comprising a human IgGl Fc polypeptide comprising the amino acid substitution G236A or G236S (EU numbering), and optionally not comprising any other amino acid substitutions relative to a wild-type human IgGl Fc polypeptide, and/or (4) a reference antibody comprising a human IgGl Fc polypeptide comprising the amino acid substitutions A330L and I332E (EU numbering), and optionally not comprising any other amino acid substitutions relative to a wild-type human IgGl Fc polypeptide; (vi) is capable of promoting signaling through a FcyRa in a host cell, wherein, optionally, (a) signaling is optionally increased as compared to the signaling promoted by a reference polypeptide and/or (b) the FcyRa comprises FcyRIIa H131, FcyRIIa R131, FcyRIIIa V158, FcyRIIIa F158, or any combination thereof; (vii) at least when comprised in an antibody, is capable of promoting antibody-dependent cellular cytotoxicity (ADCC); (viii) at least when comprised in an antibody, is capable of promoting antibody-dependent phagocytosis (ADCP); (ix) at least when comprised in an antibody, is capable of promoting complement-dependent cytotoxicity (CDC); (x) at least when comprised in an antibody, is capable of forming an immune complex; or (xi) any combination of (i)-(x).

In any of the presently disclosed embodiments, the variant can further comprise one or more modification that enhances or further enhances binding to a human FcRn as compared to (1) a reference antibody that comprises a wild-type human IgGl Fc polypeptide and/or to (2) the antibody without the one or more modification. In some embodiments, the one or more modification that enhances binding to the human FcRn comprises the amino acid substitutions: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; (viii) M428L/N434A; or (ix) any combination of (i)-(viii).

In any of the presently disclosed embodiments, the variant Fc polypeptide may comprise no additional mutations as compared to the reference IgG Fc polypeptide or fragment thereof, the IgG hinge-CH2 polypeptide, or the IgG hinge-Fc polypeptide or fragment thereof, respectively. In other embodiments, the variant of an IgG Fc polypeptide comprises, at most: 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 additional amino acid substitution(s) relative to a wild-type or parental IgG Fc polypeptide, wherein one or more of the additional amino acid substitution(s) optionally comprises a conservative amino acid substitution. In other embodiments, the variant of an IgG Fc polypeptide has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at leat 92%, at least 93%, at least 94%, at least 95%, at least 96%, or at least 97% identity to a wild-type or parental IgG Fc polypeptide.

In certain embodiments, the present disclosure provides an antibody comprising (i) a heavy chain variable region (VH) that comprises a CDRH1 amino acid sequence according to SEQ ID NO.:34, a CDRH2 amino acid sequence according to SEQ ID NO.:35 or SEQ ID NO.:36, and a CDRH3 amino acid sequence according to SEQ ID NO.:37, wherein the CDRs are defined according to the CCG numbering system ; (ii) a light chain variable region (VL) that comprises a CDRL1 amino acid sequence according to SEQ ID NOs.:40, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:43, 42, and 45, and a CDRL3 amino acid sequence according to SEQ ID NO.:46, wherein CDRs are defined according to the CCG numbering system; and (iii) a variant of an IgG Fc polypeptide or a fragment thereof comprising: (a) an alanine (A) at EU position 236 and a leucine (L) at EU position 300; (b) an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295; (c) an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300; or (d) an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377; wherein, optionally, the VL comprises a R60N substitution mutation, a R60K substitution mutation, or any combination thereof, relative to SEQ ID NO.:47 and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:47, and further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ ID NO.:47, and still further optionally wherein the antibody is afucosylated, and wherein the antibody is capable of binding to the antigenic loop region of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof and/or hepatitis D virus (HDV). In certain embodiments, the variant IgG Fc polypeptide or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 61, 63, 66, or 67.

In some embodiments, the antibody comprises: (i) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences are according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 43, and 46, respectively; (ii) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences according to SEQ ID NOs.:34, 35, and 37, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.: 40, 42, and 46, respectively, and the VL comprises a R60N substitution mutation relative to SEQ ID NO.:47; (iii) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 42, and 46, respectively, and the VL comprises a R60K substitution mutation relative to SEQ ID NO.:47; or (iv) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences are according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 45, and 46, respectively, wherein CDRs are according to CCG.

In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.: (i) 34, 35, 37, 40, 43, and 46, respectively; (ii) 34, 35, 37, 40, 42, and 46, respectively; (iii) 34, 35, 37, 40, 45, and 46, respectively; or (iv) 34, 35, 37, 40, 42, and 46, respectively, wherein CDRs are according to CCG, optionally wherein the VL further comprises a R60N substitution mutation or a R60K substitution mutation relative to SEQ ID NO.:47, and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:47, and further optionally wherein the VL does not comprise any other mutation(s) relative to SEQ ID NO.:47.

In certain embodiments, the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of: HBC34-v40; HBC34-v44; HBC34-v45; or HBC34- v50, wherein the CDRs are according to CCG, optionally wherein the VL further comprises a R60N substitution mutation or a R60K substitution mutation relative to SEQ ID NO.:47, and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:47, and further optionally wherein the VL does not comprise any other mutation(s) relative to SEQ ID NO.:47.

Table 3 provides the CDR amino acid SEQ ID NOs. of certain antibodies, wherein CDRs are defined according to IMGT (short and long versions of CDRH2 and CDRL2 are disclosed).

Table 3. CDR (IMGT numbering) amino acid SEQ ID NOs. of Certain Antibodies

In certain embodiments, the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of: HBC34-v40; HBC34-v44; HBC34-v45; or HBC34- v50, wherein the CDRs are according to IMGT, optionally wherein the VL further comprises a R60N substitution mutation or a R60K substitution mutation relative to SEQ ID NO.:47 and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:47, and further optionally wherein the VL does not comprise any other mutation(s) relative to SEQ ID NO. :47.

Table 4 provides the VH and VL amino acid SEQ ID NOs. of certain antibodies.

Table 4. VH and VL amino acid SEQ ID NOs. of Certain Antibodies

In certain embodiments, an antibody comprises the VH and VL amino acid sequences of: HBC34-v40; HBC34-v44; HBC34-v45; or HBC34-v50.

In certain embodiments, the antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein: (i) the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or (ii) the VL comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52. In particular embodiments, the VH and the VL comprise, consists essentially of, or consist of amino acid sequences having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any non-integer value therebetween) identity to the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 49, respectively; (ii) 38 and 50, respectively; (iii) 38 and 51, respectively; or (iv) 38 and 52, respectively. As a non-limiting example, in certain embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO.:38 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO.:49.

In some embodiments, the VH comprises, consist essentially of, or consists of the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or the VL comprises, consist essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52. In particular embodiments, the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 49, respectively; (ii) 38 and 50, respectively; (iii) 38 and 51, respectively; or (iv) 38 and 52, respectively.

In some embodiments, the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO. : 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In some embodiments, the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:66. In some embodiments, the VL comprises, consists essentially of, or consists of an amino acid sequence having at least amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52.

In some embodiments, the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO. : 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295. In some embodiments, the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:61. In some embodiments, the VL comprises, consists essentially of, or consists of an amino acid sequence having at least amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52.

In some embodiments, the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO. : 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:67. In some embodiments, the VL comprises, consists essentially of, or consists of an amino acid sequence having at least amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52.

In some embodiments, the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO. : 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377. In some embodiments, the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:63. In some embodiments, the VL comprises, consists essentially of, or consists of an amino acid sequence having at least amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:49; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 61.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 50; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 61.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 61. In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 52; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 61.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 49; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 63.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 50; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 63.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 63.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 52; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 63. In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 49; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 66.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 50; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 66.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 66.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth SEQ ID NO.: 52; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 66.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 49; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 67. In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 50; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 67.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 67.

In some embodiments, disclosed herein is an antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 52; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 67.

In some embodiments, disclosed herein is an antibody comprising: (i) a heavy chain variable region (VH) that comprises a CDRH1 amino acid sequence according to SEQ ID NO.:34, a CDRH2 amino acid sequence according to SEQ ID NO.:35 or SEQ ID NO.:36, and a CDRH3 amino acid sequence according to SEQ ID NO.:37; (ii) a light chain variable region (VL) that comprises a CDRL1 amino acid sequence according to SEQ ID NOs.:40, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:43, 42, and 45, and a CDRL3 amino acid sequence according to SEQ ID NO.:46; wherein CDRs are defined according to the CCG numbering system; and (iii) a variant of an IgG Fc polypeptide or a fragment thereof comprising an alanine (A) at EU position 236; wherein the antibody is afucosylated, and wherein the antibody is capable of binding to the antigenic loop region of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof and/or hepatitis D virus (HDV).

In some embodiments, disclosed herein is an antibody, comprising: (a) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (b) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 49; and (c) a variant IgG Fc polypeptide or a fragment thereof, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; (v) M428L, N434S, G236A, L328V, and Q295E, wherein the polypeptide or antibody is afucosylated; (vi) M428L, N434S, G236A, R292P, and I377N, wherein the polypeptide or antibody is afucosylated; (vii) M428L, N434S, G236A, and Y300L, wherein the polypeptide or antibody is afucosylated; or (viii) M428L, N434S, G236A, R292P, and Y300L, wherein the antibody is afucosylated. In some embodiments, the variant of an (e.g. IgGl) IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain.

In some embodiments, disclosed herein is an antibody, comprising: (a) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (b) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 50; and (c) a variant IgG Fc polypeptide or a fragment thereof, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; (v) M428L, N434S, G236A, L328V, and Q295E, wherein the polypeptide or antibody is afucosylated; (vi) M428L, N434S, G236A, R292P, and I377N, wherein the polypeptide or antibody is afucosylated; (vii) M428L, N434S, G236A, and Y300L, wherein the polypeptide or antibody is afucosylated; or (viii) M428L, N434S, G236A, R292P, and Y300L, wherein the antibody is afucosylated. In some embodiments, the variant of an (e.g. IgGl) IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain. In some embodiments, disclosed herein is an antibody, comprising: (a) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (b) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 51; and (c) a variant IgG Fc polypeptide or a fragment thereof, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; (v) M428L, N434S, G236A, L328V, and Q295E, wherein the polypeptide or antibody is afucosylated; (vi) M428L, N434S, G236A, R292P, and I377N, wherein the polypeptide or antibody is afucosylated; (vii) M428L, N434S, G236A, and Y300L, wherein the polypeptide or antibody is afucosylated; or (viii) M428L, N434S, G236A, R292P, and Y300L, wherein the antibody is afucosylated. In some embodiments, the variant of an (e.g. IgGl) IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain.

In some embodiments, disclosed herein is an antibody, comprising: (a) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (b) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 52; and (c) a variant IgG Fc polypeptide or a fragment thereof, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; (v) M428L, N434S, G236A, L328V, and Q295E, wherein the polypeptide or antibody is afucosylated; (vi) M428L, N434S, G236A, R292P, and I377N, wherein the polypeptide or antibody is afucosylated; (vii) M428L, N434S, G236A, and Y300L, wherein the polypeptide or antibody is afucosylated; or (viii) M428L, N434S, G236A, R292P, and Y300L, wherein the antibody is afucosylated. In some embodiments, the variant of an (e.g. IgGl) IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain.

In some embodiments, the polypeptide or antibody comprises an IgGl isotype. In certain embodiments, the polypeptide or antibody comprises an IgGlml7 allotype, an IgGl ml 7, 1 allotype, an IgGl m3 allotype, or an IgGl m3, 1 allotype.

In some embodiments, the variant of an IgG Fc polypeptide does not comprise any other amino acid substitution mutations relative to a wild-type or parental IgG Fc polypeptide. In other embodiments, the variant of an IgG Fc polypeptide comprises, at most: 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 additional amino acid substitution s) relative to a wild-type or parental IgG Fc polypeptide, wherein one or more of the additional amino acid substitution(s) optionally comprises a conservative amino acid substitution. In other embodiments, the variant of an IgG Fc polypeptide has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at leat 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, or at least 97% identity to a wild-type or parental IgG Fc polypeptide.

In certain embodiments, the VH and the variant of an IgG Fc polypeptide are comprised in a heavy chain, and the heavy chain comprises VH-CH1-CH2-CH3. In certain embodiments, the VL is comprised in a light chain that further comprises an (e.g. IgGl) kappa light chain. In other embodiments, the VL is comprised in a light chain that further comprises an (e.g. IgGl) lambda light chain. In any of the presently disclosed fantibodies, the variant Fc or fragment thereof can be derived from an IgGl isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype. In certain embodiments, the variant is derived from a human Fc or a fragment thereof, or from a human antibody heavy chain or a fragment thereof. In further embodiments, the variant is derived from a human IgGl isotype, a human IgG2 isotype, a human IgG3 isotype, or a human IgG3 isotype. In particular embodiments, the variant is derived from a human IgGl isotype.

An antibody, Fc, Fc fragment or portion may be of any allotype or combination of allotypes. “Allotype” refers to the allelic variation found among the IgG subclasses. For example, an allotype may comprise Glml (or Glm(a)), Glm2 (or Glm(x)), Glm3 (or Glm(f)), Glml7 (or Gm(z))m), Glm27, and/or Glm28 (Glm27 and Glm28 have been described as “alloallotypes”).

The Glm3 and Glml 7 allotypes are located at the same position in the CHI domain (position 214 according to EU numbering). Glm3 comprises R214 (EU), while Glml7 comprises K214 (EU). The Glml allotype is located in the CH3 domain (at positions 356 and 358 (EU)) and refers to the replacements E356D and M358L. The Glm2 allotype refers to a replacement of the alanine in position 431 (EU) by a glycine. Glm allotypes, alloallotypes, and features thereof are known in the art and described at, for example, www.imgt.org/IMGTrepertoire/Proteins/allotypes/human/ IGH/IGHC7G I m_allotypes.html and Lefranc, M.-P. and Lefranc, G. Human Gm, Km and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism In: B. Tait, F. Christiansen (Eds.), Immunogenetics, chap. 34, Humana Press, Springer, New York, USA. Methods Mol. Biol. 2012; 882, 635-680. PMID: 22665258, LIGM: 406, the contents and allotypes and allotype information of which are incorporated herein by reference.

The Glml allotype may be combined, for example, with the Glm3, Glml7, Glm27, Glm2, and/or Glm28 allotype. In some embodiments, an allotype is Glm3 with no Glml (Glm3,-1). In some embodiments, an allotype is Glml7,l allotype. In some embodiments, an allotype is Glm3,l. In some embodiments, an allotype is Glml7 with no Glml (Glml7,-1). Optionally, these allotypes may be combined (or not combined) with the Glm2, Glm27 or Glm28 allotype. For example, an allotype may be Glml7,l,2.

In some embodiments, an antibody of the present disclosure comprises a Glm3 allotype or a Glm3,l allotype. In some embodiments, an antibody of the present disclosure comprises a Glm3 allotype and comprises M428L and N434S or M428L and N434A mutations or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In some embodiments, an antibody of the present disclosure comprises a Glm3,l allotype and comprises M428L and N434S or M428L and N434A mutations or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In some embodiments, an antibody of the present disclosure comprises a Glml7, 1 allotype. In some embodiments, a an antibody of the present disclosure comprises a Glml7, 1 allotype and comprises M428L and N434S or M428L and N434A mutations or any other mutation(s) that enhance binding to a human FcRn, as described further herein.

In some embodiments, the antibody or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs.:61-79, or a variant thereof, e.g., that further comprises one or more mutation that enhances binding to a human FcRn, such as M428L and N434S mutations or M428L and N434A mutations.

In any of the presently disclosed embodiments, a variant Fc of an antibody may not comprise any additional mutations as compared to a reference wild-type IgG Fc.

As discussed further herein in, presently disclosed antibodies have a reduced propensity to form aggregates (e.g., dimers), and/or have improved productivity (e.g., higher titer) in a host cell, and/or have similar or substantially identical or even improved: binding to HBsAg; HBV neutralization; and/or thermostability, as compared to a reference antibody disclosed herein.

As a non-limiting example, presently disclosed antibodies produce fewer aggregates (e.g., in the form of antibody: antibody dimers, antibody: antigen-binding fragment dimers, or antigen-binding fragment: antigen-binding fragment dimers), and/or have a higher production titer in a host cell, as compared to a reference antibody or antigen-binding fragment, respectively. In this context, a dimer is a complex or aggregate comprising two antibody or antigen-binding fragment molecules (e.g., an antibody: antibody dimer, a Fab:Fab dimer, or an antibody:Fab dimer). As discussed further herein, dimerization in this context is distinct from typical associations between antibody heavy chain and light chain components, or between two antibody heavy chain polypeptides, that occur in the formation of an intact tetrameric antibody, Fv, or Fab and may involve associations between two monomers. Accordingly, it will be understood that in the present context, a "dimer" or does not refer to the association of an antibody heavy chain with an antibody light chain to provide a half-antibody comprising a functional Fab, and also does not include association of two heavy chains of an antibody (e.g., hinge-hinge and Fc-Fc) or VH-VL associations (e.g. that occur via disulfide bonds), such as in a Fv or in a Fab.

In certain embodiments, a dimer is formed by association between the VLs of two discrete antibody or antigen-binding fragment molecules. Such dimerization can, for example, reduce binding valency and/or binding affinity and/or avidity and/or neutralization potency of one or both of the antibody or antigen-binding fragment molecules comprised therein. In general, an increased presence of such dimers in a composition comprising a plurality of antibodies or antigen-binding fragments reduces the overall binding and/or neutralizing potency of the composition.

Antibody or antigen-binding fragment dimers can be identified using, known techniques, such as, for example, size-exclusion chromatography. A dimer will have a molecular weight that is higher than the molecular weight of each individual (monomer) subunit thereof, and will typically equal or approximate the sum of the molecular weights of each individual subunit thereof. For example, a homo-dimer (z.e., which comprises two antibody molecules that are identical or substantially identical in their amino acid sequences) will generally have a molecular weight that is about twice the molecular weight of each monomeric subunit thereof. For example, a typical human IgGl immunoglobulin molecule has a molecular weight of around 150 kilodaltons (for example, with each of the two heavy chains weighing around 50 kilodaltons, and each of the two light chains weighing around 25 kilodaltons), and a dimer comprising two such immunoglobulin molecules will have a molecular weight of around 300 kilodaltons. Of course, it will be understood that one antibody may have a slightly or somewhat different molecular weight than a different antibody of the same general structure and isotype, e.g., due at least in-part to any differences in the respective amino acid sequences.

As another, non-limiting, example, an antibody molecule may have a molecular weight of between 140 kilodaltons and 160 kilodaltons, and an antibody dimer comprising two antibody molecules may have a molecular weight of between 280 kilodaltons and 320 kilodaltons. Dimers may be referred-to as "high-molecular weight species" or "HMWS".

The presence of dimers in a composition or sample comprising a plurality of antibody molecules can be evaluated using, for example, absolute size exclusion chromatography (aSEC). The amount of dimer in a composition or sample can be expressed as the percentage of total antibody in the composition or sample that are comprised in a dimer. By way of illustration, for an antibody composition comprising 12% dimers, 88% of the total antibody molecules in the sample are present as monomers.

In any of the presently disclosed embodiments, in a sample comprising a plurality of the antibody (z.e., a plurality of antibody molecules), less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of the plurality is comprised in a dimer when the sample has been incubated for about 120 to about 168 hours at about 40°C, wherein, optionally, the presence of dimer is determined by absolute size-exclusion chromatography.

In any of the presently disclosed embodiments, incubation of a plurality of presently disclosed antibody results in reduced formation of dimers as compared to incubation of a plurality of a reference antibody or antigen-binding fragment molecules, wherein the reference antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 40, 42, and 56, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47, and wherein, optionally, the presence of antibody dimer is determined by absolute size-exclusion chromatography. Such a reference antibody can form dimers that, in some embodiments, collectively comprise more than 2%, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, or up to 12% of the antibody molecules in a sample (e.g., when incubated for about 120 to about 168 hours at about 40°C). In other words, in some embodiments, up to 12% or more of reference antibody molecules are comprised in a dimer, while a lesser percentage, preferably 2% or less, of presently disclosed antibody molecules are comprised in a dimer.

In some embodiments, a presently disclosed antibody forms a lower amount of dimer, and/or forms dimers at a reduced frequency and/or as a lower percentage of total antibody molecules in a sample or composition, (e.g., as determined using Size Exclusion Chromatography) as compared to a reference antibody: (i) in a 5-day, a 15- day, and/or a 32-day incubation at 4°C; (ii) in a 5-day, a 15-day, and/or a 32-day incubation at 25°C; and/or (iii) in a 5-day, a 15-day, and/or a 32-day incubation at 40°C, wherein the reference antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 40, 42, and 56, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47.

In some embodiments, the percentage of presently disclosed antibody molecules in a composition that are comprised in a dimer is less than 4/5, less than 3/4, less than 1/2, less than 1/3, less than 1/4, less than 1/5, less than 1/6, less than 1/7, less than 1/8, less than 1/9, or less than 1/10 the percentage of the reference antibody molecules in a composition that are present in a dimer, respectively. As a non-limiting example, following a 32-day (768-hour) incubation at 40°C, 22% or more of the reference antibody molecules in a composition can be comprised in a dimer, while 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of presently disclosed antibody molecules in a composition are comprised in a dimer, respectively.

In some embodiments, a host cell (e.g., a CHO cell such as an ExpiCHO cell) transfected with a polynucleotide encoding a presently disclosed antibody provides 1.5x or more, 2x or more, 3x or more, or 4x or more the amount of antibody, respectively, (e.g., measured as a concentration in mg/mL) than a reference host cell transfected with a polynucleotide encoding a reference antibody, wherein the reference antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 40, 42, and 56, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47.

In some embodiments, a presently disclosed antibody is produced in transfected cells at a higher titer as compared to a reference antibody is produced in reference transfected cells, wherein the reference antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 40, 42, and 56, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47.

In some embodiments, a presently disclosed antibody is produced in transfected cells at titers of at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 4-fold, higher than the titer at which a reference antibody is produced, wherein the reference antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 40, 42, and 56, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47.

In any of the presently disclosed embodiments, the antibody is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of 3.5 or less, about 3.2 or less, less than 3.0, less than 2.5, less than 2.0, less than 1.5, or less than 1.0. In some embodiments, the antibody is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of less than 3.5, less than 3.4, less than 3.3, less than 3.2, less than 3.1, less than 3.0, less than 2.9, less than 2.8, less than 2.7, less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.1, less than 2.0, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, or less than 1.0. In some embodiments, the antibody is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of between 0.9 and 2.0, or of between 0.9 and 1.9, or of between 0.9 and 1.8, or of between 0.9 and 1.7, or of between 0.9 and 1.6, or of between 0.9 and 1.5, or of between 0.9 and 1.4, or of between 0.9 and 1.3, or of between 0.9 and 1.2, or of between 0.9 and 1.1, or of between 0.9 and 1.0, or of between 1.0 and 2.0. In certain embodiments, the antibody is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of 2.0 or less. In some embodiments, a binding EC50 is determined by ELISA (e.g., direct antigen-binding ELISA assay, with binding curves determined by fitting the curves using Graphpad prism).

In any of the presently disclosed embodiments, the antibody is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50 of less than 20 ng/ml, preferably 15 ng/ml or less, more preferably 10 ng/mL or less. In some embodiments, the antibody thereof is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50 of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 ng/mL. In some embodiments, the antibody is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50 that is lower than the neutralization of infection EC50 (using the same assay) of a reference antibody that comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 40, 42, and 56, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47. In some embodiments, a neutralization of infection EC50 is determined following incubation of cultured cells, e.g., differentiated HepaRG cells, with a fixed amount of HBV in the presence or absence of the antibody to be tested. In such an embodiment, incubation may be carried out, for example, for 16 hours at 37°C. That incubation may be performed in a medium (e.g. supplemented with 4% PEG 8000). After incubation, cells may be washed and further cultivated. To measure virus infectivity, the levels of hepatitis B surface antigen (HBsAg) and/or hepatitis B e antigen (HBeAg) secreted into the culture supernatant, e.g. from day 7 to day 11 post-infection, may be determined by enzyme-linked immunosorbent assay (ELISA). Levels of HBsAg and/or HBeAg from treated cells can be compared to those of untreated cells to determine the presence and extent of neutralization.

"Single-chain Fv" also abbreviated as "sFv" or "scFv", are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for antigen binding. Such a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art. Additionally or alternatively, Fv can have a disulfide bond formed between and stabilizing the VH and the VL. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269- 315 (1994); Borrebaeck 1995, infra. In certain embodiments, the antibody or antigenbinding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain. In particular embodiments, a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker-VL orientation or in a VL-linker-VH orientation. Any scFv of the present disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some embodiments, a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both.

Peptide linker sequences may be chosen, for example, based on: (1) their ability to adopt a flexible extended conformation; (2) their inability or lack of ability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides and/or on a target molecule; and/or (3) the lack or relative lack of hydrophobic or charged residues that might react with the polypeptides and/or target molecule. Other considerations regarding linker design (e.g., length) can include the conformation or range of conformations in which the VH and VL can form a functional antigen-binding site. In certain embodiments, peptide linker sequences contain, for example, Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala, may also be included in a linker sequence. Other amino acid sequences which may be usefully employed as linker include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233, and U.S. Pat. No. 4,751,180. Other illustrative and non-limiting examples of linkers may include, for example, those disclosed by Chaudhary et al., Proc. Natl. Acad. Sci. USA 87: 1066-1070 (1990), and Bird et al., Science 242:423-426 (1988)) and a pentamer of four glycine residues linked in series, the C-terminal glycine of the series being linked to a single serine, when present in a single iteration or repeated 1 to 5 or more times, or more. Any suitable linker may be used, and in general can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 15 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker), and will preferably be biologically inert and/or have a low risk of immunogenicity in a human. scFv can be constructed using any combination of the VH and VL sequences or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein. In some embodiments, linker sequences are not required; for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

In some embodiments, an antibody comprises a light chain constant region (or a portion or fragment thereof), a heavy chain constant region (or a portion or fragment thereof), or both. The term "CL" refers to an "immunoglobulin light chain constant region" or a "light chain constant region," i.e., a constant region from an antibody light chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or a "heavy chain constant region," which is further divisible, depending on the antibody isotype into CHI, CH2, and CH3 (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM). The Fc region of an antibody heavy chain is described further herein. In any of the presently disclosed embodiments, an antibody or antigen-binding fragment of the present disclosure comprises any one or more of CL, a CHI, a CH2, and a CH3. In certain embodiments, a CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.: 100.

It will be understood that, for example, production in a mammalian cell line can remove one or more C-terminal lysine of an antibody heavy chain (see, e.g., Liu et al. mAbs 6(5): 1145-1154 (2014)). Accordingly, an antibody or antigen-binding fragment of the present disclosure can comprise a heavy chain, a CH1-CH3, a CH3, or an Fc polypeptide wherein a C-terminal residue is present or is absent; in other words, encompassed are embodiments wherein the C-terminal residue of a heavy chain, a CH1-CH3, or an Fc moiety is not a lysine, and embodiments where a lysine is the C- terminal residue. In certain embodiments, a composition comprises a plurality of an antibody and/or an antigen-binding fragment of the present disclosure, wherein one or more antibody or antigen-binding fragment does not comprise a lysine residue at the C- terminal end of the heavy chain, CH1-CH3, or Fc moiety, and wherein one or more antibody or antigen-binding fragment comprises a lysine residue at the C-terminal end of the heavy chain, CH1-CH3, or Fc moiety.

A "Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, z.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Both the Fab and F(ab’)2 are examples of "antigenbinding fragments." Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. Fab fragments may be joined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as "scFab." In these embodiments, an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain. A heavy chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VH + CHI, or "Fd") and a light chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VL + CL) may be linked in any arrangement to form a scFab. For example, a scFab may be arranged, in N-terminal to C-terminal direction, according to (heavy chain Fab fragment - linker - light chain Fab fragment) or (light chain Fab fragment - linker - heavy chain Fab fragment). Peptide linkers and exemplary linker sequences for use in scFabs are discussed in further detail herein.

In any of the presently disclosed embodiments, the antibody, or the antigenbinding fragment thereof, comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, a Fv, or a scFv.

Fragments of the antibodies described herein can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by cloning and expression of part of the sequences of the heavy or light chains. The present disclosure encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody as described herein, including, for example, an scFv comprising the CDRs (and, optionally, the variable regions) from an antibody according to the present description, heavy or light chain monomers and dimers (z.e., VH-VL dimer, HC-LC dimer, HC-HC dimer), single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, in which the heavy and light chain variable domains or regions are joined by a peptide linker. In certain embodiments, an antibody according to the present disclosure, or an antigen binding fragment thereof, comprises a purified antibody, a monoclonal antibody, a single chain antibody, Fab, Fab’, F(ab')2, Fv or scFv.

Antibodies and antigen binding fragments of the present disclosure may, in embodiments, be multispecific (e.g., bispecific, trispecific, tetraspecific, or the like), and may be provided in any multispecific format, as disclosed herein. In certain embodiments, an antibody or antigen-binding fragment of the present disclosure is a multispecific antibody, such as a bispecific or trispecific antibody. Formats for bispecific antibodies are disclosed in, for example, Spiess etal., Mol. Immunol. 67(2):95 (2015), and in Brinkmann and Kontermann, mAbs 9(2): 182-212 (2017), which bispecific formats and methods of making the same are incorporated herein by reference and include, for example, Bispecific T cell Engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH Common Light- Chain antibodies, TandAbs, Triple Bodies, TriBi Minibodies, Fab-scFv, scFv-CH-CL- scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y assemblies, Fcabs, Kk-bodies, orthogonal Fabs, DVD-IgGs, 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 DVLIgG (four-in-one). A bispecific or multispecific antibody may comprise a HBV- and/or HDV-specific binding domain of the instant disclosure in combination with another HBV- and/or HDV-specific binding domain of the instant disclosure, or in combination with a different binding domain that specifically binds to HBV and/or HDV (e.g., at a same or a different epitope), or with a binding domain that specifically binds to a different antigen.

Antibody fragments of the disclosure may impart monovalent or multivalent interactions and be contained in a variety of structures as described above. For instance, scFv molecules may be synthesized to create a trivalent "triabody" or a tetravalent "tetrabody". The scFv molecules may include a domain of the Fc region resulting in bivalent minibodies. In addition, the sequences of the disclosure may be a component of multispecific molecules in which the sequences of the disclosure target the epitopes of the disclosure and other regions of the molecule bind to other targets. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Antibodies or antigen-binding fragments thereof such as those described herein, including but not limited to scFv, may, in certain embodiments, be comprised in a fusion protein that is capable of specifically binding to an antigen as described herein.

As used herein, "fusion protein" refers to a protein that, in a single chain, has at least two distinct domains or motifs, wherein the domains or motifs are not naturally found together, or in the given arrangement, in a protein. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized.

Throughout this disclosure, antibodies, antigen binding fragments thereof, and fusion proteins may individually or collectively (e.g., in any combination) be referred to as "binding proteins".

Binding proteins according to the present disclosure may be provided in purified form. For example, an antibody may be present in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.

Binding proteins according to the present disclosure may be immunogenic in human and/or in non-human (or heterologous) hosts; e.g., in mice. For example, an antibody may have an idiotope that is immunogenic in non-human hosts, but not in a human host. Antibodies of the disclosure for human use include those that are not typically isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, or the like, and in some instances are not obtained by humanization or from xeno-mice. Also contemplated herein are variant forms of the disclosed antibodies, which are engineered so as to reduce known or potential immunogenicity and/or other potential liabilities, or to confer a desired structure and/or functionality of the antibody in a non- human animal, such as a mouse (e.g., a "murinized" antibody wherein one or more human amino acid residue, sequence, or motif is replaced by a residue, sequence, or motif that has reduced or abrogated immunogenicity or other liability, or has a desired structure and/or function, in a mouse; e.g., for model studies using a mouse).

As used herein, a "neutralizing antibody" (or antigen binding fragment, or fusion protein) is one that can neutralize, z.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host (e.g., host organism or host cell). The terms "neutralizing antibody" and "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used alone, or in combination (e.g., two or more of the presently disclosed antibodies in a combination, or an antibody of the present disclosure in combination with another agent, which may or may not be an antibody agent, including an antibody that is capable of neutralizing an HBV B and/or HBV D infection), as prophylactic or therapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool as described herein. Accordingly, presently disclosed antibodies or antigen-binding fragments are capable of neutralizing infection by a HBV, a HDV, or both.

As used herein, "specifically binds" or "specific for" refers to an association or union of a binding protein (e.g., an antibody or antigen binding fragment thereof) or a binding domain to a target molecule with an affinity or Ka (ie., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10⁵ M'¹ (which equals the ratio of the on-rate [K_on] to the off rate [Koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding proteins or binding domains may be classified as "high-affinity" binding proteins or binding domains or as "low-affinity" binding proteins or binding domains. "High-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of at least 10⁷ M'¹, at least 10⁸ M'¹, at least 10⁹ M'¹, at least IO¹⁰ M'¹, at least 10¹¹ M'¹, at least 10¹² M" or at least 10¹³ M'¹. "Low-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of up to 10⁷ M'¹, up to 10⁶ M'¹, or up to 10⁵ M'¹. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10'⁵ M to 10'¹³ M). The terms "binding" and "specifically binding" and similar references do not encompass non-specific sticking.

Binding of a binding protein can be determined or assessed using an appropriate assay, such as, for example, Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform); isothermal titration calorimetry (ITC), or the like, an antigen-binding ELISA (e.g., direct or indirect) with imaging by, e.g., optical density at 450nm, or by flow cytometry, or the like.

In certain embodiments, binding proteins according to the present disclosure can bind to the antigenic loop region of HBsAg. The envelope of the hepatitis B virus generally contains three "HBV envelope proteins" (also known as "HBsAg", "hepatitis B surface antigen"): S protein (for "small", also referred to as S-HBsAg), M protein (for "middle", also referred to as M-HBsAg) and L protein (for "large", also referred to as L- HBsAg). S-HBsAg, M-HBsAg and L-HBsAg share the same C-terminal extremity (also referred to as "S domain", 226 amino acids), which corresponds to the S protein (S- HBsAg) and which is crucial for virus 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 comprises four predicted transmembrane (TM) domains, whereby both the N-terminus as well as the C- terminus of the S domain are exposed to the lumen. The transmembrane domains TM1 and TM2 are both believed necessary for cotranslational protein integration into the ER membrane and the transmembrane domains TM3 and TM4 are located in 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, whereby the antigenic loop region comprises amino acids 101 - 172 of the S domain, which contains 226 amino acids in total (Salisse J. and Sureau C., 2009, Journal of Virology 83: 9321-9328). A determinant of infectivity resides in the antigenic loop region of HBV envelope proteins. In particular, residues between 119 and 125 of the HBsAg contain a CXXC motif, which is considered to be important for the infectivity of HBV and HDV (Jaoude GA, Sureau C, Journal of Virology, 2005;79: 10460-6).

When positions in the amino acid sequence of the S domain of HBsAg are referred to herein, such positions are made with reference to the amino acid sequence as set forth in SEQ ID NO: 3 (shown below) or to natural or artificial sequence variants thereof.

MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLG ONSOSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYOGMLPV CPLIPGSSTTSTGPCRTCMTTAOGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFL WEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLP LLPIFFCLWVYI

(SEQ ID NO: 3; amino acids 101 - 172 are shown underlined)

For example, the expression "amino acids 101 - 172 of the S domain" refers to the amino acid residues from positions 101 - 172 of the polypeptide according to SEQ ID NO: 3. However, a person skilled in the art understands that mutations or variations (including, but not limited to, substitution, deletion and/or addition, for example, HBsAg of a different genotype or a different HBsAg mutant as described herein) may occur naturally in the amino acid sequence of the S domain of HBsAg or be introduced artificially into the amino acid sequence of the S domain of HBsAg without affecting its biological properties. Therefore, 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 its natural or artificial mutants. In addition, when sequence fragments of the S domain of HBsAg are described herein (e.g. amino acids 101 - 172 or amino acids 120 -130 of the S domain of HBsAg), they include not only the corresponding sequence fragments of SEQ ID NO: 3, but also the corresponding sequence fragments of its natural or artificial mutants. For example, the phrase "amino acid residues from positions 101 - 172 of the S domain of HBsAg" encompasses amino acid residues from positions 101 - 172 of SEQ ID NO: 3 and the corresponding fragments of its mutants (natural or artificial mutants). As used herein, the phrases "corresponding sequence fragments" and "corresponding fragments" refer to fragments that are located in equal positions of sequences when the sequences are subjected to optimized alignment, namely, the sequences are aligned to obtain a highest percentage of identity.

The M protein (M-HBsAg) corresponds to the S protein extended by an N- terminal domain of 55 amino acids called "pre-S2". The L protein (L-HBsAg) corresponds to the M protein extended by an N-terminal domain of 108 amino acids called "pre-Sl" (genotype D). The pre-Sl and pre-S2 domains of the L protein can be present either at the inner face of viral particles (on the cytoplasmic side of the ER), and is believed to play a crucial role in virus assembly, or on the outer face (on the luminal side of the ER), available for the interaction with target cells and important for viral infectivity. Moreover, HBV surface proteins (HBsAgs) are not only incorporated into virion envelopes but also can spontaneously bud from ER-Golgi intermediate compartment membranes to form empty "subviral particles" (SVPs) that are released from the cell by secretion.

In some embodiments, a binding protein binds to the antigenic loop region of HBsAg, and is capable of binding to all of S-HBsAg, M-HBsAg and L-HBsAg.

In some embodiments, a binding protein neutralizes infection with hepatitis B virus and hepatitis delta virus. In some embodiments, the binding protein, reduces viral infectivity of hepatitis B virus and hepatitis delta virus.

To study and quantitate virus infectivity (or "neutralization") in the laboratory, standard "neutralization assays" may be utilized. For a neutralization assay, animal viruses are typically propagated in cells and/or cell lines. A neutralization assay wherein cultured cells are incubated with a fixed amount of HBV or HDV in the presence (or absence) of the antibody (or antigen-binding fragment or fusion protein) to be tested may be used. In such an assay, the levels of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant may be used and/or HBeAg staining may be assessed to provide a readout. For HDV, for example, delta antigen immunofluorescence staining may be assessed.

In a particular embodiment of an HBV neutralization assay, cultured cells, for example 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 and embodiment, incubation may be carried out, for example, for 16 hours at 37°C. That incubation may be performed in a medium (e.g. supplemented with 4% PEG 8000). After incubation, cells may be washed and further cultivated. To measure virus infectivity, the levels of hepatitis B surface antigen (HBsAg) and/or hepatitis B e antigen (HBeAg) secreted into the culture supernatant, e.g. from day 7 to day 11 postinfection, may be determined by enzyme-linked immunosorbent assay (ELISA). Additionally, HBeAg staining may be assessed in an immunofluorescence assay. In an embodiment of a HDV neutralization assay, essentially the same assay as for HBV may be used, with the difference that sera from HDV carriers may be used as HDV infection inoculum on differentiated HepaRg cells (instead of HBV). For detection, delta antigen immunofluorescence staining may be used as a readout.

Embodiments of the binding proteins of the disclosure have high neutralizing potency. In certain embodiments, the concentration of an antibody as described herein required for 50% neutralization of hepatitis B virus (HBV) and hepatitis delta virus (HDV), is, for example, about 10 pg/ml or less. In other embodiments, the concentration of a binding protein required for 50% neutralization of HBV and HDV is about 5 pg/ml. In other embodiments, the concentration of a binding protein as described herein required for 50% neutralization of HBV and HDV is about 1 pg/ml. In still other embodiments, the concentration of a binding protein required for 50% neutralization of HBV and HDV is about 750 ng/ml. In yet further embodiments, the concentration of a binding protein as described herein required for 50% neutralization of HBV and HDV is 500 ng/ml or less. In such embodiments, the concentration of a binding protein as 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, 50 ng/ml or less, or less than 20 ng/ml, preferably 15 ng/ml or less, more preferably 10 ng/ml or less, such as 7 ng/ml or less. Binding proteins according to the present disclosure, which can neutralize both HBV and HDV, are useful in the prevention and treatment of hepatitis B and hepatitis D. Infection with HDV typically occurs simultaneously with or subsequent to infection by HBV (e.g., inoculation with HDV in the absence of HBV does not cause hepatitis D since HDV requires the support of HBV for its own replication) and hepatitis D is typically observed in chronic HBV carriers.

Embodiments of disclosed binding proteins promote clearance of HBsAg and HBV. In particular embodiments, binding proteins promote clearance of both HBV and subviral particles of hepatitis B virus (SVPs). Clearance of HBsAg or of subviral particles may be assessed by measuring the level of HBsAg for example in a blood sample, e.g. from a hepatitis B patient. Similarly, clearance of HBV may be assessed by measuring the level of HBV for example in a blood sample, e.g. from a hepatitis B patient.

In the sera of patients infected with HBV, in addition to infectious particles (HBV), there is typically an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins (HBsAg) in the form of relatively smaller spheres and filaments of variable length. Subviral particles have been shown to strongly enhance intracellular viral replication and gene expression of HBV (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). This is also relevant in the context of infectivity of sera containing HBV, since the 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). Moreover, an excess of subviral particles can serve as a decoy by absorbing neutralizing antibodies and therefore delay the clearance of infection. Achievement of hepatitis B surface antigen (HBsAg) loss is considered in some instances to be an ideal endpoint of treatment and the closest outcome to cure chronic hepatitis B (CHB).

Embodiments of binding proteins of the present disclosure may promote clearance of HBsAg. In certain embodiments, the binding proteins may promote clearance of subviral particles of hepatitis B virus. In some embodiments, the binding proteins may be used to treat chronic hepatitis B. In any of the presently disclosed embodiments, a binding protein of the present disclosure is capable of binding an HBsAg of a genotype selected from the HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.

In certain embodiments, binding proteins of the present disclosure are capable of binding to any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. Examples of different HBsAg genotypes of include the following: GenBank accession number J02203 (HBV-D, ayw3); GenBank accession number FJ899792.1 (HBV-D, adw2); GenBank accession number AM282986 (HBV-A); GenBank accession number D23678 (HBV-B1 Japan); GenBank accession number AB117758 (HBV-C1 Cambodia); GenBank accession number AB205192 (HBV-E Ghana); GenBank accession number X69798 (HBV-F4 Brazil); GenBank accession number AF 160501 (HBV-G USA); GenBank accession number AY090454 (HBV-H Nicaragua); GenBank accession number AF241409 (HBV-I Vietnam); and GenBank accession number AB486012 (HBV-J Borneo). Exemplary amino acid sequences of the antigenic loop region of the S domain of HBsAg of different genotypes are described herein (e.g., SEQ ID NOs.: 5 - 15).

In some embodiments, a binding protein is capable of binding to one or more, and in some cases at least 6 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In certain embodiments, a binding protein is capable of binding to at least 8 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In some embodiments, a binding protein is capable of binding to all 10 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. HBV is differentiated into several genotypes, according to genome sequence. To date, eight well-known genotypes (A-H) of the HBV genome have been defined. Moreover, two other genotypes, I and J, have also been identified (Sunbul M., 2014, World J Gastroenterol 20(18): 5427-5434). The genotype is known to affect the progression of the disease and differences between genotypes in response to antiviral treatment have been determined.

In some embodiments, a binding protein according to the present disclosure is capable of binding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the HBsAg mutants having mutations in the antigenic loop region, with such mutant(s) being selected from one or more of 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. These mutants are naturally occurring mutants based on the S domain of HBsAg Genotype D, Genbank accession no. FJ899792 (SEQ ID NO.: 4). The mutated amino acid residue(s) in each of the mutants noted herein are indicated in the name.

SEQ ID NO.: 4:

MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLG QNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPy CPLIPGSSTTGTGPCRTCTTPAOGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFL WEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLP LLPIFFCLWVYI

(the antigenic loop region, i.e. amino acids 101 - 172, is shown underlined).

Amino acid sequences of the antigenic loop region of the S domain of HBsAg of different mutants are shown in SEQ ID NOs.: 16 - 33.

In certain embodiments, a binding protein as disclosed herein is capable of binding to at one or more, and in some cases 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, a binding protein is capable of binding 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, a binding protein is capable of binding 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.

In certain embodiments, the binding protein (e.g., including an antibody or antigen binding fragment thereof) is capable of reducing a serum concentration of HBV DNA in a mammal having an HBV infection. In certain embodiments, the binding protein is capable of reducing a serum concentration of HBsAg in a mammal having an HBV infection. In certain embodiments, the binding protein is capable of reducing a serum concentration of HBeAg in a mammal having an HBV infection. In certain embodiments, the binding protein is capable of reducing a serum concentration of HBcrAg in a mammal having an HBV infection. In some embodiments, the binding protein is capable of reducing the serum concentration of HBV DNA, HBsAg, HBeAg, and/or HBcrAg in the mammal for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days following a single administration of the binding protein.

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

In some embodiments, a binding protein is capable of binding to an epitope comprising at least one, at least two, at least three, or at least four amino acids of the antigenic loop region of HbsAg. In certain embodiments, a binding protein is capable of binding 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, a binding protein is capable of binding 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, a binding protein is capable of binding 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 position of the amino acids (e.g. 115 - 133, 120 - 133, 120 - 130) refers to the S domain of HBsAg as described above, which is 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.

The term "formed by" as used herein in the context of an epitope, means that the epitope to which the binding protein binds to may be linear (continuous) or conformational (discontinuous). A linear or a sequential epitope is an epitope that is recognized by an antibody according to its linear sequence of amino acids, or primary structure. A conformational epitope may be recognized according to a three- dimensional shape and protein structure. Accordingly, if the epitope is a linear epitope and comprises more than one amino acid located at positions selected from amino acid positions 115 -133 or from amino acid positions 120 -133 of the S domain of HBsAg, the amino acids comprised by the epitope may be located in adjacent positions of the primary structure (e.g., are 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 epitope and, thus, the amino acids forming the epitope may be or may be not located in adjacent positions of the primary structure (i.e. maybe or may be not consecutive amino acids in the amino acid sequence).

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

Amino acids to which a presently disclosed antibody, antigen binding fragment, or fusion protein binds (i.e. the amino acids forming the epitope), which are not located in adjacent positions of the primary structure, are in some cases spaced apart by one or more amino acids, to which the antibody, antigen binding fragment, or fusion protein does not bind. 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 two of the amino acids not located in adjacent positions comprised by the epitope.

In certain embodiments, a binding protein binds to an epitope comprising at least amino acids Pl 20, C121, R122 and Cl 24 of the S domain of HBsAg. In other embodiments, a binding protein of the present disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 127:

PCRXC wherein X is any amino acid or no amino acid; X is any amino acid; X is T, Y, R, S, or F; X is T, Y or R; or X is T or R.

In other embodiments, a binding protein of the present disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 119:

TGPCRTC or to an amino acid sequence sharing at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO.: 119.

In other embodiments, a binding protein of the present disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 124:

STTSTGPCRTC or to an amino acid sequence sharing at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO.: 124. In certain embodiments, a binding protein of the present disclosure binds to an epitope comprising an amino acid sequence comprising at least amino acids 145 - 151 of the S domain of HBsAg:

GNCTCIP

(SEQ ID NO.: 120).

In still other embodiments, a binding protein of the present disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO: 119 and an amino acid sequence according to SEQ ID NO.: 120.

In other embodiments, a binding protein of the present disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 124 and/or an amino acid sequence according to SEQ ID NO.: 126.

As described above, an epitope to which a binding protein of the present disclosure binds may be linear (continuous) or conformational (discontinuous). In some embodiments, a binding protein of the disclosure binds to a conformational epitope, and in certain such embodiments, the conformational epitope is present only under nonreducing conditions.

In certain embodiments, binding protein of the present disclosure, binds to a linear epitope. In certain such embodiments, the linear epitope is present under both, non-reducing conditions and reducing conditions.

In particular embodiments, a binding protein of the present disclosure binds to an epitope in the antigenic loop of HBsAg formed by an amino acid sequence according to SEQ ID NO.: 1 :

Xi X₂ X₃ TC X₄ X₅ X₆A X₇G wherein Xi, X₂, X₃, X₄, Xs, Xe and X₇ may be any amino acid (SEQ ID NO.: 1).

In some embodiments, Xi, X₂, X₃, X₄, Xs, Xe and X₇ are amino acids, which are conservatively substituted in comparison to amino acids 120 - 130 of SEQ ID NO.: 3. In some embodiments, Xi, X₂, X₃, X₄, Xs, Xe and X₇ are amino acids, which are conservatively substituted in comparison to amino acids 20 - 30 of any of SEQ ID NOs.: 5 - 33. In specific embodiments, Xi of SEQ ID NO.: 1 Xi is a small amino acid. A "small" amino acid, as used herein, 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, Xi is proline, serine or threonine.

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

In some embodiments, X3 of SEQ ID NO.: 1 is a charged amino acid or an aliphatic amino acid. A "charged" amino acid, as used herein, refers to any amino acid selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid and histidine. A "aliphatic" amino acid, as used herein, 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.

In some embodiments, X4 of SEQ ID NO.: 1 is a small amino acid and/or a hydrophobic amino acid. A "hydrophobic" amino acid, as used herein, 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.

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

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

In some embodiments, X7 of SEQ ID NO.: 1 is a polar amino acid or an aliphatic amino acid. A "polar" amino acid, as used herein, 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 such embodiments, X7 is glutamine, histidine or leucine. In some embodiments, a binding protein according to the present disclosure binds to an epitope in the antigenic loop of HBsAg formed by an amino acid sequence according to SEQ ID NO.: 2:

Xi X₂ X₃ TC X₄ X₅ X₆A X₇G wherein

Xi 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,

Xe is T, P or L, and

X₇ is Q, H or L

(SEQ ID NO.: 2).

With regard to the epitopes formed by the amino acid sequences according to SEQ ID NO. : 1 or 2, it is noted that the term "formed by" as used herein is not intended to imply that a disclosed binding protein necessarily binds to each and every amino acid of SEQ ID NO.: 1 or 2. In particular, a binding protein may bind only to some of the amino acids of SEQ ID NO.: 1 or 2, whereby other amino acid residues may act as "spacers".

In particular embodiments, a binding protein according to the present disclosure binds to an epitope in the 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 below in Table 5.

In some embodiments, binding protein 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 shown below in Table 5, or to a sequence variant thereof. In certain embodiments, a binding protein 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 shown below in Table 5. Table 5: Amino acid sequences of the antigenic loop region of the S domain of HBsAg (residues 101-172 of the S domain of HBsAg - except for SEQ ID NO: 16, which refers to residues 100-172 of the S domain of HBsAg in order to include the relevant mutation) of the different genotypes and mutants as used herein.

Nucleic acid molecules/Polynucleotides

In another aspect, the disclosure provides a nucleic acid molecule comprising a polynucleotide encoding an antibody, antigen binding fragment, or fusion protein according to the present disclosure. It will be understood that, for example, a first nucleic acid molecule can encode a heavy chain of an antibody, and a second nucleic acid molecule can encode a light chain of an antibody; these first and second nucleic acid molecules can still be referred-to as "a polynucleotide" or "a nucleic acid molecule" that encodes the antibody. In other words, a polynucleotide or nucleic acid molecule includes embodiments, wherein portions (e.g., chains) of an antibody or antigen-binding fragment are encoded by separate nucleic acid molecules and/or by separate portions of nucleic acid molecules. Exemplary polynucleotide sequences are provided in SEQ ID NOs. : 101 -111. In some embodiments, a polynucleotide encoding an antibody heavy chain comprises or consists of the polynucleotide sequence set forth in SEQ ID NO.: 102, and a polynucleotide encoding an antibody VL or LC comprises the polynucleotide sequence set forth in any one of SEQ ID NOs. : 106-111.

Due to the redundancy of the genetic code, the present disclosure also comprises sequence variants of these nucleic acid sequences and in particular such sequence variants, which encode the same amino acid sequences.

In certain embodiments, a polynucleotide or nucleic acid molecule comprises a nucleotide sequence sharing at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleotide sequence according to any one of SEQ ID NOs.: 101-111, wherein the nucleotide sequence is codon optimized for expression by a host cell. In particular 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: SEQ ID NOs.: 101-l l l.

In certain embodiments, a polynucleotide comprises a Vu-encoding nucleotide sequence having at least 50% identity to the amino acid sequence set forth in SEQ ID NO.: 102 and a Vr-encoding nucleotide sequence having at least 50% identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 106-109.

Vectors

Further included within the scope of the disclosure are vectors, for example, expression vectors, that comprise a nucleic acid molecule according to the present disclosure.

The term "vector" refers to a construct comprising a nucleic acid molecule. A vector in the context of the present disclosure is suitable for incorporating or harboring a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a desired antibody or antibody fragment thereof according to the present description.

As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the 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 optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. Any of the elements of an expression vector that contribute to transcription of a nucleic acid molecule of interest may be heterologous to the vector. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid," "expression plasmid," "virus" and "vector" are often used interchangeably.

A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bacteriophage vector.

A transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. A vector in the context of the present disclosure may be, e.g., an RNA vector or a DNA vector. A vector may be a DNA molecule. For example, a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication.

In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a lentiviral vector or a y-retroviral vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

"Retroviruses" are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

"Lentiviral vectors" include HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

In certain embodiments, the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors. In other embodiments, the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. HIV-l-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing transgenes are known in the art and have been previous described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14: 1155, 2003; Frecha et al., Mol. Ther. 18: 1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998). Other vectors that can be used with the compositions and methods of this disclosure include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).

When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi ci str onic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.

Plasmid vectors, including DNA-based antibody or antigen-binding fragmentencoding plasmid vectors for direct administration to a subject, are described further herein.

Cells

In a further aspect, the present disclosure also provides a cell (also referred to as a "host cell") expressing an antibody, antigen-binding fragment, or fusion protein according to the present disclosure; or comprising a vector or polynucleotide according the present disclosure.

Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In certain such embodiments, the cells are a mammalian cell line such as CHO cells (e.g., DHFR- CHO cells (Urlaub et al., PNAS 77:4216 (1980), CHO-KSV, ExpiCHO), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells. NSO cells, human liver cells, e.g. Hepa RG cells, myeloma cells or hybridoma cells. Other examples of mammalian host cell lines include mouse sertoli cells (e.g, TM4 cells); monkey kidney CV1 line transformed by SV40 (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 liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Mammalian host cell lines suitable for antibody production also include those described in, for example, Yazaki and

Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

In certain embodiments, a host cell is a prokaryotic cell, such as an E. coli. The 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 may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523.

Insect cells useful expressing a binding protein 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 SfSWTOl “Mimic™” cells. See, e.g., Palmberger et al., J. Biotechnol. 753(3-4): 160-166 (2011). Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expressing protein-encoding vectors, and include fungi and yeast strains with “humanized” glycosylation pathways, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat.

Biotech. 22: 1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006).

Plant cells can also be utilized as hosts for expressing a binding protein of the present disclosure. For example, PLANTIBODIES™ technology (described in, for example, U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429) employs transgenic plants to produce antibodies.

In some embodiments, a fusion protein is expressed at a cell surface by an immune cell, e.g., a T cell, NK cell, or NK-T cell, or any subtype thereof.

Any protein expression system compatible with the disclosure may be used to produce the disclosed binding proteins. Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

In particular embodiments, the cell may be transfected with a vector according to the present description with an expression vector. The term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, such as into eukaryotic cells. In the context of the present description, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into eukaryotic cells, including into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g., based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine etc. In certain embodiments, the introduction is non-viral.

Moreover, cells of the present disclosure may be transfected stably or transiently with the vector according to the present description, e.g. for expressing an antibody, or an antigen binding fragment thereof, according to the present description. In such embodiments, the cells are stably transfected with the vector as described herein encoding a binding protein. Alternatively, cells may be transiently transfected with a vector according to the present disclosure encoding a binding protein according to the present description. In any of the presently disclosed embodiments, a polynucleotide may be heterologous to the host cell.

In a related aspect, the present disclosure provides methods for producing an antibody, antigen-binding fragment, or fusion protein, wherein the methods comprise culturing a host cell of the present disclosure under conditions and for a time sufficient to produce the antibody, antigen-binding fragment, or fusion protein.

Accordingly, the present disclosure also provides recombinant host cells that heterologously express an antibody, antigen-binding fragment, or fusion protein of the present disclosure. For example, the cell may be of a species that is different to the species from which the antibody was fully or partially obtained (e.g., CHO cells expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not express the antibody or antigen binding fragment in nature. Moreover, the host cell may impart a post-translational modification (PTM; e.g., glycosylation or fucosylation) on the binding protein that is not present in a native state of the binding protein (or in a native state of a parent binding protein from which the subject binding protein was engineered or derived). Such a PTM may result in a functional difference (e.g., reduced immunogenicity). Accordingly, a binding protein of the present disclosure that is produced by a host cell as disclosed herein may include one or more post-translational modification that is distinct from the binding protein or parent binding protein in its native state (e.g., a human antibody produced by a CHO cell can comprise a post-translational modification that is distinct from the antibody when isolated from the human and/or produced by the native human B cell or plasma cell).

Production of antibodies

Antibodies according to the disclosure can be made by any method known in the art. For example, the general methodology for making monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983). In one embodiment, the EBV immortalization method described in W02004/076677 is used.

In one embodiment, antibodies are produced using a method described in WO 2004/076677. In such methods, B cells producing the antibody are transformed with EBV and a polyclonal B cell activator. Additional stimulants of cellular growth and differentiation may optionally be added during the transformation step to further enhance the efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not essential. The immortalized B cells produced using these methods can then be cultured using methods known in the art and antibodies isolated therefrom.

Another method for producing antibodies is described in WO 2010/046775. 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 cultures. Further, from the plasma cell cultures, RNA can be extracted and PCR can be performed using methods known in the art. The VH and VL regions of the antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into an expression vector that is then transfected into HEK293T cells or other host cells. The cloning of nucleic acid in expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art.

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

Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies, antigen-binding fragments, or fusion proteins of the present description. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody or fusion protein molecules of the present disclosure or fragments thereof. Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab’)2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs. Eukaryotic, e.g., mammalian, host cell expression systems may be used for production of larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells include, but are not limited to, those exemplary host cells and cell lines disclosed herein.

The present disclosure also provides a process for the production of an antibody according to the present disclosure comprising culturing a host cell comprising 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 description, and isolating the antibody molecule.

An antibody molecule or antibody fragment may comprise only a heavy or light chain polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cells. For production of products comprising both heavy and light chains, the cell line may 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 may be used, the vector including sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the disclosure may be produced by (i) expressing a nucleic acid sequence according to the disclosure in a host cell, e.g. by use of a vector according to the present description, and (ii) isolating the expressed desired product. Additionally, the method may include (iii) purifying the isolated antibody, antigen-binding fragment, or fusion protein. Transformed B cells and cultured plasma cells may be screened for those producing antibodies, antigen-binding fragments, or fusion proteins of the desired specificity or function.

Screening may be carried out by any immunoassay, e.g., ELISA, by staining of tissues or cells (including transfected cells), by neutralization assay or by one of a number of other methods known in the art for identifying desired specificity or function. The assay may select on the basis of simple recognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigen-binding antibodies, to select antibodies that can change characteristics of targeted cells, such as their signaling cascades, their shape, their growth rate, their capability of influencing other cells, their response to the influence by other cells or by other reagents or by a change in conditions, their differentiation status, or the like.

Individual transformed B cell clones may then be produced from the positive transformed B cell culture. The cloning step for separating individual clones from the mixture of positive cells may be carried out using limiting dilution, micromanipulation, single cell deposition by cell sorting or another method known in the art. 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.

The immortalized B cell clones or the transfected host-cells of described herein can be used in various ways e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, etc.

Pharmaceutical Compositions Comprising Antibodies, Antigen-Binding Fragments, Fusion Proteins, Polynucleotides, Vectors, and/or Host Cells

The present disclosure also provides a pharmaceutical composition comprising an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure and/or a cell according to the present disclosure.

Pharmaceutical compositions may also contain a pharmaceutically acceptable carrier, diluent and/or excipient. Although the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. In general, pharmaceutically acceptable carriers in a pharmaceutical composition according to the present disclosure may be active components or inactive components.

Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject.

Pharmaceutical compositions of the disclosure may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to Synagis™ and Herceptin™, for reconstitution with sterile water containing a preservative). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.

In particular embodiments, the active ingredient in a composition according to the present disclosure is an antibody molecule, an antibody fragment or variant or derivative thereof, in particular the active ingredient in the composition is an antibody, an antibody fragment, a fusion protein, or variants and derivatives thereof, as described herein. As such, it may be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition may contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the disclosure may have a pH between 5.5 and 8.5, and in some embodiments this may be between 6 and 8. In other embodiments, the pH of a pharmaceutical composition as described herein may be about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen free. The composition may be isotonic with respect to humans. In certain embodiments, pharmaceutical compositions of the disclosure are supplied in hermetically sealed containers.

Within the scope of the disclosure are compositions present in several forms of administration; the forms include, but are not limited to, those forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid. A vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the present description. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies according to the present description. Once formulated, the compositions of the present disclosure can be administered directly to the subject. In one embodiment the compositions are adapted for administration to mammalian, e.g., human subjects.

The pharmaceutical compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the description. In specific embodiments, the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be utilized, e.g. that the pharmaceutical composition is in lyophilized form. For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient can be provided be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.

A composition may be in the form of a solid or liquid. In some embodiments, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi solid, semi liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

The composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

Liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oral administration should contain an amount of an antibody or antigen-binding fragment as herein disclosed such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the antibody or antigen-binding fragment in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the antibody or antigen-binding fragment. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of antibody or antigen-binding fragment prior to dilution.

The composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

A composition may include various materials which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The composition in solid or liquid form may include an agent that binds to the antibody or antigen-binding fragment of the disclosure and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome. The composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi phasic, or tri phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation, may determine preferred aerosols.

It will be understood that compositions of the present disclosure also encompass carrier molecules for polynucleotides, as described herein (e.g., lipid nanoparticles, nanoscale delivery platforms, and the like).

In certain embodiments, a composition comprises a first vector comprising a first plasmid, and a second vector comprising a second plasmid, wherein the first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL of the antibody or antigen-binding fragment thereof. In certain embodiments, a composition comprises a polynucleotide (e.g., mRNA) coupled to a suitable delivery vehicle or carrier. Exemplary vehicles or carriers for administration to a human subject include a lipid or lipid-derived delivery vehicle, such as a liposome, solid lipid nanoparticle, oily suspension, submicron lipid emulsion, lipid microbubble, inverse lipid micelle, cochlear liposome, lipid microtubule, lipid microcylinder, or lipid nanoparticle (LNP) or a nanoscale platform (see, e.g., Li et al. Wilery Interdiscip Rev. Nanomed Nanobiotechnol. 77(2):el530 (2019)). Principles, reagents, and techniques for designing appropriate mRNA and and formulating mRNA-LNP and delivering the same are described in, for example, Pardi et al. (J Control Release 277345-351 (2015)); Thess et al. Mol Ther 23: 1456-1464 (2015)); Thran et al. (EMBO Mol Med 9(10): 1434-1448 (2017); Kose et al. (Set. Immunol. 4 eaaw6647 (2019); and Sabnis et al. (Mol. Ther. 26: 1509-1519 (2018)), which techniques, include capping, codon optimization, nucleoside modification, purification of mRNA, incorporation of the mRNA into stable lipid nanoparticles (e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid:distearoyl PC:cholesterol:polyethylene glycol lipid), and subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intratracheal administration of the same, are incorporated herein by reference.

The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a composition that comprises an antibody, antigen-binding fragment thereof, or other composition as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the peptide composition so as to facilitate dissolution or homogeneous suspension of the antibody or antigen-binding fragment thereof in the aqueous delivery system. Whether it is a polypeptide, peptide, or nucleic acid molecule, cell, or other pharmaceutically useful compound according to the present disclosure that is to be given to an individual, administration is generally in a "prophylactically effective amount" or a "therapeutically effective amount" or an "effective amount" (as the case may be), this being sufficient to show a benefit to the individual (e.g., improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner). When referring to an individual active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially, sequentially, or simultaneously.

Compositions are administered in an effective amount (e.g., to treat a HBV and/or HDV infection), which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. In certain embodiments, following administration of therapies according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated as compared to placebo-treated or other suitable control subjects. In some embodiments, the following administration of therapies according to the formulations and methods of this disclosure, subjects will exhibit a functional cure. As used herein, a “functional cure” refers to the suppression of HBV DNA for at least 6 months following a final treatment with the antibody and no longer receiving treatment (e.g., off-treatment). The functional cure can refer to the suppression of HBV DNA for 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, and/or 24 months. In certain embodiments, the functional cure can include lack of detectable HBsAg in samples from the subject. In certain embodiments, the lack of detectable HBsAg occurs with or without HBsAg seroconversion in the subject after a finite duration of therapy. Without wishing to be limited by theory, it is believed that a functional cure may be obtain in a subject via an immune response (e.g., cell-mediated immune response) induced by the Fc variants disclosed herein. The immune response may be induced by, for example, dendritic cell activation, antigen presentation by APCs, activation of CD4+ T cells, and/or activation of CD8+ T cells. In particular embodiments, a functional cure is induced by activation of T cells and/or B cells. In certain embodiments, a functional cure is induced by activation of T cells and B cells.

Generally, a therapeutically effective daily dose of an antibody or antigen binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (z.e., 0.07 mg) to about 100 mg/kg (z.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (z.e., 0.7 mg) to about 50 mg/kg (z.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (z.e., 70 mg) to about 25 mg/kg (z.e., 1.75 g). Other doses for antibodies or antigenbinding fragments are provided herein.

For polynucleotides, vectors, host cells, and related compositions of the present disclosure, a therapeutically effective dose may be different than for an antibody or antigen-binding fragment.

The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the present disclosure may be provided for example in a pre-filled syringe.

Pharmaceutical compositions as disclosed herein may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, i.e. the inventive transporter cargo conjugate molecule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

The pharmaceutical compositions according to the present description may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g., including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the pharmaceutical composition may be formulated in a suitable ointment, containing the inventive pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the present description, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Doses may be expressed in relation to the bodyweight. Thus, a dose which is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg etc.) bodyweight", even if the term "bodyweight" or "body weight" is not explicitly mentioned.

In specific embodiments, in a single dose, e.g. a daily, weekly or monthly dose, the amount of the antibody, or the antigen binding fragment thereof, in the pharmaceutical composition does not exceed 1 g. In certain such embodiments, the single dose does not exceed a dose selected from 500 mg, 250 mg, 100 mg, and 50 mg. Further embodiments of doses are provided herein. In particular embodiments the single dose of the pharmaceutical composition comprises up to 6 mg, up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 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, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 3000 mg, or in a range from 5 mg to 3000 mg, or in a range from 10 mg to 3000 mg, or in a range from 25 mg to 3000 mg, or in a range from 30 mg to 3000 mg, or in a range from 50 mg to 3000 mg, or in a range from 60 mg to 3000 mg, or in a range from 75 mg to 3000 mg, or in a range from 90 mg to 3000 mg, or in a range from 100 mg to 3000 mg, or in a range from 150 mg to 3000 mg, or in a range from 200 mg to 3000 mg, or in a range from 300 mg to 3000 mg, or in a range from 500 mg to 3000 mg, or in a range from 750 mg to 3000 mg, or in a range from 900 mg to 3000 mg, or in a range from 1500 mg to 3000 mg, or in a range from 2000 mg to 3000 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 900 mg, or in a range from 5 mg to 900 mg, or in a range from 10 mg to 900 mg, or in a range from 25 mg to 900 mg, or in a range from 30 mg to 900 mg, or in a range from 50 mg to 900 mg, or in a range from 60 mg to 900 mg, or in a range from 75 mg to 900 mg, or in a range from 90 mg to 900 mg, or in a range from 100 mg to 900 mg, or in a range from 150 mg to 900 mg, or in a range from 200 mg to 900 mg, or in a range from 300 mg to 900 mg, or in a range from 500 mg to 900 mg, or in a range from 750 mg to 900 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 500 mg, or in a range from 5 mg to 500 mg, or in a range from 10 mg to 500 mg, or in a range from 25 mg to 500 mg, or in a range from 30 mg to 500 mg, or in a range from 50 mg to 500 mg, or in a range from 60 mg to 500 mg, or in a range from 75 mg to 500 mg, or in a range from 90 mg to 500 mg, or in a range from 100 mg to 500 mg, or in a range from 150 mg to 500 mg, or in a range from 200 mg to 500 mg, or in a range from 300 mg to 500 mg, or in a range from 400 mg to 500 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 300 mg, or in a range from 5 mg to 300 mg, or in a range from 10 mg to 300 mg, or in a range from 25 mg to 300 mg, or in a range from 30 mg to 300 mg, or in a range from 50 mg to 300 mg, or in a range from 60 mg to 300 mg, or in a range from 75 mg to 300 mg, or in a range from 90 mg to 300 mg, or in a range from 100 mg to 300 mg, or in a range from 150 mg to 300 mg, or in a range from 200 mg to 300 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 200 mg, or in a range from 5 mg to 200 mg, or in a range from 10 mg to 200 mg, or in a range from 25 mg to 200 mg, or in a range from 30 mg to 200 mg, or in a range from 50 mg to 200 mg, or in a range from 60 mg to 200 mg, or in a range from 75 mg to 200 mg, or in a range from 90 mg to 200 mg, or in a range from 100 mg to 200 mg, or in a range from 150 mg to 200 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 100 mg, or in a range from 5 mg to 100 mg, or in a range from 10 mg to 100 mg, or in a range from 25 mg to 100 mg, or in a range from 30 mg to 100 mg, or in a range from 50 mg to 100 mg, or in a range from 60 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 90 mg to 100 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 25 mg, or in a range from 5 mg to 25 mg, or in a range from 10 mg to 25 mg, or in a range from 15 mg to 25 mg, or in a range from 20 mg to 25 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 50 mg, or in a range from 1 mg to 25 mg, or in a range from 5 mg to 50 mg, or in a range from 5 mg to 25 mg, or in a range from 10 to 50 mg, or in a range from 10 to 25 mg, or in a range from 1 to 15 mg, or in a range from 5 mg to 15 mg, or in a range from 10 mg to 15 mg, or wherein the single dose of the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,

195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275,

280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,

365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445,

450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530,

535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615,

620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700,

705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785,

790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870,

875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955,

960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg, or more, of the antibody, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 mg, less than 25 mg, or less than 10 mg, but is more than 1 mg, more than 2 mg, more than 3 mg, more than 4 mg, or more than 5 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody at a concentration in a range from 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.

In certain embodiments, the single dose of the pharmaceutical composition comprises about 75 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises about 90 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg of the antibody. In certain embodiments, the method comprises administering the single dose by subcutaneous injection, optionally wherein the single dose comprises or consists of 6 mg of the antibody or 18 mg of the antibody.

In certain embodiments, a method comprises administering the antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition to the subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.

In certain embodiments, a method comprises administering the antibody, antigen-binding fragment, or composition to the subject a plurality of times, wherein a second or successive administration is performed at about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 24, about 48, about 74, about 96 hours, or more, following a first or prior administration, respectively.

In certain embodiments, a method comprises administering the antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition at least one time prior to the subject

Compositions comprising an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising an antibody or antigen-binding fragment of the disclosure and each active agent in its own separate dosage formulation. For example, an antibody or antigenbinding fragment thereof as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, an antibody or antigen-binding fragment as described herein and the other active agent can be administered to the subject together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions comprising an antibody or antigen-binding fragment and one or more additional active agents can be administered at essentially the same time, z.e., concurrently, or at separately staggered times, z.e., sequentially and in any order; combination therapy is understood to include all these regimens.

In some embodiments, a composition or kit as described herein further comprises (i) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (ii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (iii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-Ll 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 (i)-(iv). In some embodiments, a kit comprises a composition or combination as described herein, and further comprises instructions for using the component to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection.

In certain embodiments, a composition of the present disclosure (e.g., antibody, antigen-binding fragment, host cell, nucleic acid, vector, or pharmaceutical omposition) is 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. In certain embodiments, a composition of the present disclosure is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof. In certain embodiments, a composition of the present disclosure is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of CTLA4. In particular embodiments, an a composition of the present disclosure is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof. In certain embodiments, a composition of the present disclosure is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. An anti-B7-H3 antibody binding fragment may be a scFv or fusion protein comprising the same, as described in, for example, Dangaj et al., Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos.

WO /201640724A1 and WO 2013/025779A1. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of CD244. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of BLTA, HVEM, CD 160, or any combination thereof. Anti CD- 160 antibodies are described in, for example, PCT Publication No. WO 2010/084158. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of TIM3. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of Gal9. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of A2aR. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015). In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFP) or Treg development or activity. In certain embodiments, a composition of the present disclosure is used in combination with an IDO inhibitor, such as levo- 1 -methyl tryptophan, epacadostat (INCB024360; Liu et al., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. 9:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (l-MT)-tira-pazamine, or any combination thereof. In certain embodiments, a composition of the present disclosure is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-1- arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2- boronoethyl)-L-cysteine (BEC), or any combination thereof. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.). In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both. In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described in, for example, PCT Publication No. WO 2016/134333. Anti-PVRL2 antibodies are described in, for example, PCT Publication No. WO 2017/021526. In certain embodiments, a composition of the present disclosure is used in combination with a LAIR1 inhibitor. In certain embodiments a composition of the present disclosure is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

In certain embodiments, a composition of the present disclosure is used in combination with an agent that increases the activity (z.e., is an agonist) of a stimulatory immune checkpoint molecule. For example, a composition of the present disclosure can be used in combination with a CD137 (4-1BB) agonist (such as, for example, urelumab), a CD134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-1127), a CD28 agonist (such as, for example, TGN1412, CD80, or CD86), a CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodies described in PCT Patent Publication No. WO 2016/054638), an agonist of ICOS (CD278) (such as, for example, GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof). In any of the embodiments disclosed herein, a method may comprise administering a composition of the present disclosure with one or more agonist of a stimulatory immune checkpoint molecule, including any of the foregoing, singly or in any combination.

An antibody according to the present disclosure can be present either in the same pharmaceutical composition as the additional active component or, the antibody according to the present disclosure may be included in a first pharmaceutical composition and the additional active component may be included in a second pharmaceutical composition different from the first pharmaceutical composition.

Uses

In a further aspect, the present disclosure provides methods for the use of an antibody, a nucleic acid, a vector, a cell, a pharmaceutical composition, a combination, or a kit according to the present disclosure in the (i) prophylaxis, treatment or attenuation of hepatitis B and/or hepatitis D; or in (ii) diagnosis of hepatitis B and/or hepatitis D (e.g., in a human subject).

Methods of diagnosis e.g., in vitro, ex vivo) may include contacting an antibody, antibody fragment (e.g., antigen binding fragment), or fusion protein with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood. The methods of diagnosis may also include the detection of an antigen/antibody or antigen/fusion protein complex, in particular following the contacting of an antibody, antibody fragment, or fusion protein with a sample. Such a detection step is typically performed at the bench, i.e. without any contact to the human or animal body. Examples of detection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay).

The disclosure also provides the use of (i) an antibody, an antibody fragment, fusion protein, or variants and derivatives thereof according to the disclosure, (ii) host cell (which can be an immortalized B cell) according to the disclosure, (iii) a nucleic acid or a vector according to the present disclosure (iv) a pharmaceutical composition of the present disclosure or (v) a combination in (a) the manufacture of a medicament for the prevention, treatment or attenuation of hepatitis B and/or hepatitis D or for (b) diagnosis of hepatitis B and/or hepatitis D.

The term "disease" as used herein is intended to be generally synonymous, and is used interchangeably with, the terms "disorder" and "condition" (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the affected human or animal to have a reduced duration or quality of life.

As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration and therapy, and refers to medical management of a disease, disorder, or condition of a subject. Benefits of treatment can include improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof. The terms "subject" or "patient" are used interchangeably herein to mean all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In certain embodiments, the patient is a human. The subjects can be male or female and can be any suitable age, including infantjuvenile, adolescent, adult, and geriatric subjects.

The disclosure also provides an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure a pharmaceutical composition according, and/or a combination of the present disclosure for use as a medicament for the prevention or treatment of hepatitis B and/or hepatitis D. It also provides the use of an antibody of the disclosure in the manufacture of a medicament for treatment of a subject and/or diagnosis in a subject. It also provides a method for treating a subject (e.g., a human subject), comprising administering to the subject an effective amount of a composition or combination as described herein. In some embodiments, the subject may be a human. One way of checking efficacy of therapeutic treatment involves monitoring disease symptoms after administration of the composition. Treatment can be a single dose schedule or a multiple dose schedule.

In one embodiment, an antibody, pharmaceutical composition, or combination according to the disclosure is administered to a subject in need of such treatment. Such a subject includes, but is not limited to, one who is particularly at risk of or susceptible to hepatitis B and/or hepatitis D.

Antibodies, polynucleotides, vectors, host cells, pharmaceutical compositions, and combinations of the same, according to the present disclosure may also be used in a kit for the prevention, treatment, attenuation, and/or diagnosis of hepatitis B and/or hepatitis D. In some embodiments, a kit further comprises instructions for using the component to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection. Further, the epitope in the antigenic loop region of HBsAg, which is capable of binding an antibody, antigen binding fragment, or fusion protein of the disclosure as described herein may be used in a kit for monitoring the efficacy of application procedures by detecting the presence or determining the titer of protective anti-HBV antibodies.

In certain embodiments, a composition or a kit of this disclosure further comprises: a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (ii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (iii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-Ll 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)-(xii).

In some embodiments, an antibody according to the present disclosure, a nucleic acid according to the present disclosure, the vector according to the present disclosure, a cell according to the present disclosure, a pharmaceutical composition according to the present disclosure, and/or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery system (e.g., an RNAi agent) of the present disclosure is used in treatment or attenuation of chronic hepatitis B infection.

In particular embodiments, an antibody according to the present disclosure (i) neutralizes HBV infection, (ii) binds to L-HBsAg (the large HBV envelope protein, which is present in infectious HBV particles), thereby preventing spreading of HBV,

(iii) binds to S-HBsAg, thereby promoting clearance of subviral particles (SVP) and/or

(iv) can induce seroconversion, i.e. an active immune response to the virus.

In particular embodiments, an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure, or a pharmaceutical composition according to the present disclosure, may be used in prevention of hepatitis B (reinfection after liver transplantation in particular for hepatitis B induced liver failure.

In further embodiments an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the description provided herein, a cell according to the present disclosure, a pharmaceutical composition, and/or a combination according to the present disclosure, may be used in prevent! on/prophyl axis of hepatitis B in non-immunized subjects. This is for example in case of (an assumed) accidental exposure to HBV (post-exposure prophylaxis). The term "non-immunized subjects" includes subjects, who never received a vaccination and are, thus, not immunized, and subjects, who did not show an immune response (e.g., no measurable anti-hepatitis B antibodies) after vaccination.

In some embodiments, an antibody to the present disclosure, the nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure, a pharmaceutical composition according to the present disclosure, or a combination of the present disclosure, is used in prophylaxis of hepatitis B in haemodialysed patients. In some embodiments, an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure, a pharmaceutical composition according to the present disclosure, or a combination of the present disclosure, is used in prevention of hepatitis B in a newborn. In such embodiments, an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure, a pharmaceutical composition according to the present disclosure, or a combination of the present disclosure, may be administered at birth or as soon as possible after birth. The administration may be repeated until seroconversion following vaccination.

Moreover, the present disclosure also provides the use of an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure or a pharmaceutical composition according to the present disclosure in the diagnosis (e.g. in vitro, ex vivo, or in vivo) of hepatitis B and/or hepatitis D.

In addition, the use of an antibody to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure or a pharmaceutical composition according to the present disclosure in determining whether an isolated blood sample is infected with hepatitis B virus and/or hepatitis delta virus is provided.

As described above, methods of diagnosis may include contacting an antibody or fusion protein with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood. The methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody with a sample. Such a detection step is typically performed at the bench, i.e. without any contact to the human or animal body. Examples of detection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay). The present disclosure also provides a method of treating, preventing and/or attenuating hepatitis B and/or hepatitis D in a subject, wherein the method comprises administering to the subject an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure, a pharmaceutical composition according to the present disclosure, and/or a combination of the present disclosure. In certain embodiments, a method further comprises administering to the subject one or more of: (vii) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (viii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (ix) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-Ll antibody or antigen binding fragment thereof, and/or an anti-CTLA4 antibody or antigen binding fragment thereof; (x) an agonist of a stimulatory immune checkpoint molecule; or (xi) any combination of (vii)-(x).

In some embodiments, the hepatitis B infection is a chronic hepatitis B infection. In some embodiments, the subject has received a liver transplant. In some embodiments, the subject is non-immunized against hepatitis B. In certain embodiments, the subject is a newborn. In some embodiments, the subject is undergoing or has undergone hemodialysis.

The present disclosure also provides a method of treating a subject who has received a liver transplant comprising administering to the subject who has received the liver transplant an effective amount of an antibody according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure, a cell according to the present disclosure, a pharmaceutical composition according to the present disclosure, or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery system (e.g., an RNAi agent) of the present disclosure.

Also provided herein are methods for detecting the presence or absence of an epitope in a correct conformation in an anti-hepatitis-B and/or an anti-hepatitis-D vaccine, wherein the methods comprise: (i) contacting the vaccine with an antibody of any one of the present disclosure; and (ii) determining whether a complex comprising an antigen and the antibody has been formed.

The term "vaccine" as used herein is typically understood to be a prophylactic or therapeutic material providing at least one antigen, such as an immunogen. The antigen or immunogen may be derived from any material that is suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as from bacteria particles, virus particles, a tumor (including a solid or liquid tumor), or other cancerous tissue. The antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response. In certain embodiments, an "antigen" or an "immunogen" refers to a substance which may be recognized by the immune system, e.g. by the adaptive immune system, and which is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigenspecific T cells as part of an adaptive immune response. In some embodiments, an antigen may be or may comprise a peptide or protein which may be presented by an MHC complex e.g., MHC class I; MHC class II) to T cells. In certain embodiments, the antigen comprises a HBV and/or HDV antigen; e.g., an HBsAg antigen.

Some embodiments of the present disclosure provide methods of treating chronic HBV infection or an HBV-associated disease in a subject in need thereof, comprising: (i) administering to the subject an agent that reduces HBV antigenic load; and (ii) administering to the subject an anti -HBV antibody. In certain embodiments, the agent that reduces HBV antigenic load is administered before the anti-HBV antibody or antigen-binding fragment thereof. In certain embodiments, administering the agent that reduces HBV antigenic load before the anti-HBV antibody or antigen-binding fragment thereof causes the viral load to be reduced when the anti-HBV antibody thereof is administered. In certain embodiments, the therapeutically effective amount of the anti- HBV antibody fragment thereof of the combination therapy is less than a therapeutically effective amount of the anti-HBV antibody thereof delivered when the agent that reduces HBV antigenic load has not been administered to the subject (e.g., when the anti-HBV antibody thereof is administered alone as a monotherapy). In certain embodiments, the present disclosure provides a method of treating a chronic HBV infection or HBV-associated disease in a subject in need thereof, comprising: administering to the subject an agent that reduces HBV antigenic load; and administering to the subject an anti-HBV antibody; and further comprising measuring the amount of HBsAg present in a blood sample from the subject before and after administering the agent that reduces HBV antigenic load, wherein a decrease in HBsAg indicates reduced expression of the at least one HBV gene.

In certain embodiments, the present disclosure provides an agent that reduces HBV antigenic load for use in the treatment of a chronic HBV infection or an HBV- associated disease in a subject, wherein the subject is subsequently administered an anti-HBV antibody. In certain other embodiments, the present disclosure provides an anti-HBV antibody for use in the treatment of a chronic HBV infection or an HBV- associated disease in a subject, and the subject has been previously administered an agent that reduces HBV antigenic load. In further embodiments, expression of at least one HBV gene is reduced after administration of the agent that reduces HBV antigenic load, and the anti-HBV antibody is administered to the subject when expression of the at least one HBV gene is reduced.

In certain embodiments, the present disclosure provides the use of an agent that reduces HBV antigenic load and/or an anti-HBV antibody in the manufacture of a medicament for the treatment of a chronic HBV infection or an HBV-associated disease.

In any of the above methods, compositions for use, or uses in manufacture, the methods and compositions may be used for treating a chronic HBV infection.

In certain embodiments, administering the anti-HBV antibody or antigenbinding fragment thereof comprises administering the anti-HBV or antigen-binding fragment thereof antibody twice per week, once per week, every other week, every two weeks, or once a month.

In certain embodiments, administering the anti-HBV antibody or antigenbinding fragment thereof comprises administering at least two doses of a therapeutically effective amount of the anti-HBV antibody or antigen-binding fragment thereof. In certain further embodiments, the at least two doses are administered twice per week, once per week, every other week, every two weeks, or once a month.

In particular embodiments of the above methods, compositions for use, or uses in manufacture, the anti-HBV antibody may recognize HBV genotypes A, B, C, D, E, F, G, H, I, and J.

In particular embodiments of the above methods, compositions for use, or uses in manufacture, the anti-HBV antibody thereof may be a human antibody; a monoclonal antibody or antigen-binding fragment thereof; or a bispecific antibody or antigenbinding fragment thereof, with a first specificity for HBsAg and a second specificity that stimulates an immune effector (e.g., a second specificity that stimulates cytotoxicity or a vaccinal effect). In certain other embodiments of the above methods, compositions for use, or uses in manufacture disclosed herein, the anti-HBV antibody is a monoclonal antibody.

In certain embodiments, the anti-HBV antibody is administered in at least two separate doses. In particular embodiments, the at least two doses are administered twice per week, once per week, every other week, every two weeks, or once a month.

In certain embodiments, the subject is a human and a therapeutically effective amount of the anti-HBV antibody is administered; wherein the therapeutically effective amount is from about 3 mg/kg to about 30 mg/kg.

In particular embodiments of the above methods, compositions for use, or uses in manufacture, the single dose of the pharmaceutical composition comprises up to 6 mg, up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 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, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 3000 mg, or in a range from 5 mg to 3000 mg, or in a range from 10 mg to 3000 mg, or in a range from 25 mg to 3000 mg, or in a range from 30 mg to 3000 mg, or in a range from 50 mg to 3000 mg, or in a range from 60 mg to 3000 mg, or in a range from 75 mg to 3000 mg, or in a range from 90 mg to 3000 mg, or in a range from 100 mg to 3000 mg, or in a range from 150 mg to 3000 mg, or in a range from 200 mg to 3000 mg, or in a range from 300 mg to 3000 mg, or in a range from 500 mg to 3000 mg, or in a range from 750 mg to 3000 mg, or in a range from 900 mg to 3000 mg, or in a range from 1500 mg to 3000 mg, or in a range from 2000 mg to 3000 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 900 mg, or in a range from 5 mg to 900 mg, or in a range from 10 mg to 900 mg, or in a range from 25 mg to 900 mg, or in a range from 30 mg to 900 mg, or in a range from 50 mg to 900 mg, or in a range from 60 mg to 900 mg, or in a range from 75 mg to 900 mg, or in a range from 90 mg to 900 mg, or in a range from 100 mg to 900 mg, or in a range from 150 mg to 900 mg, or in a range from 200 mg to 900 mg, or in a range from 300 mg to 900 mg, or in a range from 500 mg to 900 mg, or in a range from 750 mg to 900 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 500 mg, or in a range from 5 mg to 500 mg, or in a range from 10 mg to 500 mg, or in a range from 25 mg to 500 mg, or in a range from 30 mg to 500 mg, or in a range from 50 mg to 500 mg, or in a range from 60 mg to 500 mg, or in a range from 75 mg to 500 mg, or in a range from 90 mg to 500 mg, or in a range from 100 mg to 500 mg, or in a range from 150 mg to 500 mg, or in a range from 200 mg to 500 mg, or in a range from 300 mg to 500 mg, or in a range from 400 mg to 500 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 300 mg, or in a range from 5 mg to 300 mg, or in a range from 10 mg to 300 mg, or in a range from 25 mg to 300 mg, or in a range from 30 mg to 300 mg, or in a range from 50 mg to 300 mg, or in a range from 60 mg to 300 mg, or in a range from 75 mg to 300 mg, or in a range from 90 mg to 300 mg, or in a range from 100 mg to 300 mg, or in a range from 150 mg to 300 mg, or in a range from 200 mg to 300 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 200 mg, or in a range from 5 mg to 200 mg, or in a range from 10 mg to 200 mg, or in a range from 25 mg to 200 mg, or in a range from 30 mg to 200 mg, or in a range from 50 mg to 200 mg, or in a range from 60 mg to 200 mg, or in a range from 75 mg to 200 mg, or in a range from 90 mg to 200 mg, or in a range from 100 mg to 200 mg, or in a range from 150 mg to 200 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 100 mg, or in a range from 5 mg to 100 mg, or in a range from 10 mg to 100 mg, or in a range from 25 mg to 100 mg, or in a range from 30 mg to 100 mg, or in a range from 50 mg to 100 mg, or in a range from 60 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 90 mg to 100 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 25 mg, or in a range from 5 mg to 25 mg, or in a range from 10 mg to 25 mg, or in a range from 15 mg to 25 mg, or in a range from 20 mg to 25 mg, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 50 mg, or in a range from 1 mg to 25 mg, or in a range from 5 mg to 50 mg, or in a range from 5 mg to 25 mg, or in a range from 10 to 50 mg, or in a range from 10 to 25 mg, or in a range from 1 to 15 mg, or in a range from 5 mg to 15 mg, or in a range from 10 mg to 15 mg, or wherein the single dose of the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,

790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg, or more, of the antibody, or the single dose of the pharmaceutical composition comprises the antibody in an amount that is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 mg, less than 25 mg, or less than 10 mg, but is more than 1 mg, more than 2 mg, more than 3 mg, more than 4 mg, or more than 5 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises about 75 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises about 90 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody. In certain embodiments, the single dose of the pharmaceutical composition comprises about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg of the antibody. In certain embodiments, the method comprises administering the single dose by subcutaneous injection, optionally wherein the single dose comprises or consists of 6 mg of the antibody or 18 mg of the antibody. In some instances, elements of the antibodies, antigen-binding fragments, fusion proteins, nucleic acids, cells, compositions, combinations, uses, and methods provided herein are described or listed with reference to embodiments or examples. However, it should be understood that the examples and embodiments described herein may be combined in various ways to create additional embodiments.

EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the disclosure are presented. However, the present disclosure shall not to be limited in scope by the specific embodiments described herein.

EXAMPLE 1 : DESIGN OF ANTI-HBV ANTIBODY FC VARIANTS

Anti-HBV antibodies are disclosed in PCT Publication No. WO 2017/060504. Engineering anti-HBV antibody "HBC34-v7" produced, inter alia, antibody "HBC34- v35" (PCT Publication No. WO 2020/132091), having VH and VL amino acid sequences according to SEQ ID NOs.:38 and 47, respectively. HBC34-v35 binds to HBsAg with picomolar affinity and potently neutralizes ten (10) HBV genotypes and Hepatitis D virus, binding to a conserved conformational epitope. Engineered variants of anti-HBV antibody “HBC34-v35” were produced, inter alia, antibodies "HBC34- v40," "HBC34-v44," "HBC34-v45," and "HBC34-v50" (PCT Publication No. WO 2021/262840), having mutations in L-CDR2 and/or in framework sequence relative to HBC34-v35 were generated. The HBC34-v40, HBC34-v44, HBC34-v45, and HBC34- v50 antibodies had similar or even improved neutralizing activity (EC50) as compared to HBC34-v35 and exhibited low generation high molecular weight species (HMWS) corresponding to an antibody dimer. In other words, the HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 antibodies were determined to have a low propensity for aggregation while maintaining potent binding. HBC34-v40 comprises E49Q, V50D, and K51S mutations in L-CDR2 (CCG numbering) compared to parental HBC34-v35. These mutations change from hydrophobic interaction to electrostatic repulsion and the loss of a salt bridge.

HBC34-v44 comprises an E49A mutation in L-CDR2 as compared to HBC34- v35. This mutation results in loss of a salt bridge.

HBC34-v45 and HBC34-v50 comprise framework mutations at R60 relative to HBC34-v35. R60N and R60K mutations in HBC34-v45 and HBC34-v50, respectively, reduced dimer formation.

Binding of HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50, to HBsAg from ten ((A)-(J)) genotypes was tested by FACS. HBC34-v35 was included as reference. HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50, to HBsAg bind to HBsAg, with HBC34-v40 showing the most potent binding (data not shown). HBC34- v40, HBC34-v44, HBC34-v45, and HBC34-v50 bind to ten HBsAg-genotype D mutants, as previously tested by FACS (data not shown).

In addition, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 demonstrate similar or even improved neutralizing activity (EC50) as compared to HBC34-v35. Neutralization was assessed previously by measuring the levels of HBeAg (genotype D) in the cell culture supernatant of HBV-infected HepG2 cells expressing NTCP. Results from the neutralization assay are provided in Figure 38, and show that several antibodies (HBC34-v40-HBC34-v44, HBC34-v45, and HBC34-v50) had similar or even improved neutralizing activity (EC50) as compared to HBC34-v35.

Additional neutralization assays were previously performed to assess the neutralization capacity of antibodies against HDV by measuring the levels of HBsAg (genotype A) in the cell culture supernatant of HDV-infected cells. Results from the neutralization assay are provided in Figure 39, and show that HBC34-v40 and HBC34- v44 had similar neutralizing activity (EC50) as compared to HBC34-v35.

EXAMPLE 2

DESIGN OF NOVEL FC VARIANTS Human IgGl Fc regions were engineered as described in PCT Publication No. WO 2022/251119 (hereby incorporated by reference in entirety) for improved function, such as to potentially promote prophylactic, therapeutic, or vaccinal effects by activating certain FcyRs (e.g. FcyRIIA, FcyRIIIA, FcyRIIB). Enhancing activation of FcyRIIA in early infection may promote antibody-dependent cellular phagocytosis (ADCP) and viral neutralization. Enhancing activation of FcyRIIA and/or FcyRIIIA in late or established infection may promote ADCP and/or antibody-dependent cellular cytotoxicity (ADCC), facilitate clearance of virally infected cells, and block viral spread. Enhancing activation of FcyRIIA and/or FcyRIIIA at any time during infection may provide a vaccinal effect by promoting antigen presentation and adaptive immunity.

Briefly, Fc variants were assessed and new variants were developed using an iterative discovery workflow. An initial set of approximately 2500 Fc point mutations was generated, and functional data was collected and analyzed. Functional data included binding interactions (e.g. to FcyRI, FcyRIIA (R131), FcyRIIB, FcyRIIC, FcyRIIIA (VI 58), FcRn, and Clq), signaling via FcyRs, thermostability, expressability, polyreactivity, and half-life extendibility. A machine learning and multi-factor prediction-based algorithm was developed to assist in designing further variants. Fc variants were expressed as IgGl antibodies (Kallewaard et al. Cell 766(3):596-608 (2016)) in CHO cells, titered using high-performance liquid chromatography (HPLC), and purified using protein A columns. A first plate (2 x 96, with or without 2-deoxy-2- fluoro-L-fucose (2FF), which inhibits fucosylation) contained wells for measuring effects of known mutations (as reference) and wells for measuring effects of novel mutations (single or combination).

Fc variants were analyzed using various assays to evaluate biophysical, biochemical, and biological properties. These included aggregation (e.g. by sizeexclusion chromatography), thermostability, glycosylation, structure, signaling, and binding (e.g. using surface plasmon resonance or meso scale discovery-based assays). Effector functions were also tested, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Binding characteristics of single Fc mutations were evaluated, combinations of up to three mutations were identified that had the highest effect on increasing the IIA/IIB ratio, and additional variations included. The resulting further variants were analyzed. Characteristics of interest included increased affinity for FcyRIIa with reduced affinity for FcyRIIb, or vice versa. Using unbiased cluster analysis and radar plotting with manual analysis, nine clusters of Fc variants having strongly increased, increased, similar or the same, decreased, or strongly decreased affinity for various FcyRs and FcRn were identified.

EXAMPLE 3

FURTHER TESTING OF FC VARIANTS

Based on results obtained from the first plate of variants described in Example 2, a second plate (2 x 20, with and without 2FF) of variants was generated. The twenty Fc variant antibodies were expressed and purified to evaluate titer and yield. Variants were expressed without or with 2FF to determine the effect of fucosylation on titer and yield (data not shown). Mean titer was higher for variants expressed in cells without 2FF. Fc variants were purified using two elutions and combined prior to determining yield. Average yield was higher in purified Fc variants expressed without 2FF.

Binding of (No 2FF) Fc variants to FcyRIIA-H (high affinity), FcyRIIA- R (low affinity), FcyRIIB, FcyRIIIA-V (high affinity), FcyRIIIA-F (low affinity), and FcRn (at pH 6) was tested and expressed as fold-change relative to wild-type Fc (Figure 2A). The ratio of FcyRIIA-H/FcyRIIB binding, as well Clq binding and complementdependent cytotoxicity (CDC) data, are also shown in Figure 2B. Variants shown in Figure 3 were not treated with 2FF. Antibody signaling through different FcyRs was measured using a reporter assay (Promega™ luciferase reporter cells; average of 3 experiments). Fucosylated Fc variants were tested for signalling through all four FcyR receptors shown (Figure 4A), while afucosylated variants were tested for signalling through FcyRIIIA-V and FcyRIIIA-F (Figure 4B). A number of variant Fes were selected for further characterization. A summary of characteristics of these variants (both fucosylated and afucosylated), as well as of comparator variant Fes comprising known mutations (e.g., G236A S239D A330L I332E (“GASDALIE”); G236A A330L I332E (“GAALIE”) is shown in Figures 5A-5C.

Several variants demonstrated improved attributes over the GAALIE variant, including increased IIA/IIB ratio, increased stability (Tm), balanced binding to FcyR alleles, and increased Clq binding and complement activation. Dose-dependent FcyR signaling through FcyRIIA-H (high affinity, Figure 8A) and FcyRIIB (Figure 8B) for one such Fc variant, "G236A R292P Y300L" was measured using a reporter cell assay.

FcyR binding versus signaling through FcyRIIA-H (high affinity, Figure 9A) and FcyRIIB (Figure 9B) by Fc variants is shown in Figures 9A and 9B. FcyR binding was measured using a Meso Scale Discovery binding assay and FcyR signaling was measured using a reporter cell assay.

Additional experiments were conducted using an anti-HBsAg antibody (HBC34-v35), with results shown/summarized in Figures 10A-1 ID. These results showed, inter alia, that afucosylated Fc variant antibodies bearing the G236A (“GA”) mutation have stronger binding to FcyRs IIA and IIIA as compared to fucosylated Fc variant antibodies bearing G236A A330L I332E (“GAALIE”), and induce comparable signalling via FcyRIIIA, and potentially stronger signalling via FcyRIIA, as compared to fucosylated GAALIE-bearing Fc variant antibodies. Additionally, GA- afucosylated Fc variant antibodies had improved melting temperature versus fucosylated GAALIE-bearing Fc variant antibodies. GA-afucosylated Fc variant antibodies induced NK cell-mediated ADCC against target cells. GA-afucosylated Fc variant antibodies retained partial Clq binding (0.3x compared to reference IgGl antibody bearing M428L and N434S mutations only), while GAALIE mutations caused abrogation of Clq binding.

EXAMPLE 4

CHARACTERIZATION OF HBC35-V40-FC VARIANTS NanoDSF Melting Temperature (Tm)

HBC34-v40-LS and variants were diluted to 1 mg/mL in formulation buffer and loaded into capillaries compatible with the NanoTemper Prometheus instrument. The Prometheus instrument recorded fluorescence while heating the capillaries from 20°C to 95°C at a rate of 1.0°C/minute. Temperatures corresponding to protein unfolding were identified using inflection points in the fluorescence signal.

Aggregation (25 mg/mL)

Each antibody was concentrated to 25 mg/mL using Amicon spin concentrators. The concentrated samples were transferred to glass vials and incubated at 40°C for one month. An aliquot was taken each week and diluted to 2.5 mg/mL. Ten micrograms of the diluted aliquots were injected onto a Tosoh TSKGel size exclusion column with 50 mM Sodium Phosphate, 300 mM NaCl pH 7.4 mobile phase. The relative peak areas of high molecular weight species were evaluated for each timepoint.

Glycan Release

A fifteen microgram aliquot of each antibody was denatured with surfactant and heat. A deglycosylating enzyme, PNGase F, was added to the samples. The glycans were then labeled with a fluorescent tag from Waters (RapiFluor). Protein was separated from the labeled glycans using solid phase extraction, and then the extracted glycans were injected onto a hydrophilic interaction chromatography column (MPA: 50 mM ammonium sulfate pH 4.0/MPB: ACN). The glycans were identified using standards purchased from Waters. The glycan composition of each sample was determined by the relative peak area of each glycan.

FcRn Affinity Chromatography

A thirty microgram aliquot of each antibody was injected onto a column packed with human FcRn. The pH of the mobile phase was adjusted from pH 5.5 to 8.8 using a linear gradient of 20 mM MES, 140 mM NaCl pH 5.5 and 20 mM Tris, 140 mM NaCl pH 8.8 for 80 minutes. The elution pH of the antibodies was reported.

Table 6: Properties of HBC34-v40 mAbs.

All antibodies are in the background of LS and use the Glm3 allotype, unless otherwise indicated. * Measurement was performed in the background of LS and Glml7,l allotype. HMWS - High molecular weight species, N.D. - not determined

EXAMPLE 5

BINDING OF HBC34-V40-FC VARIANTS TO FcyR

The binding affinity of HBC34-v40-Fc variants was determined by surface plasmon resonance (SPR) using a Biacore T200 instrument (see Figure 6A-B). A Series S sensor CAP chip and the Biotin CAPture reagent (Cytiva, Cat#: 28920234) was used for surface capture of biotinylated FcyRs. Running buffer was HBS-EP+ pH 7.4 (Cytiva, Cat #: BRI 00669) and measurements were performed at 25 °C. Experiments were performed with a 3-fold dilution series of IgGs at 819, 273, 91, 30.3, and 10.1 nM and were run as single-cycle kinetics with 600s association time and 100s dissociation time. To quantify binding affinity (KD), data were double reference-subtracted and fit to a steady-state binding model using Biacore Insight software. For measurements where FcgR2b was the ligand, most IgG analytes had low binding activity so Rmax from the binding of HBC34-v40-rIgGlm3-LS-S239D-H268E-G236A was applied to all IgG as a constant.

Table 7: Fold-change results for Fc variant antibodies

EXAMPLE 6

ANTIBOD Y-FC- VARIANT-DEPENDENT ACTIVATION OF FcyRIlA AND FcyRIIlA SIGNALING IN JURKAT REPORTER T CELLS

Antibody-dependent activation of human FcyRIIa or FcyRIIIa was examined. HBsAg was produced in PLC/PRF/5 cells and serially diluted 3-fold in Assay buffer from 1,000 U/ml to 4.12 U/ml. Simultaneously HBC34-v40 and HBC34-v40-Fc variants were serially diluted 5-fold respectively in Assay buffer from 100 pg/ml to 0.000256 pg/ml respectively (or as indicated in Figures 13-14). Serial dilutions of the target antigens were added in a white flat bottom 96-well plate in 25 pl, then serially diluted HBC34-v40 or HBC34-v40-Fc variants added to each well (25 pl per well), and the antib ody/antigen was incubated for 20 minutes at room temperature. Jurkat cells expressing FcyRIIa or FcyRIIIa on their surface and stably transfected with NFAT- driven luciferase gene (Promega) were thawed and added at a cell density of 5 x 10⁴/well or 7.5 x 10⁴/well in 25 pl. Control wells were also included that were used to measure antibody-independent activation (containing HBsAg and Jurkat cells but no antibody) and spontaneous luminescence of the plate (wells containing the Assay buffer only). Plates were incubated for 18 hours at 37°C with 5% CO2. Activation of human FcgRIIa (H131 allele) and FcyRIIIa (V158 allele) in this bioassay results in the NFAT- mediated expression of the luciferase reporter gene. Luminescence was measured using the Bio-Gio™ Luciferase Assay Reagent according to the manufacturer's instructions. A diagram summarizing the reporter cell assay described in this Example is shown in Fig. 7.

Results

G326R-L328R (GRLR) mutations abrogated FcyR binding and served as the negative control. The afucosylation (afuc) Fc modification, which results from the removal of fucose from the N297 glycan, specifically increased binding to FcyRIIIa. Immune complexes including HBC34-v40-LS did not induce activation of FcyRs Ila or Illa. Immune complexes including HBC34-v40-LS-GAALIE activated FcgRIIa but not Illa. Immune complexes including HBC34-v40-LS-GAYL or -LS-GARPYL activated FcgRIIa more than the WT (LS) Fc variant or LS-GAALIE (see Figs. 12B and 13A-D ). Immune complexes including HBC34-v40-LS-GAYL-afuc or -LS-GARPYL-afuc activated FcgRIIIa more than the WT (LS), LS-GAALIE, GAYL or GARPYL (see Figs. 12A and 14A-F). Therefore, the results indicate that the HBC34-v40-LS- GARPYL and -GAYL Fc mutations in immune complexes with HBsAg increase activation via FcyRIIa, while HBC34-v40-LS-GARPYL-afuc and -GAYL-afuc Fc mutations increase activation via FcyRIIIa.

EXAMPLE 7

MONOCYTE-DERIVED DENDRITIC CELL (MODCS) ACTIVATION BY IMMUNE COMPLEXES OF ANTI-HBV ANTIBOD Y-FC VARIANTS AND HBSAG An in vitro model was used to assay induction of primary human monocyte- derived dendritic cell (moDCs) activation by immune complexes of HBC34-v40-Fc variants and HBsAg. Blood from healthy donors was obtained from the Swiss Blood Donation Center of Lugano (Switzerland), with informed consent from the Swiss Red Cross and authorization from the Comitato Etico Canton Ticino. Human mononuclear cells (PBMCs) were freshly isolated from peripheral blood using Ficoll-Paque PLUS and density gradient centrifugation. Monocytes (CD14+ cells) were further isolated by positive selection using magnetic microbeads. Monocytes were cultured in complete medium (RPMI 1640, 10% FBS, 1% NEAA, 1% Glutamine, 1% Pen/Strep, 1% Sodium Pyruvate, P-mercaptoethanol 50pM) in a 12-well plate flat bottom at a concentration of 500,000 cells/mL. To induce the differentiation of monocytes into monocyte-derived dendritic cells (moDCs), the complete medium was enriched by a cocktail of cytokines including GM-CSF (50 ng/mL) and IL-4 (1,000 lU/mL). Monocytes were differentiated into immature moDCs for 6 days.

Each stimulating condition was prepared in a 96-well round bottom plate, containing HBC34-v40-Fc variants alone (50 pg/mL), HBsAg alone (30, 100, 300 or 1000 lU/mL), or HBC34-v40-Fc variants (50 pg/mL) in combination with HBsAg (30, 100, 300 or 1000 lU/mL). As source of HBsAg, the serum of patients with chronic HBV infection was used. LPS (100 ng/mL) was used as positive control of moDCs activation. The plate was then incubated at 37 °C for at least 1 hour to allow the formation of immune complexes between HBsAg and mAbs. After the incubation, 100,000 or 200,000 moDCs were gently added to each well. moDCs together with immune complexes were incubated at 37 °C for 21-24 hours before assessing the moDCs activation state.

After 21-24 hours of incubation at 37 °C, to assess moDCs activation, cells were stained with anti-CD14, anti-CD83, anti-CD86 and anti-HLA-DR antibodies conjugated to different fluorochromes. To determine cell viability cells were stained with Zombie Aqua. Data were acquired using the ZE5 Cell Analyzer (Bio-Rad). Figure 15 depicts a diagram of differentiation of monocytes to moDCs and activation with HBsAg + HBC34-v40 Fc variants. Results

The results demonstrate that immune complexes of HBsAg and HBC34-v40- LS-GAALIE, -GA, -GAYL, -GARPYL, and -GALVQE activate human moDCs more than the WT Fc. Compared to non- stimulated moDCs, HBC34-v40-LS in complex with HBsAg from serum of patients with CHB increased the surface expression of the activation marker CD83, the co-stimulatory marker CD86, as well as HLA-DR (data not shown). When stimulated with HBsAg HBC34-v40-LS-GAALIE, -GA, -GAYL, - GARPYL, or -GALVQE, moDCs became significantly more activated than with HBC34-v40-LS. These results were confirmed over a wide range of HBsAg concentrations (30, 100, 300, 1000 lU/mL (see Fig. 18A-B). In contrast, HBsAg alone (data not shown) or HBsAg in complex with HBC34-GRLR (abrogated binding to FcyRs) did not activate moDCs. The activation of moDCs by HBC34-v40-LS- GAALIE, -GA, -GAYL, -GARPYL, or -GALVQE demonstrate that the HBC34-v40-Fc variants successfully complete an initial step towards a vaccinal effect.

EXAMPLE 8

POLYCLONAL STIMULATION BY MODCS OF HBSAG-SPECIFIC CD4+ MEMORY T CELLS FROM A HUMAN VACCINEE

A diagram summarizing the process described in this Example can be found in Figure 23. PBMCs were freshly isolated from whole blood of HBV vaccinees following the same protocol as described in Example 7. Monocytes (CD14+ cells) were further isolated by positive selection using magnetic microbeads and put in differentiation into moDCs for 6 days with GM-CSF and IL-4 as described above. The negative fraction (PBMCs minus monocytes) was stored in liquid nitrogen for the following isolation of CD4+ memory T cells. Each stimulating condition was prepared in a 96-well flat bottom plate, containing HBC34-v40-Fc variants alone (50 pg/mL), HBsAg alone (30 or 100 lU/mL), or HBC34-v40-Fc variants (50 pg/mL) in combination with HBsAg (30 or 100 lU/mL). Staphylococcal enterotoxin B (SEB) (1 pg/mL) was used as positive control of T lymphocytes activation and proliferation. The plate was then incubated at 37 °C for at least 1 hour to allow the formation of immune complexes between HBsAg and mAbs. After the incubation, 10,000 moDCs were gently added to each well. moDCs together with immune complexes were incubated at 37 °C for 21-24 hours to prepare for co-culture with autologous total CD4+ memory T cells.

Starting from frozen negative fraction (PBMCs minus monocytes), total CD4+ T cells were enriched by positive selection using magnetic microbeads and then memory CD4+ T cells were sorted based on the expression of the following surface markers: CD4+CD25-CD45RA- (CCR7+/-). Cells were sorted using the Sony cell sorter SH800SFP. To follow the proliferation in co-culture with activated autologous moDCs, sorted CD4+ memory T cells were labelled with carboxyfluorescein succinimidyl ester (CFSE). Finally, 100,000 CFSE-labelled CD4+ memory T cells were put in co-culture with autologous moDCs stimulated the day before (24 hours) as described above. The cells were incubated for 5 days at 37 °C.

After 5 days of co-culture, to assess the expression of activation markers, CD4+ memory T cells were stained with anti-CD4, anti-CD25, anti-CD45RA, anti-CCR7, anti-ICOS, anti-HLA-DR antibodies conjugated to different fluorochromes. To determine cell viability cells were stained with Zombie Aqua. Data were acquired using the ZE5 Cell Analyzer (Bio-Rad).

Results

The data demonstrate that immune complexes of HBC34-v40-LS-GAYL, - GAYL-afuc or -GARPYL-afuc and HBsAg increase the activation of HBsAg-specific CD4+ memory T cells from human HBV vaccinees. To develop long-lasting adaptive immunity against HBV via vaccinal effect, activated dendritic cells need to present HBsAg-derived peptides to T cells. Polyclonal CD4+ memory T cells isolated from HBV vaccinees were co-cultured with autologous moDCs that have been stimulated with ICs of HBsAg from HBV+ patient sera and HBC34-v40-LS-GAALIE Fc variants. After 5 days of co-culture, HBC34-v40-LS, -GA, -GAALIE, -GAYL, and -GARPYL induced the proliferation (% of CFSE low cells) of HBsAg-specific CD4⁺ memory T cells (see Figs. 24A-B and 25A-B) and expression of activation markers CD25, ICOS, and HLA-DR (data not shown). The proliferation induced by ICs with HBC34-v40-LS- GAYL, -GAYL-afuc and -GARPYL-afuc was substantially higher compared to the other Fc variants. In contrast, when moDCs were stimulated with HBsAg in complex with HBC34-GRLR (abrogated binding to FcyRs), CD4⁺ memory T cells proliferation (see Figs. 24A-B and 25A-B) and activation was significantly lower and comparable to stimulation with HBsAg alone (data now shown), underlining an FcyR-mediated effect. Overall, the proliferation of HBsAg-specific CD4⁺ memory T cells is evidence for an mAb-mediated vaccinal effect, thus potentially contributing to long-term adaptive immunity against HB V.

EXAMPLE 9

RESTIMULATION OF CD8+ T CELL IN TOTAL PBMCS FROM PATIENTS WITH CHRONIC HEPATITIS B

A diagram summarizing the process described in this Example can be found in Fig. 27. Frozen PBMCs from CHB patients were thawed, put in culture in complete medium enriched with 50 ng/mL of Flt3-L and incubated overnight at 37 °C. Each stimulating condition was prepared in a 96-well flat bottom plate, containing HBC34- v40-Fc variants alone (50 pg/mL), HBsAg alone (30 ZU/mL), or HBC34-v40-Fc variants (50 pg/mL) in combination with HBsAg (30 ZU/mL). SEB (1 pg/mL) was used as positive control of T lymphocytes activation and proliferation. The plate was then incubated at 37 °C for at least 1 hour to allow the formation of immune complexes between HBsAg and mAbs. In order to follow their proliferation upon stimulation, in a separate container total PBMCs from CHB patients were labelled with carboxyfluorescein succinimidyl ester (CFSE). After the incubation, 300,000 CFSE- labelled PBMCs were gently added to each well. PBMCs together with immune complexes were incubated at 37 °C for 5 days to allow stimulation of CD8+ T cells by APCs.

After 5 days of stimulation, to assess the expression of activation markers, PBMCs were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD69 antibodies conjugated to different fluorochromes. To determine cell viability cells were stained with Zombie Aqua. Data were acquired using the ZE5 Cell Analyzer (Bio-Rad) and analyzed using FlowJo vl0.8.1 software. Results

T cells in patients with CHB are considered to be anergic or not protective. We used PBMCs from patients with CHB to assess the possibility to restimulate CD8+ T cells within these samples. When incubated with immune complexes of HBsAg from patient serum and HBC34-v40-LS Fc variants GRLR, LS, GA, GAALIE or GAYL, no substantial proliferation of CD8+ T cells was observed. In contrast, immune complexes with HBC34-v40-LS-GARPYL induces slight and HBC34-v40-LS-GARPYL-afuc or GAYL-afuc substantial proliferation of CD8+ T cells within PBMCs of patients with CHB (see Fig. 28A-B). This data indicate that Fc engineering that increases signaling of FcyRIIa as well as FcyRIIIa (e.g., GAYL-afuc or GARPYL-afuc) is capable of overcoming anergy and re-activating CD8+ T cells from patients with CHB.

EXAMPLE 10

RESTIMULATION OF POLYCLONAL MEMORY CD4+ T CELLS FROM MICE IMMUNIZATION WITH HBV VACCINE

Next, the re-activation of HBsAg-specific CD4+ memory T cells was examined ex vivo in T cells that were isolated from mice transgenically expressing the full set of human FcyRs (huFcyRs I, IIA, IIIA, and IIB on the C57BL/6 background) (Smith et al. 2012). This model allows to investigate engineered human Fc variants, which specifically modulate binding to human but not to mouse FcyRs. To induce T cell memory, HuFcyR or wild-type C57BL/6 mice were vaccinated with Engerix B20 (HBV vaccine) and boosted after two weeks. Two months after the booster, CD44+ CD4+ memory T cells were FACS sorted from LNs and spleens and labelled with CFSE. In parallel, bone marrow derived dendritic cells (BMDCs) were generated from naive HuFcyR mice and stimulated with HBC34-v40 Fc variants in complex with HBsAg produced in PLC hepatoma cells. After 6 days of co-culture with stimulated BMDCs, proliferation of HBsAg-specific CD4+ memory T cells was assessed by flow cytometry, tracking CFSE dilution. A diagram summarizing the process described in this Example can be found in Fig. 29. The in vivo work was authorized by the local IACUC authority (license n. TI- 42/2020). Eight to 10 weeks old HuFcgR mice were primed with 2 pg HBsAg/mouse of the Engerix B20 vaccine (100 pl s.c. + 100 pl i.p.). Two weeks post first immunization, HuFcyR mice were boosted with additional 2 pg/mouse of the Engerix B20 vaccine as above. In one experiment (ELN X012940), C57BL/6 mice were immunized with the same protocol and used as memory T cells donors.

For the generation and stimulation of bone-marrow derived dendritic cells (BMDCs), huFcyR mice femurs were collected, and bone marrow (BM) was isolated by flushing 30 ml of PBS into the lumen of the bone. BM cells were centrifugated 5 min at 500g (4°C) and re-suspended in 1 ml of pre-warmed BMDCs differentiation medium. Seven million BM cells were then seeded in one 10-cm Petri culture dish after resuspension with 10 ml of pre-warmed BMDCs differentiation medium and further incubated for 8 days at 37°C. The stimulation conditions from BMDC listed below were prepared in a volume of 100 pL complete medium in a 96-well, flat-bottom plate. The used stimulation conditions were: 1) mAbs alone (20 pg/mL), 2) HBsAg alone (1000 ZU/mL), 3) IC mAb-HBsAg (HBC34-v40 Fc variants 20 pg/mL, complexed in the culture plate for 1 hour at 37°C with HBsAg at 1000 lU/mL). SEB (1 pg/mL) was used as positive control of T cell proliferation. LPS (100 ng/mL) was used as positive control for BMDCs activation (data not shown). Fifty thousand BMDCs resuspended in 100 pl CM, were gently added to each plate well containing 100 pL CM with IC or control stimuli and incubated at 37 °C for 21-24 hr.

For the isolation of polyclonal memory CD4+ T cells from mice immunized with Engerix HB V vaccine, two months post immunization, mice were euthanized to allow organ collection (axillary, brachial and inguinal LNs and spleens). A single cell suspension was obtained via gently smashing the lymphoid organs with a 5-mL syringe plunger on 70 mm cell strainer in 20 ml FACS buffer. The obtained cell suspension was pelleted at 400g for 5 min 4°C, resuspended with 5 ml ACK buffer for 5 min to eliminate red blood cells, washed once with FACS buffer, and finally re-suspended in complete medium. CD4⁺ T cells were isolated by negative selection using magnetic microbeads from the CD4⁺ T Cell Isolation Kit. Briefly, 100 pl of biotin-antibody cocktail were added to the cell suspension and incubated 5 min on ice. Two hundred mL of anti -biotin microbeads were added and incubated 10 min on ice. After washing the cells with MACS buffer at 400g for 5 min at 4°C, the pellet was resuspended in 3 mL of MACS buffer and added to the LS magnetic column attached to the QuadroMACS™ Separator. The CD4⁺ T cells were eluted from the LS column detached from the magnet with MACS buffer. CD4⁺ CD44⁺ memory T cells were then sorted based on the expression of surface markers. Briefly, MACS-isolated CD4⁺ T cells were washed with FACS buffer and then resuspended in 200 pL of surface staining mix containing antibodies recognizing the markers mentioned above conjugated to different fluorochromes (CD4-APC-Cy7, CD44-PerCP) and incubated for 20 min on ice. After a washing step with FACS buffer and a spin at 400g for 5 min at 4°C, T cells were resuspended in FACS buffer, filtered with Pre- Separation Filters (30 pm) and sorted using the Sony cell sorter SH800SFP. To follow the proliferation of CD4⁺ memory T cells in co-culture with ICs-pulsed BMDCs, T cells were labeled for 8 min at 37°C with CFSE, at a final concentration of 5 pM in 1 mL of PBS, supplemented with 2% FBS. CD4⁺ T cells were then washed three times with CM at 300 g for 8 min.

For restimulation, CFSE-labeled CD4⁺ memory T cells were put in co-culture with BMDCs stimulated the day before, as described above. ICs-pulsed BMDCs in 96- well plate flat bottom were centrifuged at 450 g for 5 min and the supernatant was discarded. BMDCs were then re-suspended in 200 pl of complete medium containing CFSE-labeled CD4⁺ memory T cells for a final co-culture condition of 1 : 10 (5E04 BMDCs and 5E05 CD4⁺ memory T cells). The co-coculture was incubated for 6 days at 37 °C. After 6 days of co-culture at 37 °C, cells were stained to assess the CD4⁺ memory T cells re-stimulation and activation. Briefly, cells were stained with Zombie Aqua for 30 min at RT to determine cell viability. After a washing step with FACS buffer and a spin at 400g for 5 min at 4°C, cells were stained with a surface markers mix containing antibodies recognizing CD4-APC/Cy7, CD44-PerCP, CD69-Pe-Dazzle 594, CD62L-BV605 and incubated for 20 min on ice. After a washing step with FACS buffer and a spin at 400g for 5 min at 4°C, cells were re-suspended in FACS buffer and acquired using the ZE5 Cell Analyzer (Bio-Rad). CFSE low cells were interpreted as cells that had proliferated.

Results

Results showed that HuFcyR-BMDCs induced a significantly higher proliferation of HBsAg-specific CD4+ memory T cell when stimulated with ICs of HBsAg and HBC34-v40-GA, GALVQE or GAYL than after stimulation with ICs containing HBC34-v40 (WT), indicating that Fc engineering can augment the activation of HBsAg-specific T cells via FcyRIIa-mediated effect (see Figures 31A-B and 32). In contrast, when HuFcyR-BMDCs were stimulated with HBsAg in complex with HBC34-v40-GRLR, CD4⁺ memory T cells proliferation was low and comparable to stimulation with HBsAg alone or mAbs alone. Stimulated with SEB superantigen served as positive control. These results indicate that increased binding of engineered Fes of HBC34-v40 to FcyRIIa was essential to activate HuFcyR-BMDCs and thus allow efficient antigen presentation and re-stimulation of HBsAg-specific CD4+ T cells, further supporting the concept of a vaccinal effect.

EXAMPLE 11

RESTIMULATION OF POLYCLONAL MEMORY CD4+ T CELLS FROM HBV

IMMUNIZED MICE WITH FC VARIANTS LACKING LS MODIFICATION

Using the protocol of Example 10, experiments were performed to test the reactivation of CD4+ memory T cells with immune complexes formed with HBsAg and HBC34-v40-Fc variants lacking the LS modification, e.g., HBC34-v40- GA, -GAALIE, -GAYL, -GALVQE, and -GARPYL. The results are shown in Fig. 33A-C and demonstrate that the presence of the LS modification does not strongly influence the activation of HBsAg-specific memory T cells. Modest improvement was demonstrated for HBC34-v40-LS-GA and -LS-GAYL compared to HBC34-v40-GA and -GAYL, respectively. Further, improvement was demonstrated at the highest concentration of HBC34-v40-GALVQE compared to HBC34-v40-LS-GALVQE.

EXAMPLE 12 RESTIMULATION OF POLYCLONAL MEMORY CD4+ T CELLS FROM HBV IMMUNIZED MICE WITH AFUCOSYLATED FC VARIANTS

Using the protocol of Example 10, experiments were performed to test the reactivation of CD4+ memory T cells with immune complexes formed with HBsAg and HBC34-v40-LS-GAYL and LS-GARPYL and HBC34-v40-LS-GAYL-afuc and LS- GARPYL-afuc, respectively. Afucosylation increases FcyRIIIa signaling for the Fc variants. The results are shown in Fig. 34A-B and demonstrate the that FcyRIIIa also plays a role in re-activation of CD4+ memory T cells.

EXAMPLE 13

IN VIVO PRIMING OF NAIVE CD4+ T CELLS FROM MICE TRANSGENIC FOR A HBSAG-SPECIFIC TCR

To assess the capacity of ICs to prime naive HBs-specific CD4+ T cells in vivo, naive CD4+ Tg T cells were isolated from mice expressing transgenes for a reactive TCR specific for HBsAg residues 126-138. Isolated naive HBs-specific CD4+ T cells were transferred into HuFcyR mice that were immunized with ICs. 3 days post immunization, proliferation and activation of CD4+ Tg T cells were assessed by flow cytometry. A diagram summarizing the process described in this Example can be found in Figs. 35 and 36.

For the isolation of monoclonal naive CD4+ T cells from mice carrying a reactive TCR specific for HBV surface protein (HBsAg) in the context of I-Ad, mice were euthanized to allow organ collection (spleens). A single cell suspension was obtained via gently smashing the lymphoid organs with a 5-ml syringe plunger on 70mm cell strainer in 20 ml FACS buffer. The obtained cell suspension was pelleted at 400g for 5 min 4°C, resuspended with 5 ml ACK buffer for 5 min to eliminate red blood cells, washed once with FACS buffer, and finally re-suspended in complete medium. CD4+ T cells were isolated by negative selection using magnetic microbeads from the CD4+ T Cell Isolation Kit. Briefly, 100 pl of biotin-antibody cocktail were added to the cell suspension and incubated 5 min on ice. Two hundred ml of anti-biotin microbeads were added and incubated 10 min on ice. After washing the cells with MACS buffer at 400g for 5 min at 4°C, the pellet was resuspended in 3 mL of MACS buffer and added to the LS magnetic column attached to the QuadroMACS™ Separator. The CD4+ T cells were eluted from the LS column detached from the magnet with MACS buffer.

For in vivo CD4+ T cells transfer and mice immunization, isolated naive CD4+ T cells were re-suspended at a concentration of 200,000 CD4+ Tg T cells in 100 pl saline buffer and injected i.v. into HuFcgR recipient mice. 24 hrs post-T cells transfer, recipient mice were immunized i.p. with ICs mAb-HBsAg (HBC34-v40 Fc variants 5 pg, complexed in the culture plate for 1 hour at 37°C with 5000 IU HBsAg), saline buffer as negative control, or immunization with HBsi26-i38 peptide (HBsl26-138 peptide + adjuvant AS03) was used as a positive control.

3 days post-ICs immunization, mice were euthanized to allow organ collection (spleens). A single cell suspension was obtained via gently smashing the lymphoid organs with a 5 -ml syringe plunger on 70mm cell strainer in 20 ml FACS buffer. The obtained cell suspension was pelleted at 400g for 5 min 4°C, resuspended with 5 ml ACK buffer for 5 min to eliminate red blood cells, washed once with FACS buffer, and finally re-suspended in complete medium. Isolated cells were stained to assess the CD4⁺ T cells proliferation and activation. Briefly, cells were stained with Zombie Aqua for 30 min at RT to determine cell viability. After a washing step with FACS buffer and a spin at 400g for 5 min at 4°C, cells were stained with a surface markers mix containing antibodies recognizing CD4-APC/Cy7, CD44-PerCP, CD69-Pe-Dazzle 594, CD62L-BV605, CD45.1-BV785, CD45.2-AF700 and incubated for 20 min on ice. After a washing step with FACS buffer and a spin at 400g for 5 min at 4°C, cells were re-suspended in FIX buffer (BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit) and incubated for 20 min on ice. After a washing step with Permeabilization buffer (BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit) and a spin at 400g for 5 min at 4°C, cells were re-suspended in intracellular mix containing antibodies recognizing IFNg-BV421, IL-2-PE, TNFa-APC and incubated for 20 min on ice. After a washing step with Permeabilization buffer and a spin at 400g for 5 min at 4°C, cells were re-suspended in FACS buffer and acquired using the FACS Symphony Al (BD). Results

The results showed that while the expansion of HBs-specific Tg CD4+ T cells is not increased upon stimulation with ICs with Fc variants, the immunization with ICs of HBsAg and HBC34-v40-GARPYL or GAYL increase the activation of HBs-specific Tg CD4+ T cells compared to ICs containing HBC34-v40 (WT) or GRLR (see Fig. 37).

Listing of Embodiments

The Embodiments provided below are intended to examples of embodiments of the present disclosure. The embodiments are not intended to limit the disclosure in any way.

Embodiment 1. An antibody comprising:

(i) a heavy chain variable region (VH) that comprises a CDRH1 amino acid sequence according to SEQ ID NO.: 34, a CDRH2 amino acid sequence according to SEQ ID NO.:35 or SEQ ID NO.:36, and a CDRH3 amino acid sequence according to SEQ ID NO.:37;

(ii) a light chain variable region (VL) that comprises a CDRL1 amino acid sequence according to SEQ ID NOs.:40, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:43, 42, and 45, and a CDRL3 amino acid sequence according to SEQ ID NO.:46; and

(iii) a variant of an IgG Fc polypeptide or a fragment thereof comprising:

(a) an alanine (A) at EU position 236 and a leucine (L) at EU position 300;

(b) an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295;

(c) an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300; or

(d) an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377; wherein CDRs are defined according to the CCG numbering system; and wherein, optionally, the VL comprises a R60N substitution mutation or a R60K substitution mutation relative to SEQ ID NO.:47 and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:47, and further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ ID NO.:47, and still further optionally wherein the antibody is afucosylated, and wherein the antibody is capable of binding to the antigenic loop region of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof and/or hepatitis D virus (HDV).

Embodiment 2. The antibody of embodiment 1, wherein the variant IgG Fc polypeptide or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 61, 63, 66, or 67.

Embodiment 3. The antibody of embodiment 1 or 2, wherein:

(i) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences are according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 43, and 46, respectively;

(ii) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences according to SEQ ID NOs.:34, 35, and 37, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.: 40, 42, and 46, respectively, and the VL comprises a R60N substitution mutation relative to SEQ ID NO.:47;

(iii) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 42, and 46, respectively, and the VL comprises a R60K substitution mutation relative to SEQ ID NO.:47; or (iv) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences are according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 45, and 46, respectively.

Embodiment 4. The antibody of any one any one of embodiments 1-3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs. :

(i) 34, 35, 37, 40, 43, and 46, respectively;

(ii) 34, 35, 37, 40, 42, and 46, respectively;

(iii) 34, 35, 37, 40, 45, and 46, respectively; or

(iv) 34, 35, 37, 40, 42, and 46, respectively.

Embodiment 5. The antibody of any one of embodiments 1-4, wherein:

(i) the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or

(ii) the VL comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52.

Embodiment 6. The antibody of any one of embodiments 1-5, wherein:

(i) the VH comprises, consists essentially of, or consists of an amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or

(ii) the VL comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52.

Embodiment 7. The antibody of any one of embodiments 1-6, wherein the VH and the VL comprise, consists essentially of, or consist of amino acid sequences having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any non-integer value therebetween) identity to the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 49, respectively; (ii) 38 and 50, respectively; (iii) 38 and 51, respectively; or (iv) 38 and 52, respectively.

Embodiment 8. The antibody of any one of embodiments 1-7, wherein the VH and the VL comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOs.:

(i) 38 and 49, respectively;

(ii) 38 and 50, respectively;

(iii) 38 and 51, respectively; or

(iv) 38 and 52, respectively.

Embodiment 9. The antibody of any one of embodiments 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO. : 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300.

Embodiment 10. The antibody of embodiment 9, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:66.

Embodiment 11. The antibody of any one of embodiments 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO. : 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295.

Embodiment 12. The antibody of embodiment 11, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:61.

Embodiment 13. The antibody of any one of embodiments 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO. : 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300.

Embodiment 14. The antibody of embodiment 13, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:67.

Embodiment 15. The antibody of any one of embodiments 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377.

Embodiment 16. The antibody of embodiment 15, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:63.

Embodiment 17. The antibody of any one of embodiments 1-16, wherein the VL comprises, consists essentially of, or consists of an amino acid sequence having at least amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52. Embodiment 18. An antibody, comprising:

(i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38;

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:49; and

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non- integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 61.

Embodiment 19. An antibody, comprising:

(i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38;

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 50; and

Embodiment 20. An antibody, comprising: (i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38;

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and

Embodiment 21. An antibody, comprising:

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 52; and

Embodiment 22. An antibody, comprising:

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 49; and

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non- integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 63.

Embodiment 23. An antibody, comprising:

Embodiment 24. An antibody, comprising:

(i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any noninteger value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 63.

Embodiment 25. An antibody, comprising:

Embodiment 26. An antibody, comprising:

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 49; and

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any noninteger value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 66.

Embodiment 27. An antibody, comprising:

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non- integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 66.

Embodiment 28. An antibody, comprising:

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and (iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any noninteger value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 66.

Embodiment 29. An antibody, comprising:

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth SEQ ID NO.: 52; and

Embodiment 30. An antibody, comprising:

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any noninteger value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 67.

Embodiment 31. An antibody, comprising:

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non- integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 67.

Embodiment 32. An antibody, comprising:

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:51; and

Embodiment 33. An antibody, comprising:

(ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 52; and

Embodiment 34. An antibody comprising:

(ii) a light chain variable region (VL) that comprises a CDRL1 amino acid sequence according to SEQ ID NOs.:40, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:43, 42, and 45, and a CDRL3 amino acid sequence according to SEQ ID NO.:46; wherein CDRs are defined according to the CCG numbering system; and

(iii) a variant of an IgG Fc polypeptide or a fragment thereof comprising an alanine (A) at EU position 236; wherein, optionally, the VL comprises a R60N substitution mutation or a R60K substitution mutation relative to SEQ ID NO.:47 and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:47, and further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ ID NO.:47, and still further optionally wherein the antibody is afucosylated, wherein the antibody is afucosylated, and wherein the antibody is capable of binding to the antigenic loop region of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof and/or hepatitis D virus (HDV).

Embodiment 35. The antibody of anyone of embodiments 1-34, wherein the variant IgG Fc polypeptide has increased binding to a human FcyRIIa and/or has decreased binding to a human FcyRIIb, as compared to the binding of a reference polypeptide to the human FcyRIIa or the human FcyRIIb, respectively, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.

Embodiment 36. The antibody of embodiment 35, wherein the increased binding to a human FcyRIIa comprises more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, or at least 10-fold greater binding to the human FcyRIIa as compared to the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.

Embodiment 37. The antibody of embodiment 35 or embodiment 36, wherein the human FcyRIIa comprises Hl 31 and, optionally, the increased binding to the human FcyRIIa Hl 31 comprises at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa Hl 31 as compared to the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa H131.

Embodiment 38. The antibody of any one of embodiments 35-37, wherein the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa R131 comprises more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least

10-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa R131.

Embodiment 39. The antibody of any one of embodiments 35-38, wherein the decreased binding to a human FcyRIIb comprises less than 0.9-fold, less than 0.8- fold, less than 0.7-fold, less than 0.6-fold, or between 0.5-fold and 0.9-fold, of the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIb.

Embodiment 40. The antibody of any one of embodiments 1-39, wherein

(1) a ratio of (i) the binding of the variant IgG Fc polypeptide to a human FcyRIIa to (ii) the binding of the variant IgG Fc polypeptide, respectively, to a human FcyRIIb is greater than

(2) a ratio of (iii) the binding of a reference polypeptide to the human FcyRIIa to (iv) the binding of the reference polypeptide to the human FcyRIIb, wherein the reference polypeptide comprises a wild-type human IgG Fc polypeptide or a fragment thereof, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.

Embodiment 41. The antibody of embodiment 40, wherein the human

FcyRIIa comprises H131.

Embodiment 42. The antibody of embodiment 40 or 41, wherein the human

FcyRIIa comprises R131.

Embodiment 43. The antibody of any one of embodiments 40-42, wherein the ratio in (1) is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, or at least 14-fold greater than the ratio in (2). Embodiment 44. The antibody of any one of embodiments 1-43, wherein the variant IgG Fc polypeptide is derived from or comprises an IgGl isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype.

Embodiment 45. The antibody of any one of embodiments 1-44, wherein the variant IgG Fc polypeptide is derived from or comprises a human Fc or a fragment thereof, or from a human antibody heavy chain or a fragment thereof.

Embodiment 46. The antibody of any one of embodiments 1-45, wherein the variant IgG Fc polypeptide is derived or comprises from a human IgGl isotype, a human IgG2 isotype, a human IgG3 isotype, or a human IgG3 isotype.

Embodiment 47. The antibody of any one of embodiments 1-46, wherein the variant IgG Fc polypeptide is derived from or comprises a human IgGl isotype, optionally comprising a Glm3 allotype, a Glml7 allotype, a Glm3,l allotype, or a Glml7,l allotype.

Embodiment 48. The antibody of embodiment 47, wherein the variant IgG Fc polypeptide an comprises an IgGl isotype comprising (i) R214, E356, and M358; or (ii) K214, D356, and L358.

Embodiment 49. The antibody of any one of embodiments 1-48, wherein the antibody is capable of any one or more of the following:

(i) increasing specific lysis (e.g. via ADCC) by natural killer cells and/or PBMCs (e.g. expressing Fl 58/VI 58 or VI 58/VI 58 FcyRIIIA) against antigenexpressing target cells, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state (e.g. the antibody comprising a human IgGl Fc comprising the mutations G236A, A330L, and I332E);

(ii) increasing ADCP by monocytes (e.g. CD 14+ monocytes, optionally expressing Fl 58/VI 58 FcyRIIA and R131/H131 FcyRIIA or F158/F158 FcyRIIA and R131/H131 FcyRIIA) against antigen-expressing target cells, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state;

(iii) increasing the percentage of CD83+ cells (e.g. moDCs) and/or increasing expression of CD83 by moDCs in a sample when provided in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided in combination with the antigen;

(iv) increasing production of one or more cytokine (optionally selected from the group consisting of IL-ip, IFN-y, IL-6, and TNF-a) by moDCs in a sample when provided in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided in combination with the antigen; and

(v) increasing the ability of moDCs to stimulate antigen-specific CD4+ T cells when provided to the moDCs in combination with the antigen, as compared to the antibody comprising a reference Fc polypeptide not comprising the mutation(s) and/or fucosylation state, when provided to the moDCs in combination with the antigen, wherein, optionally, (1) the moDCs and the CD4+ T cells are from the same (optionally antigen-vaccinated) subject and/or (2) stimulation of antigen-specific CD4+ T cells is determined by an increase in CD25 expression and/or an increase in proliferation (e.g. as determined by a reduction in CFSE staining over time) and/or an increase in expression of CD69 and/or an increase in expression of NF AT and/or an increase in expression of CD44, by the antigen-specific CD4+ T cells.

Embodiment 50. The antibody of any one of embodiments 1-49, wherein the variant IgG Fc polypeptide further comprises one or more modification that enhances binding to a human FcRn as compared to (1) a reference antibody comprises a wild-type human IgGl Fc polypeptide and/or to (2) the antibody of any one of embodiments 1-49 without the one or more modification.

Embodiment 51. The antibody of embodiment 50, wherein the one or more modification that enhances binding by the human FcRn comprises the amino acid substitutions:

(i) M428L/N434S;

(ii) M428L/N434A;

(iii) T250Q/M428L; (iv) P257I/Q311I;

(v) P257EN434H;

(vi) D376V/N434H;

(vii) T307A/E380A/N434A;

(viii) M252Y/S254T/T256E; or

(ix) any combination of (i)-(viii).

Embodiment 52. The antibody of embodiment 50 or 51, wherein the one or more modification that enhances binding by the human FcRn comprises the amino acid substitutions M428L/N434S.

Embodiment 53. The antibody of embodiment 50 or 51, wherein the one or more modification that enhances binding by the human FcRn comprises the amino acid substitutions M428L/N434A.

Embodiment 54. The antibody of any one of embodiments 1-53, wherein the variant IgG Fc polypeptide does not comprise any additional mutations as compared to a reference wild-type IgG Fc.

Embodiment 55. The antibody of any one of embodiments 1-54, comprising a light chain constant region (CL) that comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.: 100.

Embodiment 56. The antibody of any one of embodiments 1-55, wherein, in a sample comprising a plurality of the antibody, less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of the plurality is comprised in a dimer when the sample has been incubated for about 120 to about 168 hours at about 40°C, wherein, optionally, the presence of dimer is determined by absolute size-exclusion chromatography.

Embodiment 57. The antibody of any one of embodiments 1-56, wherein incubation of a plurality of the antibody results in reduced formation of a dimer as compared to incubation of a plurality of a reference antibody, wherein the reference antibody comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47, and wherein, optionally, the presence of antibody dimer is determined by absolute size-exclusion chromatography.

Embodiment 58. The antibody of any one of embodiments 1-57, wherein the antibody comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody.

Embodiment 59. The antibody of any one of embodiments 1-58, wherein the antibody is a multi-specific antibody.

Embodiment 60. The antibody of any one of embodiments 1-59, wherein the antibody is a bispecific antibody.

Embodiment 61. The antibody of any one of embodiments 1-60, wherein the antibody or the antigen-binding fragment is capable of binding an HBsAg of a genotype selected from the HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.

Embodiment 62. The antibody of any one of embodiments 1-61, wherein the antibody or antigen-binding fragment is capable of reducing a serum concentration of HBV DNA in a mammal having an HBV infection.

Embodiment 63. The antibody of any one of embodiments 1-62, wherein the antibody or antigen-binding fragment is capable of reducing a serum concentration of HBsAg in a mammal having an HBV infection.

Embodiment 64. The antibody of any one of embodiments 1-63, wherein the antibody or antigen-binding fragment is capable of reducing a serum concentration of HBeAg in a mammal having an HBV infection.

Embodiment 65. The antibody of any one of embodiments 1-64, wherein the antibody or antigen binding fragment is capable of reducing a serum concentration of HBcrAg in a mammal having an HBV infection.

Embodiment 66. The antibody of any one of embodiments 1-65, which is capable of neutralizing infection by a hepatitis D virus (HDV).

Embodiment 67. A polynucleotide comprising a nucleotide sequence that encodes the antibody of any one of embodiments 1-66. Embodiment 68. The polynucleotide of embodiment 67, wherein the nucleotide sequence that encodes the antibody is codon optimized for expression in a host cell.

Embodiment 69. The polynucleotide of embodiment 68, comprising a nucleotide sequence having at least 50% identity to the nucleotide sequence according to any one of SEQ ID Nos.: 106, 107, 108, and 109.

Embodiment 70. The polynucleotide of any one of embodiments 67-69, comprising (i) the polynucleotide sequence set forth in SEQ ID NO.: 102 or SEQ ID NO. : 103, and (ii) the polynucleotide sequence set forth in any one or more of SEQ ID NOs.: 106, 107, 108, and 109.

Embodiment 71. A vector comprising the polynucleotide of any one of embodiments 67-70.

Embodiment 72. The vector of embodiment 71, wherein the vector comprises a lentiviral vector or a retroviral vector.

Embodiment 73. A host cell comprising the polynucleotide of any one of embodiments 67-70 and/or the vector of embodiment 71 or 72.

Embodiment 74. A pharmaceutical composition comprising:

(i) the antibody of any one of embodiments 1-66;

(ii) the polynucleotide according to any one of embodiments 67-70;

(iii) the vector according to embodiment 71 or 72;

(iv) the host cell of embodiment 73; or

(v) any combination of (i)-(iv), and a pharmaceutically acceptable excipient, diluent or carrier.

Embodiment 75. A kit comprising:

(a) a component selected from:

(i) the antibody or antigen-binding fragment of any one of embodiments 1- 66;

(ii) the polynucleotide according to any one of embodiments 67-70;

(iii) the vector according to embodiment 71 or 72; (iv) the host cell of embodiment 73;

(v) the pharmaceutical composition of embodiment 74; or

(vi) any combination of (i)-(vi); and

(b) (1) instructions for using the component to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection and/or (2) a means for administering the component to the subject, such as a syringe.

Embodiment 76. The composition of embodiment 74 or the kit of embodiment 75, further comprising:

(i) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof;

(ii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha;

(iii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-Ll 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 (i)-(iv).

Embodiment 77. The composition or kit of embodiment 76, wherein the polymerase inhibitor comprises lamivudine.

Embodiment 78. A method of producing the antibody of any one of embodiments 1-66, comprising culturing the host cell of embodiment 73 under conditions and for a time sufficient to produce the antibody or antigen-binding fragment.

Embodiment 79. Use of: (i) the antibody or antigen-binding fragment of any one of embodiments 1-66; (ii) the polynucleotide of any one of embodiments 67- 70; (iii) the vector of embodiment 71 or 72; (iv) the host cell of embodiment 73; and/or (v) the pharmaceutical composition of embodiment 74, in the manufacture of a medicament to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection in a subject.

Embodiment 80. A method of treating, preventing, and/or attenuating a hepatitis B and/or hepatitis D infection in a subject, comprising administering to the subject an effective amount of: (i) the antibody or antigen-binding fragment of any one of embodiments 1-66; (ii) the polynucleotide of any one of embodiments 67-70; (iii) the vector of embodiment 71 or 72; (iv) the host cell of embodiment 73; and/or (v) the pharmaceutical composition of embodiment 74.

Embodiment 81. The method of embodiment 80, further comprising administering to the subject one or more of: (vi) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (vii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (viii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-Ll antibody or antigen binding fragment thereof, and/or an anti-CTLA4 antibody or antigen binding fragment thereof; (ix) an agonist of a stimulatory immune checkpoint molecule; or (x) any combination of (vi)-(ix).

Embodiment 82. The method of embodiment 80 or 81, wherein the hepatitis B infection is a chronic hepatitis B infection.

Embodiment 83. The method of any one of embodiments 80-82, wherein the subject has received a liver transplant.

Embodiment 84. The method of any one of embodiments 80-83, wherein the subject is non-immunized against hepatitis B.

Embodiment 85. The method of any one of embodiments 80-84, wherein the subject is a newborn.

Embodiment 86. The method of any one of embodiments 80-85, wherein the subject is undergoing or has undergone hemodialysis.

Embodiment 87. The method of any one of embodiments 80-86, wherein the method comprises administering to the subject a single dose of a pharmaceutical composition comprising the antibody or antigen-binding fragment. Embodiment 88. The method of embodiment 87, wherein the single dose of the pharmaceutical composition comprises the antibody in a range from 2 to 18 mg/kg (subject body weight).

Embodiment 89. The method of embodiment 87 or 88, wherein the single dose of the pharmaceutical composition comprises up to 6 mg, up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 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, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 3000 mg, or in a range from 5 mg to 3000 mg, or in a range from 10 mg to 3000 mg, or in a range from 25 mg to 3000 mg, or in a range from 30 mg to 3000 mg, or in a range from 50 mg to 3000 mg, or in a range from 60 mg to 3000 mg, or in a range from 75 mg to 3000 mg, or in a range from 90 mg to 3000 mg, or in a range from 100 mg to 3000 mg, or in a range from 150 mg to 3000 mg, or in a range from 200 mg to 3000 mg, or in a range from 300 mg to 3000 mg, or in a range from 500 mg to 3000 mg, or in a range from 750 mg to 3000 mg, or in a range from 900 mg to 3000 mg, or in a range from 1500 mg to 3000 mg, or in a range from 2000 mg to 3000 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 900 mg, or in a range from 5 mg to 900 mg, or in a range from 10 mg to 900 mg, or in a range from 25 mg to 900 mg, or in a range from 30 mg to 900 mg, or in a range from 50 mg to 900 mg, or in a range from 60 mg to 900 mg, or in a range from 75 mg to 900 mg, or in a range from 90 mg to 900 mg, or in a range from 100 mg to 900 mg, or in a range from 150 mg to 900 mg, or in a range from 200 mg to 900 mg, or in a range from 300 mg to 900 mg, or in a range from 500 mg to 900 mg, or in a range from 750 mg to 900 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 500 mg, or in a range from 5 mg to 500 mg, or in a range from 10 mg to 500 mg, or in a range from 25 mg to 500 mg, or in a range from 30 mg to 500 mg, or in a range from 50 mg to 500 mg, or in a range from 60 mg to 500 mg, or in a range from 75 mg to 500 mg, or in a range from 90 mg to 500 mg, or in a range from 100 mg to 500 mg, or in a range from 150 mg to 500 mg, or in a range from 200 mg to 500 mg, or in a range from 300 mg to 500 mg, or in a range from 400 mg to 500 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 300 mg, or in a range from 5 mg to 300 mg, or in a range from 10 mg to 300 mg, or in a range from 25 mg to 300 mg, or in a range from 30 mg to 300 mg, or in a range from 50 mg to 300 mg, or in a range from 60 mg to 300 mg, or in a range from 75 mg to 300 mg, or in a range from 90 mg to 300 mg, or in a range from 100 mg to 300 mg, or in a range from 150 mg to 300 mg, or in a range from 200 mg to 300 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 200 mg, or in a range from 5 mg to 200 mg, or in a range from 10 mg to 200 mg, or in a range from 25 mg to 200 mg, or in a range from 30 mg to 200 mg, or in a range from 50 mg to 200 mg, or in a range from 60 mg to 200 mg, or in a range from 75 mg to 200 mg, or in a range from 90 mg to 200 mg, or in a range from 100 mg to 200 mg, or in a range from 150 mg to 200 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 100 mg, or in a range from 5 mg to 100 mg, or in a range from 10 mg to 100 mg, or in a range from 25 mg to 100 mg, or in a range from 30 mg to 100 mg, or in a range from 50 mg to 100 mg, or in a range from 60 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 90 mg to 100 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 25 mg, or in a range from 5 mg to 25 mg, or in a range from 10 mg to 25 mg, or in a range from 15 mg to 25 mg, or in a range from 20 mg to 25 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 50 mg, or in a range from 1 mg to 25 mg, or in a range from 5 mg to 50 mg, or in a range from 5 mg to 25 mg, or in a range from 10 to 50 mg, or in a range from 10 to 25 mg, or in a range from 1 to 15 mg, or in a range from 5 mg to 15 mg, or in a range from 10 mg to 15 mg, or wherein the single dose of the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,

270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350,

355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,

440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520,

525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605,

610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690,

695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775,

780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860,

865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945,

950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg, or more, of the antibody, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 mg, less than 25 mg, or less than 10 mg, but is more than 1 mg, more than 2 mg, more than 3 mg, more than 4 mg, or more than 5 mg.

Embodiment 90. The method of any one of embodiments 87-89, wherein the single dose of the pharmaceutical composition comprises the antibody at a concentration in a range from 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 91. The method of any one of embodiments 87-90, wherein the single dose of the pharmaceutical composition comprises about 75 mg of the antibody. Embodiment 92. The method of any one of embodiments 87-90, wherein the single dose of the pharmaceutical composition comprises about 90 mg of the antibody.

Embodiment 93. The method of any one of embodiments 87-90, wherein the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody.

Embodiment 94. The method of any one of embodiments 87-90, wherein the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody.

Embodiment 95. The method of any one of embodiments 87-90, wherein the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody.

Embodiment 96. The method of any one of embodiments 87-90, wherein the single dose of the pharmaceutical composition comprises about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg of the antibody.

Embodiment 97. The method of any one of embodiments 87-90, wherein the method comprises administering the single dose by subcutaneous injection, optionally wherein the single dose comprises or consists of 6 mg of the antibody or 18 mg of the antibody.

Embodiment 98. The method of any one of embodiments 87-97, wherein the method comprises administering the single dose by intravenous injection.

Embodiment 99. The method of any one of embodiments 87-98, wherein the pharmaceutical composition further comprises water, optionally USP water.

Embodiment 100. The method of any one of embodiments 87-99, wherein the pharmaceutical composition further comprises histidine, optionally at a concentration in a range from 10 mM to 40 mM, such as 20 mM, in the pharmaceutical composition.

Embodiment 101. The method of any one of embodiments 87-100, 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 102. The method of any one of embodiments 87-101, wherein the pharmaceutical composition further comprises a surfactant or a triblock copolymer, optionally a polysorbate or poloxamer-188, preferably polysorbate 80 (PS80), wherein, optionally, the polysorbate or poloxamer-188 is present in a range from 0.01% to 0.05% (w/v), preferably 0.02% (w/v).

Embodiment 103. The method of any one of embodiments 87-102, wherein the pharmaceutical composition has a pH in a range from 5.8 to 6.2, in a range from 5.9 to 6.1, or of 5.8, of 5.9, of 6.0, of 6.1, or of 6.2.

Embodiment 104. The method of embodiment 103, wherein the pharmaceutical composition comprises:

(i) the antibody at 150 mg/mL;

(ii) USP water;

(iii) 20 mM histidine;

(iv) 7% sucrose; and

(v) 0.02% PS80, wherein the pharmaceutical composition comprises a pH of 6.

Embodiment 105. The method of any one of embodiments 87-104, wherein the subject is an adult.

Embodiment 106. The method of embodiment 105, wherein the subject is in a range from 18 years of age to 65 years of age.

Embodiment 107. The method of any one of embodiments 87-106, wherein the subject weighs from 40 kg to 125 kg and/or the subject has a body mass index (BMI) from 18 to 35 kg/m².

Embodiment 108. The method of any one of embodiments 87-107, wherein the subject has a chronic HBV infection; e.g., defined by positive serum HBsAg, HBV DNA, and/or HBeAg on 2 occasions, wherein the 2 occasions are at least 6 months apart.

Embodiment 109. The method of any one of embodiments 87-108, wherein the subject does not have cirrhosis. Embodiment 110. The method of embodiment 109, wherein absence of cirrhosis is determined by:

Fibroscan evaluation (e.g., within 6 months prior to administering the single dose of the pharmaceutical composition); or liver biopsy (e.g., within 12 months prior to administering the single dose of the pharmaceutical composition), wherein, preferably the absence of cirrhosis is determined by the absence of Metavir F3 fibrosis or the absence of F4 cirrhosis.

Embodiment 111. The method of any one of embodiments 87-110, wherein the subject has received a nucleos(t)ide reverse transcriptase inhibitor (NRTI), optionally within 120 days, further optionally within 60 days, prior to the single dose being administered.

Embodiment 112. The method of embodiment 111, wherein the NRTI comprises one or more of: tenofovir; tenofovir disoproxil (e.g., tenofovir disproxil fumarate); tenofovir alafenamide; Entecavir; Lamivudine; Adefovir; and adefovir dipivoxil.

Embodiment 113. The method of any one of embodiments 87-112, wherein the subject has a serum HBV DNA concentration of less than 100 lU/mL no more than 28 days prior to the single dose being administered.

Embodiment 114. The method of any one of embodiments 87-113, wherein the subject has a serum HBsAg concentration of less than 3,000 lU/mL prior to the single dose being administered, and optionally less than 1,000 lU/mL prior to the single dose being administered.

Embodiment 115. The method of any one of embodiments 87-114, wherein the subject has a serum HBsAg concentration of greater than or equal to 3,000 lU/mL no more than 28 days prior to the single dose being administered, and optionally greater than or equal to 1,000 lU/mL no more than 28 days prior to the single dose being administered. Embodiment 116. The method of any one of embodiments 87-115, wherein the subject was HB e-antigen (HBeAg)-negative no more than 28 days prior to the single dose being administered.

Embodiment 117. The method of any one of embodiments 87-116, wherein the subject was negative for anti-HB antibodies no more than 28 days prior to the single dose being administered.

Embodiment 118. The method of any one of embodiments 87-117, wherein the subject, prior to administration of the single dose:

(i) does not have fibrosis and/or does not have cirrhosis; and/or

(ii) has alanine aminotransferase (ALT) < 2 x Upper Limit of Normal (ULN).

Embodiment 119. The method of any one of embodiments 87-118, wherein at 56 days following administration of the single dose, the subject has a > 2-fold reduction in serum HBsAg (e.g., concentration of HBsAg in serum, e.g., as determined using an Abbott ARCHITECT assay) as compared to the subject’s serum HBsAg at from 0 days to 28 days prior to administration of the single dose.

Embodiment 120. The method of any one of embodiments 87-119, wherein following administration of the single dose (e.g., at 56 days following administration of the single dose), the subject has:

(i) has reduced or less severe intrahepatic spread of HBV as compared to a reference subject; and/or

(ii) comprises an adaptive immune response against HBV.

Embodiment 121. The method of any one of embodiments 87-120, wherein the subject is male.

Embodiment 122. The method of any one of embodiments 87-121, wherein the subject is female.

Embodiment 123. A pharmaceutical composition comprising the antibody of any one of embodiments 1-66 at a concentration ranging from 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, and a pharmaceutically acceptable carrier, excipient, or diluent.

Embodiment 124. The pharmaceutical composition of embodiment 123, wherein 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 125. The pharmaceutical composition of embodiment 123 or 124, wherein the pharmaceutical composition comprises about 75 mg of the antibody.

Embodiment 126. The pharmaceutical composition of embodiment 123 or 124, wherein the pharmaceutical composition comprises about 90 mg of the antibody.

Embodiment 127. The pharmaceutical composition of embodiment 123 or 124, wherein the pharmaceutical composition comprises about 300 mg of the antibody.

Embodiment 128. The pharmaceutical composition of embodiment 123 or 124, wherein the pharmaceutical composition comprises about 900 mg of the antibody.

Embodiment 129. The pharmaceutical composition of embodiment 123 or 124, wherein the pharmaceutical composition comprises about 3,000 mg of the antibody.

Embodiment 130. The pharmaceutical composition of embodiment 123 or 124, wherein the pharmaceutical composition comprises about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg of the antibody.

Embodiment 131. The pharmaceutical composition of any one of embodiments 123-130, wherein the pharmaceutical composition comprises water, optionally USP water.

Embodiment 132. The pharmaceutical composition of any one of embodiments 123-131, wherein the pharmaceutical composition comprises histidine, optionally at a concentration from 10 mM to 40 mM, such as 20 mM, in the pharmaceutical composition.

Embodiment 133. The pharmaceutical composition of any one of embodiments 123-132, wherein the pharmaceutical composition comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8%, or 9%, preferably about 7% (w/v). Embodiment 134. The pharmaceutical composition of any one of embodiments 123-133, wherein the pharmaceutical composition comprises a surfactant, optionally a polysorbate, preferably polysorbate 80 (PS80), wherein, optionally, the polysorbate is present in a range from 0.01% to 0.05% (w/v), preferably 0.02% (w/v). Embodiment 135. The pharmaceutical composition of any one of embodiments 123-134, wherein the pharmaceutical composition has a pH ranging from 5.8 to 6.2, ranging from 5.9 to 6.1, or of 5.8, of 5.9, of 6.0, of 6.1, or of 6.2.

Embodiment 136. The pharmaceutical composition of any one of embodiments 123-135, wherein the pharmaceutical composition comprises: (i) the antibody at 150 mg/mL;

(ii) USP water;

(iii) 20 mM histidine;

(iv) 7% sucrose; and

(v) 0.02% PS80, wherein the pharmaceutical composition comprises a pH of 6.

TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING):

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. Provisional Patent Application no. 63/344,909, filed on May 23, 2022, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above- detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. An antibody comprising:

(iii) a variant of an IgG Fc polypeptide or a fragment thereof comprising:

(a) an alanine (A) at EU position 236 and a leucine (L) at EU position 300;

2. The antibody of claim 1, wherein the variant IgG Fc polypeptide or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 61, 63, 66, or 67.

3. The antibody of claim 1 or 2, wherein:

(iii) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 42, and 46, respectively, and the VL comprises a R60K substitution mutation relative to SEQ ID NO.:47; or

(iv) in the VH, the CDRH1, CDRH2, CDRH3 amino acid sequences are according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.:40, 45, and 46, respectively.

4. The antibody of any one any one of claims 1-3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.: (i) 34, 35, 37, 40, 43, and 46, respectively;

(ii) 34, 35, 37, 40, 42, and 46, respectively;

(iii) 34, 35, 37, 40, 45, and 46, respectively; or

(iv) 34, 35, 37, 40, 42, and 46, respectively.

5. The antibody of any one of claims 1-4, wherein:

6. The antibody of any one of claims 1-5, wherein:

7. The antibody of any one of claims 1-6, wherein the VH and the VL comprise, consists essentially of, or consist of amino acid sequences having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any non-integer value therebetween) identity to the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 49, respectively; (ii) 38 and 50, respectively; (iii) 38 and 51, respectively; or (iv) 38 and 52, respectively.

8. The antibody of any one of claims 1-7, wherein the VH and the VL comprise, consist essentially of, or consist of the amino acid sequences set forth in SEQ ID NOs.:

(i) 38 and 49, respectively;

(ii) 38 and 50, respectively;

(iii) 38 and 51, respectively; or

(iv) 38 and 52, respectively.

9. The antibody of any one of claims 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300.

10. The antibody of claim 9, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:66.

11. The antibody of any one of claims 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295.

12. The antibody of claim 11, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:61.

13. The antibody of any one of claims 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300.

14. The antibody of claim 13, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:67.

15. The antibody of any one of claims 1-8, wherein the VH comprises, consists essentially of, or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and the variant IgG Fc polypeptide or a fragment thereof comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377.

16. The antibody of claim 15, wherein the variant IgG Fc polypeptide or a fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.:63.

17. The antibody of any one of claims 1-16, wherein the VL comprises, consists essentially of, or consists of an amino acid sequence having at least amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 49, 50, 51, and 52.

18. An antibody, comprising:

19. An antibody, comprising:

(i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 50; and

(iii) a variant IgG Fc polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any noninteger value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 61.

20. An antibody, comprising:

21. An antibody, comprising:

(i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 52; and

22. An antibody, comprising:

23. An antibody, comprising:

24. An antibody, comprising:

25. An antibody, comprising:

26. An antibody, comprising:

27. An antibody, comprising:

28. An antibody, comprising:

29. An antibody, comprising:

(i) a VH comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38; (ii) a VL comprising, consisting essentially of, or consisting of an amino acid sequence having at least 90% (z.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth SEQ ID NO.: 52; and

30. An antibody, comprising:

31. An antibody, comprising:

32. An antibody, comprising:

33. An antibody, comprising:

34. An antibody comprising:

35. The antibody of anyone of claims 1-34, wherein the variant IgG Fc polypeptide has increased binding to a human FcyRIIa and/or has decreased binding to a human FcyRIIb, as compared to the binding of a reference polypeptide to the human FcyRIIa or the human FcyRIIb, respectively, wherein, optionally, binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery.

36. The antibody of claim 35, wherein the increased binding to a human FcyRIIa comprises more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold greater binding to the human FcyRIIa as compared to the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa.

37. The antibody of claim 35 or claim 36, wherein the human FcyRIIa comprises Hl 31 and, optionally, the increased binding to the human FcyRIIa Hl 31 comprises at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa H131 as compared to the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa H131.

38. The antibody of any one of claims 35-37, wherein the human FcyRIIa comprises R131 and, optionally, the increased binding to the human FcyRIIa R131 comprises more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater binding to the human FcyRIIa R131 as compared to the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIa R131.

39. The antibody of any one of claims 35-38, wherein the decreased binding to a human FcyRIIb comprises less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, or between 0.5-fold and 0.9-fold, of the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcyRIIb.

40. The antibody of any one of claims 1-39, wherein

41. The antibody of claim 40, wherein the human FcyRIIa comprises H131.

42. The antibody of claim 40 or 41, wherein the human FcyRIIa comprises R131.

43. The antibody of any one of claims 40-42, wherein the ratio in (1) is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13 -fold, or at least 14-fold greater than the ratio in (2).

44. The antibody of any one of claims 1-43, wherein the variant IgG Fc polypeptide is derived from or comprises an IgGl isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype.

45. The antibody of any one of claims 1-44, wherein the variant IgG Fc polypeptide is derived from or comprises a human Fc or a fragment thereof, or from a human antibody heavy chain or a fragment thereof.

46. The antibody of any one of claims 1-45, wherein the variant IgG Fc polypeptide is derived or comprises from a human IgGl isotype, a human IgG2 isotype, a human IgG3 isotype, or a human IgG3 isotype.

47. The antibody of any one of claims 1-46, wherein the variant IgG Fc polypeptide is derived from or comprises a human IgGl isotype, optionally comprising a Glm3 allotype, a Glml7 allotype, a Glm3,l allotype, or a G1 ml 7,1 allotype.

48. The antibody of claim 47, wherein the variant IgG Fc polypeptide an comprises an IgGl isotype comprising (i) R214, E356, and M358; or (ii) K214, D356, and L358.

49. The antibody of any one of claims 1-48, wherein the antibody is capable of any one or more of the following:

50. The antibody of any one of claims 1-49, wherein the variant IgG Fc polypeptide further comprises one or more modification that enhances binding to a human FcRn as compared to (1) a reference antibody comprises a wild-type human IgGl Fc polypeptide and/or to (2) the antibody of any one of claims 1-49 without the one or more modification.

51. The antibody of claim 50, wherein the one or more modification that enhances binding by the human FcRn comprises the amino acid substitutions:

(i) M428L/N434S;

(ii) M428L/N434A;

(iii) T250Q/M428L;

(iv) P257EQ311I;

(v) P257I/N434H;

(vi) D376V/N434H;

(vii) T307A/E380A/N434A;

(viii) M252Y/S254T/T256E; or

(ix) any combination of (i)-(viii).

52. The antibody of claim 50 or 51, wherein the one or more modification that enhances binding by the human FcRn comprises the amino acid substitutions M428L/N434S.

53. The antibody of claim 50 or 51, wherein the one or more modification that enhances binding by the human FcRn comprises the amino acid substitutions M428L/N434A.

54. The antibody of any one of claims 1-53, wherein the variant IgG Fc polypeptide does not comprise any additional mutations as compared to a reference wild-type IgG Fc.

55. The antibody of any one of claims 1-54, comprising a light chain constant region (CL) that comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.: 100.

56. The antibody of any one of claims 1-55, wherein, in a sample comprising a plurality of the antibody, less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of the plurality is comprised in a dimer when the sample has been incubated for about 120 to about 168 hours at about 40°C, wherein, optionally, the presence of dimer is determined by absolute size-exclusion chromatography.

57. The antibody of any one of claims 1-56, wherein incubation of a plurality of the antibody results in reduced formation of a dimer as compared to incubation of a plurality of a reference antibody, wherein the reference antibody comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:47, and wherein, optionally, the presence of antibody dimer is determined by absolute size-exclusion chromatography.

58. The antibody of any one of claims 1-57, wherein the antibody comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody.

59. The antibody of any one of claims 1-58, wherein the antibody is a multi-specific antibody.

60. The antibody of any one of claims 1-59, wherein the antibody is a bispecific antibody.

61. The antibody of any one of claims 1-60, wherein the antibody or the antigen-binding fragment is capable of binding an HBsAg of a genotype selected from the HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.

62. The antibody of any one of claims 1-61, wherein the antibody or antigenbinding fragment is capable of reducing a serum concentration of HBV DNA in a mammal having an HBV infection.

63. The antibody of any one of claims 1-62, wherein the antibody or antigenbinding fragment is capable of reducing a serum concentration of HBsAg in a mammal having an HBV infection.

64. The antibody of any one of claims 1-63, wherein the antibody or antigenbinding fragment is capable of reducing a serum concentration of HBeAg in a mammal having an HBV infection.

65. The antibody of any one of claims 1-64, wherein the antibody or antigen binding fragment is capable of reducing a serum concentration of HBcrAg in a mammal having an HBV infection.

66. The antibody of any one of claims 1-65, which is capable of neutralizing infection by a hepatitis D virus (HDV).

67. A polynucleotide comprising a nucleotide sequence that encodes the antibody of any one of claims 1-66.

68. The polynucleotide of claim 67, wherein the nucleotide sequence that encodes the antibody is codon optimized for expression in a host cell.

69. The polynucleotide of claim 68, comprising a nucleotide sequence having at least 50% identity to the nucleotide sequence according to any one of SEQ ID Nos.: 106, 107, 108, and 109.

70. The polynucleotide of any one of claims 67-69, comprising (i) the polynucleotide sequence set forth in SEQ ID NO.: 102 or SEQ ID NO.: 103, and (ii) the polynucleotide sequence set forth in any one or more of SEQ ID NOs.: 106, 107, 108, and 109.

71. A vector comprising the polynucleotide of any one of claims 67-70.

72. The vector of claim 71, wherein the vector comprises a lentiviral vector or a retroviral vector.

73. A host cell comprising the polynucleotide of any one of claims 67-70 and/or the vector of claim 71 or 72.

74. A pharmaceutical composition comprising:

(i) the antibody of any one of claims 1-66;

(ii) the polynucleotide according to any one of claims 67-70;

(iii) the vector according to claim 71 or 72;

(iv) the host cell of claim 73; or

75. A kit compri sing :

(a) a component selected from:

(i) the antibody or antigen-binding fragment of any one of claims 1-66;

(ii) the polynucleotide according to any one of claims 67-70;

(iii) the vector according to claim 71 or 72;

(iv) the host cell of claim 73;

(v) the pharmaceutical composition of claim 74; or

(vi) any combination of (i)-(vi); and (b) (1) instructions for using the component to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection and/or (2) a means for administering the component to the subject, such as a syringe.

76. The composition of claim 74 or the kit of claim 75, further comprising:

(iv) an agonist of a stimulatory immune checkpoint molecule; or

(v) any combination of (i)-(iv).

77. The composition or kit of claim 76, wherein the polymerase inhibitor comprises lamivudine.

78. A method of producing the antibody of any one of claims 1-66, comprising culturing the host cell of claim 73 under conditions and for a time sufficient to produce the antibody or antigen-binding fragment.

79. Use of: (i) the antibody or antigen-binding fragment of any one of claims 1-66; (ii) the polynucleotide of any one of claims 67-70; (iii) the vector of claim 71 or 72; (iv) the host cell of claim 73; and/or (v) the pharmaceutical composition of claim 74, in the manufacture of a medicament to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection in a subject.

80. A method of treating, preventing, and/or attenuating a hepatitis B and/or hepatitis D infection in a subject, comprising administering to the subject an effective amount of: (i) the antibody or antigen-binding fragment of any one of claims 1-66; (ii) the polynucleotide of any one of claims 67-70; (iii) the vector of claim 71 or 72; (iv) the host cell of claim 73; and/or (v) the pharmaceutical composition of claim 74.

81. The method of claim 80, further comprising administering to the subject one or more of: (vi) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (vii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (viii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-Ll antibody or antigen binding fragment thereof, and/or an anti-CTLA4 antibody or antigen binding fragment thereof; (ix) an agonist of a stimulatory immune checkpoint molecule; or (x) any combination of (vi)-(ix).

82. The method of claim 80 or 81, wherein the hepatitis B infection is a chronic hepatitis B infection.

83. The method of any one of claims 80-82, wherein the subject has received a liver transplant.

84. The method of any one of claims 80-83, wherein the subject is nonimmunized against hepatitis B.

85. The method of any one of claims 80-84, wherein the subject is a newborn.

86. The method of any one of claims 80-85, wherein the subject is undergoing or has undergone hemodialysis.

87. The method of any one of claims 80-86, wherein the method comprises administering to the subject a single dose of a pharmaceutical composition comprising the antibody or antigen-binding fragment.

88. The method of claim 87, wherein the single dose of the pharmaceutical composition comprises the antibody in a range from 2 to 18 mg/kg (subject body weight).

89. The method of claim 87 or 88, wherein the single dose of the pharmaceutical composition comprises up to 6 mg, up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 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, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 3000 mg, or in a range from 5 mg to 3000 mg, or in a range from 10 mg to 3000 mg, or in a range from 25 mg to 3000 mg, or in a range from 30 mg to 3000 mg, or in a range from 50 mg to 3000 mg, or in a range from 60 mg to 3000 mg, or in a range from 75 mg to 3000 mg, or in a range from 90 mg to 3000 mg, or in a range from 100 mg to 3000 mg, or in a range from 150 mg to 3000 mg, or in a range from 200 mg to 3000 mg, or in a range from 300 mg to 3000 mg, or in a range from 500 mg to 3000 mg, or in a range from 750 mg to 3000 mg, or in a range from 900 mg to 3000 mg, or in a range from 1500 mg to 3000 mg, or in a range from 2000 mg to 3000 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 900 mg, or in a range from 5 mg to 900 mg, or in a range from 10 mg to 900 mg, or in a range from 25 mg to 900 mg, or in a range from 30 mg to 900 mg, or in a range from 50 mg to 900 mg, or in a range from 60 mg to 900 mg, or in a range from 75 mg to 900 mg, or in a range from 90 mg to 900 mg, or in a range from 100 mg to 900 mg, or in a range from 150 mg to 900 mg, or in a range from 200 mg to 900 mg, or in a range from 300 mg to 900 mg, or in a range from 500 mg to 900 mg, or in a range from 750 mg to 900 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 500 mg, or in a range from 5 mg to 500 mg, or in a range from 10 mg to 500 mg, or in a range from 25 mg to 500 mg, or in a range from 30 mg to 500 mg, or in a range from 50 mg to 500 mg, or in a range from 60 mg to 500 mg, or in a range from 75 mg to 500 mg, or in a range from 90 mg to 500 mg, or in a range from 100 mg to 500 mg, or in a range from 150 mg to 500 mg, or in a range from 200 mg to 500 mg, or in a range from 300 mg to 500 mg, or in a range from 400 mg to 500 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 300 mg, or in a range from 5 mg to 300 mg, or in a range from 10 mg to 300 mg, or in a range from 25 mg to 300 mg, or in a range from 30 mg to 300 mg, or in a range from 50 mg to 300 mg, or in a range from 60 mg to 300 mg, or in a range from 75 mg to 300 mg, or in a range from 90 mg to 300 mg, or in a range from 100 mg to 300 mg, or in a range from 150 mg to 300 mg, or in a range from 200 mg to 300 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 200 mg, or in a range from 5 mg to 200 mg, or in a range from 10 mg to 200 mg, or in a range from 25 mg to 200 mg, or in a range from 30 mg to 200 mg, or in a range from 50 mg to 200 mg, or in a range from 60 mg to 200 mg, or in a range from 75 mg to 200 mg, or in a range from 90 mg to 200 mg, or in a range from 100 mg to 200 mg, or in a range from 150 mg to 200 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 100 mg, or in a range from 5 mg to 100 mg, or in a range from 10 mg to 100 mg, or in a range from 25 mg to 100 mg, or in a range from 30 mg to 100 mg, or in a range from 50 mg to 100 mg, or in a range from 60 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 90 mg to 100 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 25 mg, or in a range from 5 mg to 25 mg, or in a range from 10 mg to 25 mg, or in a range from 15 mg to 25 mg, or in a range from 20 mg to 25 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 50 mg, or in a range from 1 mg to 25 mg, or in a range from 5 mg to 50 mg, or in a range from 5 mg to 25 mg, or in a range from 10 to 50 mg, or in a range from 10 to 25 mg, or in a range from 1 to 15 mg, or in a range from 5 mg to 15 mg, or in a range from 10 mg to 15 mg, or wherein the single dose of the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,

90. The method of any one of claims 87-89, wherein the single dose of the pharmaceutical composition comprises the antibody at a concentration in a range from 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.

91. The method of any one of claims 87-90, wherein the single dose of the pharmaceutical composition comprises about 75 mg of the antibody.

92. The method of any one of claims 87-90, wherein the single dose of the pharmaceutical composition comprises about 90 mg of the antibody.

93. The method of any one of claims 87-90, wherein the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody.

94. The method of any one of claims 87-90, wherein the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody.

95. The method of any one of claims 87-90, wherein the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody.

96. The method of any one of claims 87-90, wherein the single dose of the pharmaceutical composition comprises about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg of the antibody.

97. The method of any one of claims 87-90, wherein the method comprises administering the single dose by subcutaneous injection, optionally wherein the single dose comprises or consists of 6 mg of the antibody or 18 mg of the antibody.

98. The method of any one of claims 87-97, wherein the method comprises administering the single dose by intravenous injection.

99. The method of any one of claims 87-98, wherein the pharmaceutical composition further comprises water, optionally USP water.

100. The method of any one of claims 87-99, wherein the pharmaceutical composition further comprises histidine, optionally at a concentration in a range from 10 mM to 40 mM, such as 20 mM, in the pharmaceutical composition.

101. The method of any one of claims 87-100, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8%, or 9%, preferably about 7% (w/v).

102. The method of any one of claims 87-101, wherein the pharmaceutical composition further comprises a surfactant or a triblock copolymer, optionally a polysorbate or poloxamer-188, preferably polysorbate 80 (PS80), wherein, optionally, the polysorbate or poloxamer-188 is present in a range from 0.01% to 0.05% (w/v), preferably 0.02% (w/v).

103. The method of any one of claims 87-102, wherein the pharmaceutical composition has a pH in a range from 5.8 to 6.2, in a range from 5.9 to 6.1, or of 5.8, of 5.9, of 6.0, of 6.1, or of 6.2.

104. The method of claim 103, wherein the pharmaceutical composition comprises:

(i) the antibody at 150 mg/mL;

(ii) USP water;

(iii) 20 mM histidine;

(iv) 7% sucrose; and (v) 0.02% PS80, wherein the pharmaceutical composition comprises a pH of 6.

105. The method of any one of claims 87-104, wherein the subject is an adult.

106. The method of claim 105, wherein the subject is in a range from 18 years of age to 65 years of age.

107. The method of any one of claims 87-106, wherein the subject weighs from 40 kg to 125 kg and/or the subject has a body mass index (BMI) from 18 to 35 kg/m².

108. The method of any one of claims 87-107, wherein the subject has a chronic HBV infection; e.g., defined by positive serum HBsAg, HBV DNA, and/or HBeAg on 2 occasions, wherein the 2 occasions are at least 6 months apart.

109. The method of any one of claims 87-108, wherein the subject does not have cirrhosis.

110. The method of claim 109, wherein absence of cirrhosis is determined by:

111. The method of any one of claims 87-110, wherein the subject has received a nucleos(t)ide reverse transcriptase inhibitor (NRTI), optionally within 120 days, further optionally within 60 days, prior to the single dose being administered.

112. The method of claim 111, wherein the NRTI comprises one or more of: tenofovir; tenofovir disoproxil (e.g., tenofovir disproxil fumarate); tenofovir alafenamide; Entecavir; Lamivudine; Adefovir; and adefovir dipivoxil.

113. The method of any one of claims 87-112, wherein the subject has a serum HBV DNA concentration of less than 100 lU/mL no more than 28 days prior to the single dose being administered.

114. The method of any one of claims 87-113, wherein the subject has a serum HBsAg concentration of less than 3,000 lU/mL prior to the single dose being administered, and optionally less than 1,000 lU/mL prior to the single dose being administered.

115. The method of any one of claims 87-114, wherein the subject has a serum HBsAg concentration of greater than or equal to 3,000 lU/mL no more than 28 days prior to the single dose being administered, and optionally greater than or equal to 1,000 lU/mL no more than 28 days prior to the single dose being administered.

116. The method of any one of claims 87-115, wherein the subject was HB e-antigen (HBeAg)-negative no more than 28 days prior to the single dose being administered.

117. The method of any one of claims 87-116, wherein the subject was negative for anti-HB antibodies no more than 28 days prior to the single dose being administered.

118. The method of any one of claims 87-117, wherein the subject, prior to administration of the single dose:

(i) does not have fibrosis and/or does not have cirrhosis; and/or

(ii) has alanine aminotransferase (ALT) < 2 x Upper Limit of Normal (ULN).

119. The method of any one of claims 87-118, wherein at 56 days following administration of the single dose, the subject has a > 2-fold reduction in serum HBsAg (e.g., concentration of HBsAg in serum, e.g., as determined using an Abbott ARCHITECT assay) as compared to the subject’s serum HBsAg at from 0 days to 28 days prior to administration of the single dose.

120. The method of any one of claims 87-119, wherein following administration of the single dose (e.g., at 56 days following administration of the single dose), the subject has:

(ii) comprises an adaptive immune response against HBV.

121. The method of any one of claims 87-120, wherein the subject is male.

122. The method of any one of claims 87-121, wherein the subject is female.

123. A pharmaceutical composition comprising the antibody of any one of claims 1-66 at a concentration ranging from 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, and a pharmaceutically acceptable carrier, excipient, or diluent.

124. The pharmaceutical composition of claim 123, wherein 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.

125. The pharmaceutical composition of claim 123 or 124, wherein the pharmaceutical composition comprises about 75 mg of the antibody.

126. The pharmaceutical composition of claim 123 or 124, wherein the pharmaceutical composition comprises about 90 mg of the antibody.

127. The pharmaceutical composition of claim 123 or 124, wherein the pharmaceutical composition comprises about 300 mg of the antibody.

128. The pharmaceutical composition of claim 123 or 124, wherein the pharmaceutical composition comprises about 900 mg of the antibody.

129. The pharmaceutical composition of claim 123 or 124, wherein the pharmaceutical composition comprises about 3,000 mg of the antibody.

130. The pharmaceutical composition of claim 123 or 124, wherein the pharmaceutical composition comprises about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg of the antibody.

131. The pharmaceutical composition of any one of claims 123-130, wherein the pharmaceutical composition comprises water, optionally USP water.

132. The pharmaceutical composition of any one of claims 123-131, wherein the pharmaceutical composition comprises histidine, optionally at a concentration from 10 mM to 40 mM, such as 20 mM, in the pharmaceutical composition.

133. The pharmaceutical composition of any one of claims 123-132, wherein the pharmaceutical composition comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8%, or 9%, preferably about 7% (w/v).

134. The pharmaceutical composition of any one of claims 123-133, wherein the pharmaceutical composition comprises a surfactant, optionally a polysorbate, preferably polysorbate 80 (PS80), wherein, optionally, the polysorbate is present in a range from 0.01% to 0.05% (w/v), preferably 0.02% (w/v).

135. The pharmaceutical composition of any one of claims 123-134, wherein the pharmaceutical composition has a pH ranging from 5.8 to 6.2, ranging from 5.9 to 6.1, or of 5.8, of 5.9, of 6.0, of 6.1, or of 6.2.

136. The pharmaceutical composition of any one of claims 123-135, wherein the pharmaceutical composition comprises:

(i) the antibody at 150 mg/mL;

(ii) USP water;

(iii) 20 mM histidine;

(iv) 7% sucrose; and

(v) 0.02% PS80, wherein the pharmaceutical composition comprises a pH of 6.