CN117042797A - Compositions and methods for treating hepatitis b virus infection - Google Patents

Abstract

The present disclosure relates to pharmaceutical compositions comprising antibodies that neutralize Hepatitis B Virus (HBV) infection. Furthermore, the present disclosure relates to the use of the pharmaceutical composition in the treatment of HBV infection.

Description

Compositions and methods for treating hepatitis b virus infection

Statement regarding sequence listing

The sequence listing relevant to the present application is provided in text format to replace paper copies and is hereby incorporated by reference into this specification. The text file containing the sequence listing is named 930485_433 wo_sequence_list. The text file was 124KB, created at 2022, month 1, 24, and submitted electronically via the EFS-Web.

The present disclosure relates to pharmaceutical antibody compositions and methods for the prevention and treatment of hepatitis b virus infection.

Background

HBV consists of: (i) An envelope comprising three related surface proteins (hepatitis b surface antigen, HBsAg) and lipids; and (ii) an icosahedral nucleocapsid encapsulating the viral DNA genome and DNA polymerase. HBV capsids are formed in the cytosol of infected cells during packaging of RNA pregenomic replication complexes and acquire budding capability during synthesis of viral DNA genome by retrosynthesis of the pregenomic in the granule lumen. The three HBV envelope proteins S-HBsAg, M-HBsAg and L-HBsAg form complex transmembrane folds at the endoplasmic reticulum and form disulfide-linked homodimers and heterodimers. During budding at the endomembrane, the short linear domain in the pre-cytosolic S region interacts with binding sites on the capsid surface. The virus particles are then secreted into the blood. In addition, surface proteins can bud in the absence of capsids and form subviral particles (SVP) that are also secreted in 3-4 log excess compared to viral particles. High levels of HBsAg may deplete HBsAg-specific T cell responses and are proposed as an important factor in viral immune tolerance in patients with Chronic Hepatitis B (CHB) (Chisari FV, isogawa M, wieland SF, pathobiology (Pathologie Biologie), 2010; 58:258-66).

Hepatitis b virus causes potentially life threatening acute and chronic liver infections. Acute hepatitis B is characterized by viremia with or without symptoms and with risk of developing fulminant hepatitis (Liang TJ, block TM, mcMahon BJ, ghany MG, guo JT, locarnini S, zoulim F, chang KM, lok AS, current and future therapies for hepatitis B from discovery to cure (Present and future therapies of hepatitis B: from discovery to cure), liver disease science (Hepatology) 2015, day 8, month 3, doi:10.1002/hep.28025.[ electronic version prior to printing plate ]). Despite the availability of effective vaccines against hepatitis b since 1982, WHO reported that there were still 2.4 million people chronically infected with hepatitis b and more than 780 000 people dying from hepatitis b complications each year. About one third of patients with Chronic Hepatitis B (CHB) suffer from cirrhosis, liver failure and hepatocellular carcinoma, resulting in 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, current and future therapies for hepatitis B: from discovery to cure, & gt, 3 months, day 2015, hepatology: doi:10.1002/hep.28025.[ electronic version prior to printing plate ]).

For HBV infected patients, serious complications may occur due to co-infection or repeated infection of HDV. According to WHO, hepatitis delta infects about 1500 tens of thousands worldwide. HDV is considered a subviral satellite because it can only propagate in the presence of HBV. HDV is one of the smallest animal viruses known (40 nm), whereby its genome is only 1.6kb and encodes S and L HDAg. All other proteins required for HDV genome replication, including RNA polymerase, are provided by the host cell, and HDV envelope is provided by HBV. When introduced into a recipient cell, the HDV RNA genome replicates and associates with multiple copies of the HDV encoded protein to assemble a Ribonucleoprotein (RNP) complex. RNP is derived from cells by HBV envelope proteins that are capable of assembling lipoprotein vesicles that bud into the lumen of the anterior golgi compartment prior to secretion. In addition, HBV envelope proteins also provide a mechanism to target HDV to uninfected cells, thereby ensuring HDV transmission.

Complications caused by HDV include a greater likelihood of liver failure occurring in acute infections and rapidly developing cirrhosis, with an increased chance of liver cancer in chronic infections. In combination with hepatitis B virus, the mortality rate of hepatitis D in all hepatitis infections is highest at 20% (Fattovich G, giustina G, christensen E, pantalena M, zagni I, realdi G, schalm SW, the effect of hepatitis D infection on the morbidity and mortality rate of compensatory cirrhosis B (Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B) [ intestinal tract (Gut) ] 3 month 2000; 46 (3): 420-6). The only approved therapy for chronic HDV infection is interferon- α. However, treatment of HDV with interferon- α is relatively inefficient and intolerant. Treatment with interferon-alpha resulted in one-fourth of the patients developing a sustained virologic response six months after treatment. Furthermore, nucleoside (nucleotide) analogs (NA) have been extensively tested in hepatitis delta, but they appear to be ineffective. Combination therapy of NA with interferon has also proven to be disappointing (Zaigham Abbas, minaam Abbas, hepatitis D management: novel treatment options are required (Management of hepatitis delta: need for novel therapeutic Options) [ journal of gastroenterology, world J journal of gastroenterol.) ] 2015, 28 th month, 21 (32): 9461-9465). Thus, new treatment options are needed.

Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

The drawings provided herein are intended to illustrate in more detail the subject matter included in the present disclosure. The drawings are not intended to limit the disclosure in any way. Throughout this disclosure, exemplary antibodies HBC34V35 (with or without Fc mutations such as MLNS and GAALIE) are also referred to as HBC34-V35 and HBC34-V35. Thus, it should be understood that HBC34V35, HBC34-V35 and HBC34-V35 have the same meaning. Similarly, exemplary antibodies HBC34V34 are also referred to as HBC34-V34 and HBC34-V34, and exemplary antibodies HBC34V7 are also referred to as HBC34-V7 and HBC34-V7. Furthermore, it should be understood that "MLNS-GAALIE" has the same meaning as "mlns_gaalie" (i.e., the m428l+n434s+g236 a+a330 l+i332e mutation in the Fc portion (EU numbering)).

FIGS. 1A-1B show the binding of HBC34-v7 and two engineered antibodies of the present disclosure ("HBC 34-v34"; "HBC34-v 35") to HBsAg adw (1A) and HBsAg adr (1B) at the indicated concentrations, as determined in a direct antigen-based ELISA assay. All antibodies were generated as IgG1 (g 1m17,1 allotype).

FIGS. 2A-2K show the binding of HBC34-v7, HBC34-v34 and HBC34-v35 to all known HBsAg genotypes ((A) - (J), respectively) and to a mock control (K). A genotype representative sequence representing the antigenic outer loop of HBsAg was used, as shown in example 5 of PCT publication No. WO 2017/060504. Staining was performed by FACS. Antibody concentrations are shown on the y-axis of the graph.

FIGS. 3A and 3B show the binding of HBC34-v7 and HBC34-v35 to the wild-type or variant Fc region to HBsAg adw in a direct antigen-based ELISA assay (2 experiments; data from "experiment 1" is shown in FIG. 3A, and data from "experiment 2" is shown in FIG. 3B). Antigen binding curves are shown in the upper panel of each figure. EC50 values (determined by fitting the curve using Graphpad prism) are shown in the middle panel of each plot. The binding to the uncoated plate (control) is shown in the lower panel of each figure. Fc region: "HBC34v7" and "HBC34-v35" =wild type; "HBC34-v35-MLNS" =M428L/N434S. "HBC34-v35-MLNS-GAALIE" =M428L/N434S/G236A/A330L/I332E. Three batches of HBCs 34-v35 were tested. Two batches of HBCs 34-v35-MLNS and two batches of HBCs 34-v35-MLNS-GAALIE were tested. A batch of HBCs 34-v7 was used.

FIGS. 4-7 show the effect of HBC34-v35 on serum HBAg levels in an in vivo mouse model of HBV infection. AAV/HBV infected SCID mice were transplanted with primary human hepatocytes and HBC34-v35 or PBS (control) was administered at 1, 5 or 15mg/kg as described in example 5. FIG. 4 shows serum HBV DNA concentrations before and after treatment. Fig. 5 shows serum HBsAg concentrations before and after treatment. Fig. 6 shows serum HBeAg concentrations before and after treatment. Fig. 7 shows serum HBcrAg concentrations before and after treatment.

FIGS. 8A-8E show the binding of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE to human Fc gamma R as assessed by Biological Layer Interferometry (BLI). His-tagged human FcgammaR ((A) FcgammaRIIa allele H131, (B) FcgammaRIIa allele R131, (C) FcgammaRIIIa allele F158, (D) FcgammaRIIIa allele V158, and (E) FcgammaRIIb) was captured on an anti-penta-His sensor for 6 minutes. Then, in the presence of 1 μg/ml of an afiniband F (ab ') 2 fragment goat anti-human IgG, i.e. a F (ab') 2 fragment specific for cross-linking a human mAb by Fab fragment, the fcγr loaded sensor was exposed to a kinetic buffer (ph 7.1) containing 2 μg/ml of each mAb (left part of the figure) for 5 minutes followed by a dissociation step in the same buffer for another 4 minutes (right part of the figure). Association and dissociation curves as a function of interference pattern were measured in real time using Octet RED96 (fortbio).

FIG. 9 shows the binding of HBC34-V35-MLNS and HBC34-V35-MLNS-GAALIE to human C1q as measured by Octet. Complete IgG1 of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE mAb was captured at 10. Mu.g/ml by Fab fragment using an anti-human Fab (CH 1) sensor for 10 min. The IgG-loaded sensor was then exposed to kinetic buffer containing 3 μg/ml purified human C1q (pH 7.1) for 4 minutes (left part of the figure), followed by a dissociation step in the same buffer for another 4 minutes (right part of the figure). Association and dissociation curves as a function of interference pattern were measured in real time using Octet RED96 (fortbio).

FIGS. 10A and 10B show in vitro activation of human FcgammaRIII using a receptor-linked activation NFAT mediated luciferase reporter in engineered Jurkat cellsa. Fcγriiia activation was tested using a validated, commercially available biological reporter assay, wherein recombinant HBsAg (Engerix B) was used as the target antigen. Serial dilutions of HBC34v35-MLNS and HBC34-v35-MLNS-GAALIE and control (Ctr) mAbs were incubated with 0.2. Mu.g/ml HBsAg for 25 min at 37 ℃. Jurkat effector cells (Promega), which express either FcgammaRIIIa low affinity allele F158 (A) or FcgammaRIIIa high affinity allele V158 (B), were resuspended in assay buffer and then added to the assay plates. After incubation at 37℃for 24 hours, bio-Glo- ^TM Luciferase assay reagent (Promega), and luminescence was quantified using a photometer (Bo Tek, bio-Tek).

FIGS. 11A and 11B show activation of human FcgammaRIIA in vitro using a receptor linked activation NFAT mediated luciferase reporter in engineered Jurkat cells. The fcγriia was activated using a validated, commercially available biological reporter assay, wherein recombinant HBsAg (Engerix B) was used as the target antigen. Serial dilutions of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE and control mAb (Ctr) were incubated with 2 (A) or 0.2 μg/ml (B) HBsAg for 25 min at 37 ℃. Jurkat effector cells (Promega) expressing the FcgammaRIIA high affinity allele H131 were resuspended in assay buffer and then added to the assay plate. After incubation at 37℃for 23 hours, bio-Glo- ^TM Luciferase assay reagent (Promega), and luminescence was quantified using a photometer (Bo Tek, bio-Tek).

FIG. 12 shows the use of receptor ligation to activate NFAT mediated luciferase reporter to activate human FcgammaRIIB in vitro in engineered Jurkat cells. Activation of human fcyriib was tested using a validated, commercially available biological reporter assay, wherein recombinant HBsAg (Engerix B) was used as target antigen. Serial dilutions of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE and control mAb (Ctr) were incubated with 1. Mu.g/ml HBsAg for 15 min at 37 ℃. The fcyriib expressing Jurkat effector cells (plagmaigb) were resuspended in assay buffer and then added to the assay plate. After incubation at 37℃for 20 hours, bio-is added Glo- ^TM Luciferase assay reagent (Promega), and luminescence was quantified using a photometer (Bo Tek, bio-Tek).

FIGS. 13A and 13B show in vitro killing of PLC/PRF/5 human hepatoma cells by human primary NK cells in the presence of HBC34-v35-MLNS and HBC34-v 35-MLNS-GAALIE. (A) ADCC was tested using freshly isolated NK cells from one donor that was previously genotyped to express heterozygous high (V158) and low (F158) affinity fcγriiia (F/V). Serial dilutions of HBC34-v35, HBC34-v35-MLNS-GAALIE, 17.1.41 and control mAb were added to HBsAg secreting hepatoma cell line PLC/PRF/5 (also known as Alexander cell). The PLC/PRF/5 cells were incubated with the antibody for 10 minutes at room temperature. NK cells were added to the assay plate (ratio of effector cells to target cells 10:1) and incubated at 37 ℃ for 4 hours. Cell death was determined by measuring Lactate Dehydrogenase (LDH) release. (B) PLC/PRF/5 human hepatoma cells were stained by HBC34v35 and 17.1.41mAb as assessed by flow cytometry. Cells were thoroughly washed, fixed with formaldehyde (4%) or fixed and permeabilized (0.5% saponins) before staining with different concentrations of HBC34-v35 and 17.1.41 mab. Using Alexa 647AffiniPure F (ab') 2 fragment goat anti-human IgG, an antibody specific for the Fcgamma fragment, was tested for binding of these human mAbs by flow cytometry.

FIGS. 14A and 14B show in vitro activation of primary human NK cells in the presence of HBC34v35-MLNS and HBC34 v35-MLNS-GAALIE and HBsAg. Activation of NK cells was tested using freshly isolated cells from two donors previously genotyped to express either (a) homozygous high (V158) or (B) low (F158) affinity fcγriiia. Serial dilutions of HBC34-V35, HBC34-V35-MLNS-GAALIE and HBC 34-V35-lalab were incubated with NK cells for 4 hours. NK cell activation was measured by flow cytometry by staining NK cells with anti-CD 107a mAb as a functional marker for identification of NK cell activity. CD107a (also known as LAMP-1) is a marker of NK cell degranulation.

Fig. 15A-15C show the evaluation schedule of healthy adult subjects in an exemplary single increment dose (SAD) clinical study comprising an exemplary pharmaceutical composition of the antibody HBC34-v35-MLNS-GAALIE as described in example 9.

Fig. 16A-16E show the evaluation schedule for subjects with chronic HBV infection without cirrhosis and receiving Nucleoside Reverse Transcriptase Inhibitor (NRTI) therapy in an exemplary clinical study described in example 9.

Figures 17A-17C show time points at which pharmacokinetic measurements were made on subjects according to the exemplary clinical study described in example 9.

Fig. 18 shows a dosing schedule according to an exemplary clinical study described in example 9.

Fig. 19 shows a clinical laboratory evaluation according to an exemplary clinical study described in example 9.

Fig. 20 shows upregulation of activation and costimulatory markers on monocyte-derived dendritic cells (modcs) stimulated by the following immune complexes: HBC34-v35-MLNS+HBsAg; or HBC34-v35-MLNS-GAALIE+HBsAg, as described in example 10.

Figure 21 shows moDC secretion cytokines stimulated by the following immune complexes: HBC34-v35-MLNS+HBsAg; or HBC34-v35-MLNS-GAALIE+HBsAg, as described in example 10.

FIGS. 22A and 22B show IFN-y release from whole blood cultures stimulated by immunocomplexes with: HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in example 10. (a) IFN- γ concentration (log 10); (B) Normalized IFN-gamma fold change (log 10), as described in example 10.

FIGS. 23A and 23B show the release of IL-2 in whole blood cultures stimulated by immunocomplexes with the following: HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in example 10. (a) IL-2 concentration (log 10); (B) Normalized fold IL-2 change (log 10), as described in example 10.

FIGS. 24A and 24B show IFN-gamma and IL-2 in whole blood cultures stimulated by immunocomplexes with: HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in example 10. (a) IFN- γ;100 μg/ml mAb; (B) IL-2; IL-2 μg/ml mAb.

Fig. 25 shows an exemplary single increment dose (SAD) cohort from the phase 1 clinical study described in example 9 and examples 11-16.

FIG. 26 shows computer modeling of predicted decrease in serum HBsAg in subjects following 60mg x 4 doses of HBC34-v 35-MLNS-GAALIE.

FIG. 27 shows a table summarizing specific demographics and baseline characteristics of subjects administered HBC34-v35-MLNS-GAALIE at 6mg (cohort 1 b), subjects administered HBC34-v35-MLNS-GAALIE at 18mg (cohort 2 b), and subjects administered HBC34-v35-MLNS-GAALIE at 75mg (cohort 3 b).

Fig. 28 shows a table summarizing the security and tolerability data of queues 1b, 2b and 3 b.

FIGS. 29A-29B show the levels of alanine Aminotransferase (ALT) over time. (A) ALT levels in cohort 1b (6 mg HBC34-v35-MLNS-GAALIE or placebo) are shown. (B) ALT levels in cohort 2b (18 mg HBC34-v35-MLNS-GAALIE or placebo) are shown.

FIGS. 30A-30B show the actual decrease in HBsAg levels in the serum of subjects after 6mg x 1 doses of S.C. or placebo (1B), 18mg x 1 doses of S.C. or placebo (2B), 75mg x 1 doses of S.C. or placebo (3B), 300mg x 1 doses of S.C. or placebo (4B), and 18mg x 1 doses of S.C. or placebo (1 c) of HBC34-v 35-MLNS-GAALIE. (A) HBsAg change over time (Log 10 IU/mL) is shown. (B) The absolute HBsAg (UI/mL) is shown as a function of time.

FIG. 31 shows the average decrease in serum HBsAg levels in subjects following HBC34-v35-MLNS-GAALIE 6mg x 1 dose S.C. or placebo, HBC34-v35-MLNS-GAALIE 18mg x 1 dose S.C. or placebo, or HBC34-v35-MLNS-GAALIE 75mg x 1 dose S.C. or placebo. Data do not include HBsAg reduction<0.2log ₁₀ IU/mL participant。

FIG. 32 shows HBsAg changes from baseline detected in HBsAg levels in serum of subjects after HBC34-v35-MLNS-GAALIE 6mg x 1 dose S.C. or placebo, HBC34-v35-MLNS-GAALIE 18mg x 1 dose S.C. or placebo, and HBC34-v35-MLNS-GAALIE 75mg x 1 dose S.C. or placebo.

FIGS. 33A-33C show free PK curves for HBC34-v35-MLNS-GAALIE in serum of subjects in cohorts 1b, 2b, 3b and 1C. (A) The free PK of HBC34-v35-MLNS-GAALIE in queue 1b (6 mg) is shown. (B) Free PKs for HBC34-v35-MLNS-GAALIE in queues 2b (HBsAg <3000IU/mL, HBeAg-) and 1c (18 mg, any HBsAg, HBeAg+/-) are shown. (C) The free PK of HBC34-v35-MLNS-GAALIE in queue 3b (75 mg) is shown.

FIG. 34 shows a summary of free PK parameters in queues 1b (6 mg HBC34-v 35-MLNS-GAALIE), 2b (18 mg HBC34-v 35-MLNS-GAALIE) and 3b (75 mg HBC34-v 35-MLNS-GAALIE). Data do not include 1 subject from 1 b.

FIGS. 35A-35C show the total PK profile of HBC34-v35-MLNS-GAALIE in serum of subjects in cohorts 1b, 2b, 3b and 1C. (A) The total PK of HBC34-v35-MLNS-GAALIE in queue 1b (6 mg) is shown. (B) The total PK for HBC34-v35-MLNS-GAALIE in queues 2b (HBsAg <3000IU/mL, HBeAg-) and 1c (18 mg, any HBsAg, HBeAg+/-) are shown. (C) The total PK of HBC34-v35-MLNS-GAALIE in queue 3b (75 mg) is shown.

FIG. 36 shows a summary of total PK parameters in queues 1b (6 mg SC HBC34-v 35-MLNS-GAALIE), 2b (18 mg SC HBC34-v 35-MLNS-GAALIE), 3b (75 mg SC HBC34-v 35-MLNS-GAALIE) and 1c (18 mg SC HBC34-v 35-MLNS-GAALIE). Data do not include 1 subject from 1 b.

Figure 37 shows a table summarizing the inclusion demographics of subjects in part a of the clinical study. Including surrogate subjects; BMI = body mass index.

Figure 38 shows a table summarizing safety and tolerability data for subjects in part a of the clinical study. Including surrogate subjects; AE = adverse event; IV = intravenous; sc=subcutaneous.

FIG. 39 showsSummarizing tables of serum pharmacokinetic parameters of HBC34-v35-MLNS-GAALIE after a single subcutaneous or intravenous dose to healthy subjects. Except t _1/2 、T _max 、T _last All parameters are expressed in terms of mean and CV% except as median (Q1, Q3). AUC = area under the curve; AUC (AUC) _inf AUC from time 0 to infinity; AUC (AUC) _last AUC of the last measurable concentration; c (C) _last =last measurable concentration; CL/F = apparent oral clearance; c (C) _max =maximum concentration; IV = intravenous; sc=subcutaneous; t (T) _last Time of last measurable concentration; t (T) _max =reach C _max Time of (2); t (T) _1/2 =half-life; V/F = apparent distribution volume.

FIG. 40 shows the serum concentration PK profile of HBC34-v35-MLNS-GAALIE after a single subcutaneous or intravenous dose in healthy subjects. HV = healthy volunteer; PK = pharmacokinetics; IV = intravenous; sc=subcutaneous.

Detailed Description

The present disclosure provides pharmaceutical compositions comprising antibodies that neutralize Hepatitis B Virus (HBV) infection and methods of using these compositions. In certain embodiments, the antibody binds to HBsAg of a genotype selected from A, B, C, D, E, F, G, H, I and J or any combination thereof. In certain embodiments, the antibodies include mutations in the heavy chain that extend the in vivo half-life of the antibody (e.g., in humans) and mutations in the heavy chain that increase binding affinity to fcγr (e.g., human fcγriia, human fcγriiia, or both).

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

Although antibodies that neutralize HBV, pharmaceutical compositions including those antibodies, and methods of using such pharmaceutical compositions are described in detail below, it is to be understood that the disclosure is not limited to the specific methods, protocols, and reagents described herein as they may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present 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.

Hereinafter, aspects of the present disclosure are described. Certain embodiments are provided, however, it should be understood that embodiments of the present disclosure may be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed to limit the disclosure to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any of the disclosed subject matter. Furthermore, any permutation and combination of the subject matter described herein should be considered disclosed by the description of the application unless the context indicates otherwise.

Throughout this disclosure, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" are used synonymously with e.g. "having", "including" and "comprising", and the term should be taken to imply the inclusion of a stated member, ratio, integer (including a portion thereof, e.g. one tenth and one hundredth of an integer, where appropriate), concentration or step, but not the exclusion of any other unexplained member, ratio, integer, concentration or step. The term "consisting essentially of (consisting essentially of)" is not equivalent to "comprising" and refers to a specified material or step of the claims, or to a material or step that does not materially affect the basic characteristics of the claimed subject matter. For example, when the amino acid sequence of a domain, region, module, or protein comprises an extension, deletion, mutation, or combination thereof (e.g., an amino acid between amino-or carboxy-terminal or domains), a protein domain, region, or module (e.g., a binding domain) or protein "consists essentially of" a particular amino acid sequence, the extension, deletion, mutation, or combination thereof binds up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the contributing domain, region, module, or protein length, and does not significantly affect (i.e., does not reduce activity by more than 50%, such as not more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain, region, module, or protein (e.g., binding protein's target binding affinity).

The term "consist" is a specific embodiment of the term "comprising" wherein any other non-stated member, integer or step is excluded. In the context of the present disclosure, the term "comprising" encompasses the term "consisting of. The term "comprising" thus encompasses "comprising" as well as "consisting of," e.g., a composition "comprising" X may consist of only X or may include additional substances, e.g., x+y.

In addition, it is to be understood that the present application discloses individual compounds or groups of compounds derived from various combinations of structures and substituents described herein to the same extent as each compound or group of compounds is set forth individually. Accordingly, the selection of a particular structure or particular substituent is within the scope of the present disclosure.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (including the context of the following 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 alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof. Recitation of ranges of values herein are intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the present disclosure as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the subject matter disclosed herein.

The word "substantially" does not exclude "complete"; for example, a composition that is "substantially free" of Y may be completely free of Y. In certain embodiments, "substantially" refers to a given amount, effect, or activity of a composition, method, or use of the present disclosure as compared to 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.

The term "about" in relation to the value x means x.+ -. 10%, for example x.+ -. 5%, or x.+ -. 7%, or x.+ -. 10%, or x.+ -. 12%, or x.+ -. 15% or x.+ -. 20%. For example, in certain embodiments, "about" means ± 20% of the indicated range, value, or structure.

"optional" or "optionally" means that the subsequently described element, component, event or circumstance may or may not occur, and that the description includes instances where the element, component, event or circumstance occurs and instances where it does not.

As used herein, the term "disease" is intended to be generally synonymous and is used interchangeably with the terms "disorder" and "condition" (e.g., medical condition), as all reflect abnormal conditions of one of the human or animal bodies or parts thereof that impair normal function, often manifest as overt signs and symptoms, and result in a reduction in the life duration or quality of life of the human or animal.

As used herein, the term "therapeutically effective" means that the pharmaceutical composition or antibody described herein is of sufficient nature or amount to provide a benefit to a subject. In the context of the present disclosure, a benefit provided to a subject is the treatment of hepatitis b virus infection. As used herein, reference to "treating" a subject or patient is intended to include prophylaxis, control, attenuation, amelioration, and therapy. Therapeutic benefits include improving clinical outcome; alleviating or alleviating symptoms associated with the disease; reducing the occurrence of symptoms; improving the quality of life; longer disease-free state; reducing the extent of the disease; stabilizing the disease state; delay disease progression; relief; survival; prolonging survival rate; or any combination thereof. The term "subject" or "patient" is used interchangeably herein to mean a person susceptible to or already infected with HBV.

The dose is typically related to body weight (i.e., the body weight of the subject). Thus, a dose expressed in [ g, mg, or other units ]/kg (or g, mg, etc.) generally refers to [ g, mg, or other units ] "per kg (or g, mg, etc.) of body weight", even though the term "body weight" is not explicitly mentioned.

As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those which are later modified, for example hydroxyproline, gamma-carboxyglutamic acid 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 alpha 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. The analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but the basic chemical structure remains the same as a naturally occurring amino acid. Amino acid mimetics refers 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 variants of these terms, refer to a molecule comprising at least two amino acids joined to each other by a (ordinary or modified) peptide bond. For example, a peptide, polypeptide or protein may be composed of a plurality of amino acids selected from the group consisting of 20 amino acids defined by the genetic code, each amino acid being linked to each other at least by a peptide bond. The peptide, polypeptide or protein may be composed of L-amino acids and/or D-amino acids. The terms "peptide", "polypeptide", "protein" also include "peptidomimetics", which are defined as peptide analogs containing non-peptide structural elements that are capable of mimicking or antagonizing the biological effects of a native parent peptide. In certain embodiments, the peptidomimetic lacks properties such as a peptide bond that is susceptible to enzymatic cleavage.

In addition to these amino acids, the peptide, polypeptide or protein may contain amino acids other than 20 amino acids defined by the genetic code, or it may be composed of amino acids other than 20 amino acids defined by the genetic code. In certain embodiments, a peptide, polypeptide, or protein in the context of the present disclosure may comprise an amino acid modified by a natural process (e.g., post-translational maturation process) or by a chemical process (e.g., synthetic process), which processes are known in the art and include the processes described herein. Such modifications may occur anywhere in the polypeptide; for example, in a peptide backbone; in the amino acid chain; or at the carboxy-terminus or the amino-terminus. The peptide or polypeptide may be branched, such as after ubiquitination, or may be cyclic, with or without branching. The terms "peptide", "polypeptide" and "protein" also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide or protein modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or nucleotide derivative, covalent fixation of a lipid or lipid derivative, covalent fixation of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including polyethylene glycol, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, selenoylation, sulfation, amino acid addition such as arginylation or ubiquitination. Such modifications have been described in the literature (see protein Structure and molecular Properties (Proteins Structure and Molecular Properties) (1993) 2 nd edition, T.E. Cright, N.Y. (Post-translational Covalent Modifications of Proteins) for Post-translational covalent modification of proteins (1983) B.C.Johnson edit, new York academy of sciences (Academic Press, new York); seifer et al (1990) protein modification and non-protein cofactor analysis (Analysis for protein modifications and nonprotein cofactors), methods in enzymology (meth. Enzymol.) (182:626-646 and Rattan et al (1992) protein synthesis: post-translational modification and ageing (Protein Synthesis: post-translational Modifications and Aging), new York academy of sciences (Ann NY Acad Sci), 663:48-62). Thus, the terms "peptide", "polypeptide", "protein" may include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like. Variants of the proteins, peptides and polypeptides of the 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 described herein.

As used herein, "(poly) peptide" and "protein" may be used interchangeably with respect to polymers of amino acid residues (e.g., multiple amino acid monomers linked by peptide bonds).

"nucleic acid molecule" or "polynucleotide" or "nucleic acid" refers to a polymeric compound comprising covalently linked nucleotides, which may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers may be linked by phosphodiester linkages or analogues of such linkages. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenates, phosphorodiselenates, phosphorothioanilines, phosphoroanilides, phosphorophosphoramidates, and the like.

Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), including cDNA, genomic DNA, and synthetic DNA, any of which may be single-stranded or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or the non-coding strand (antisense strand). Polynucleotides (including oligonucleotides) and fragments thereof may be produced, for example, by Polymerase Chain Reaction (PCR) or by in vitro translation, or by any of ligation, cleavage, endonuclease action, or exonuclease action.

Nucleic acid molecules encoding an amino acid sequence include all nucleotide sequences encoding the same amino acid sequence. Some forms of nucleotide sequences may also include introns to the extent that they may be removed by co-transcription or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence due to redundancy or degeneracy of the genetic code or by splicing or both.

Variants of the nucleic acid molecules of the present disclosure are also contemplated. The variant nucleic acid molecule is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to a nucleic acid molecule of a defined or reference polynucleotide described herein, or hybridizes to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 65-68 ℃. The nucleic acid molecule variants retain the ability to encode fusion proteins or binding domains thereof that have the functions described herein, such as specifically binding to a target molecule.

As used herein, the term "sequence variant" refers to any sequence having one or more changes as compared to a reference sequence, wherein the reference sequence is any disclosed sequence and/or the sequences listed in the "sequence listing and SEQ ID No. (sequence listing)", i.e., SEQ ID No. 1 to SEQ ID No. 120. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. In certain embodiments, the sequence variant in the context of a nucleotide sequence, the reference sequence is also a nucleotide sequence, while in certain embodiments of the sequence variant in the context of an amino acid sequence, the reference sequence is also an amino acid sequence. As used herein, a "sequence variant" may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference sequence.

"percent sequence identity" refers to a relationship between two or more sequences as determined by comparing the sequences. The method for determining sequence identity may be designed to give the best match between the compared sequences. For example, sequences may be aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences may be ignored for comparison purposes. The percent sequence identity referred to herein is calculated by reference to the length of the sequence unless otherwise indicated. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using the BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithms used in the BLAST program can be found in Altschul et al, nucleic Acids Res 25:3389-3402,1997. In the context of the present disclosure, it should be understood that when analyzing using sequence analysis software, the results of the analysis are based on "default values" of the referenced program. "default value" means any set of values or parameters that are initially loaded when the software is first initialized.

"sequence variants" in the context of nucleic acid (nucleotide) sequences have altered sequences in which one or more nucleotides in a reference sequence are deleted or substituted or one or more nucleotides are inserted into the sequence of a reference nucleotide sequence. Nucleotides are referred to herein by standard single letter designations (A, C, G or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence may or may not change the corresponding reference amino acid sequence (i.e., an amino acid "sequence variant"). In certain embodiments, the nucleotide sequence variant does not produce an amino acid sequence variant (e.g., a silent mutation). In some embodiments, nucleotide sequence variants that result in "non-silent" mutations are contemplated. In some embodiments, nucleotide sequence variants of the present disclosure encode 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 also refer to codon optimized versions of reference or wild-type nucleotide or amino acid sequences. In any of the embodiments described herein, the polynucleotides of the present disclosure may be codon optimized for a host cell containing the polynucleotide (see, e.g., scholten et al, clinical immunology (Clin. Immunol.))) 119:135-145 (2006)).

"sequence variants" in the context of amino acid sequences have altered sequences in which one or more amino acids are deleted, substituted or inserted compared to a reference amino acid sequence. As a result of the change, such sequence variants have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having no more than 10 changes (i.e., any combination of deletions, insertions, or substitutions) per 100 amino acids of the reference sequence is "at least 90% identical" to the reference sequence.

"conservative substitutions" refer to amino acid substitutions that do not significantly affect or alter the binding characteristics of a particular protein. Generally, conservative substitutions are substitutions in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include those found in one of the following groups: group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); group 3: asparagine (Asn or N), glutamine (Gln or Q); group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids may be grouped into conservative substitutions based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, for substitution purposes, the aliphatic groupings may include Gly, ala, val, leu and Ile. Other conservative substitution sets include: sulfur-containing: met and cysteine (Cys or C); acidic: asp, glu, asn and Gln; small aliphatic, non-polar or slightly polar residues: ala, ser, thr, pro and Gly; polar negatively charged residues and amides thereof: asp, asn, glu and Gln; polar positively charged residues: his, arg and Lys; large aliphatic, nonpolar residues: met, leu, ile, val and Cys; large aromatic residues: phe, tyr and Trp. Additional information can be found in Cright on (1984) Proteins (Proteins), W.H. Frieman, inc. (W.H. Freeman and Company).

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

In general, changes in sequence variants do not eliminate or significantly reduce the desired function of the corresponding reference sequence. For example, it is preferred that the variant sequences of the present disclosure do not significantly reduce or completely eliminate the function of the sequences of the antibodies or antigen binding fragments thereof to bind to the same epitope and/or to sufficiently neutralize infection of HBV and HDV as compared to antibodies or antigen binding fragments having (or encoded by) the reference sequence. Guidance in determining which nucleotide and amino acid residues, respectively, may be substituted, inserted or deleted without eliminating the desired structure or function may be found by using known computer programs.

As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide, or protein refers to the source of the nucleic acid, peptide, polypeptide, or protein. A nucleic acid sequence or amino acid sequence derived from a particular sequence may have an amino acid sequence that is substantially identical to the sequence from which it was derived or a portion thereof, wherein "substantially identical" includes sequence variants as defined above. The nucleic acid sequence or amino acid sequence derived from a particular peptide or protein may be derived from a corresponding domain in the particular peptide or protein. In this context, "corresponding" means having the same function 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). Thus, one of ordinary skill in the art can readily identify the "corresponding" portions of peptides, proteins, and nucleic acids. Likewise, a sequence that is "derived from" another (e.g., a "source") sequence may be identified by one of ordinary skill in the art as having its origin in the source sequence.

The nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be identical to the starting nucleic acid, peptide, polypeptide or protein from which it was derived. However, the nucleic acid sequence or 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 was derived, in particular the nucleic acid sequence or 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 was 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 an amino acid sequence as compared to a reference sequence, e.g., corresponding genomic, wild-type, or reference sequence. Mutations, for example compared to a reference genomic sequence, may be, for example, (naturally occurring) somatic mutations, spontaneous mutations, induced mutations (e.g. induced by enzymes, chemicals or radiation), or mutations obtained by site-directed mutagenesis (molecular biological methods for specific and deliberate alterations in nucleic acid sequences and/or amino acid sequences). Thus, the term "mutation" is understood to also include physical mutations, for example in a nucleic acid sequence or an amino acid sequence. Mutations include substitutions, deletions and insertions of one or more nucleotides or amino acids, and inversions of several consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding the amino acid sequence in order to express (recombinant) the mutant polypeptide. Mutations can be achieved, for example, by altering (e.g., by site-directed mutagenesis) the codons of a nucleic acid molecule encoding an amino acid (e.g., by altering one, two, or three nucleotide bases) to provide codons encoding different amino acids or encoding the same amino acid, or by synthesizing sequence variants.

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

As used herein, the term "recombinant" (e.g., recombinant antibody, recombinant protein, recombinant nucleic acid, or linker) refers to any molecule (antibody, protein, nucleic acid, etc.) that is recombinantly produced, expressed, produced, or isolated and that does not occur in nature. "recombinant" may be used synonymously with "engineered" or "non-natural" and may refer to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by the introduction of an exogenous nucleic acid molecule, wherein such alteration or modification is introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications that introduce expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruptions of the genetic material of the cell. Other modifications include, for example, non-coding regulatory regions, wherein the modification alters 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 the host cell or subject, or any gene, protein, compound, nucleic acid molecule or activity that has been altered that is native to the host cell or subject. 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 differ between the native and altered gene, protein, compound, or nucleic acid molecule. In certain embodiments, a heterologous, non-endogenous or exogenous gene, protein, or nucleic acid molecule (e.g., receptor, ligand, etc.) may not be endogenous to the host cell or subject, and nucleic acid encoding such gene, protein, or nucleic acid molecule may have been added to the host cell by conjugation, transformation, transfection, electroporation, etc., wherein the added nucleic acid molecule may be integrated into the host cell genome or may be present as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologue" or "homologue" 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 the 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. The non-endogenous polynucleotide or gene and the encoded polypeptide or activity may be from the same species, 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 subject.

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include primary subject cells and cultures derived therefrom, regardless of the number of metastases. It is also understood that all progeny may not be identical in DNA content, due to deliberate or inadvertent mutation. Including progeny of variants having the same or substantially the same function, phenotype, or biological activity as that screened for in the originally transformed cell. Where different naming is desired, it will be apparent from the context.

The present disclosure is based in part on the design of antibodies and antigen binding fragments capable of neutralizing hepatitis b and hepatitis d viruses. Embodiments of antibodies and antigen binding fragments according to the present disclosure may be used in methods of preventing, treating or attenuating HBV and HDV. In certain embodiments, the antibodies and antigen binding fragments described herein bind to two or more different genotypes of hepatitis b virus surface antigen and two or more different infectious mutants of hepatitis b virus surface antigen. In specific embodiments, the antibodies and antigen binding fragments described herein bind to all currently known genotypes of hepatitis b virus surface antigens and all currently known infectious mutants of hepatitis b virus surface antigens.

Antibodies and antigen binding fragments thereof

In one aspect, the present disclosure provides isolated antibodies or antigen-binding fragments thereof that bind to the antigenic loop region of HBsAg and neutralize hepatitis b virus and hepatitis d virus infection for use in the pharmaceutical compositions and methods disclosed herein.

As used herein, unless the context clearly indicates otherwise, "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (although heavy chain antibodies lacking light chains are understood to be encompassed by the term "antibody"), as well as any antigen-binding portion or fragment of an intact antibody, such as scFv, fab, or F (ab') 2 fragments, that has or retains the ability to bind to an antigen target molecule recognized by the intact antibody. Thus, the term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F (ab ') 2 fragments, fab' fragments, fv fragments, recombinant IgG (IgG) 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 immunoglobulin forms such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, bifunctional antibodies, trifunctional antibodies and tetrafunctional antibodies, tandem diavs, tandem triavs. Unless otherwise indicated, the term "antibody" is to be understood as encompassing functional antibody fragments thereof. The term also encompasses whole or full length antibodies, including antibodies of any class or subclass thereof, including IgG and subclasses thereof, igM, igE, igA and IgD.

Thus, the antibodies of the disclosure may belong to any isotype (e.g., igA, igG, igM, also referred to as the α, γ, and μ heavy chains, respectively). For example, in certain embodiments, the antibody is of the IgG type. In the IgG isotype, the antibody can be of the IgG1, igG2, igG3 or IgG4 subclass, e.g., igG1. In some embodiments, the antibody comprises amino acid sequences from two different isotypes (e.g., exchange of constant domain amino acid sequences), such as an antibody comprising a constant region comprising an amino acid sequence from an IgA antibody and an amino acid sequence from an IgG antibody. Antibodies of the disclosure may comprise kappa or lambda light chains. In some embodiments, the antibody is of the IgG1 type and comprises a kappa light chain.

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

Human antibodies are known (van Dijk, m.a. and van de Winkel, j.g.), latest view of chemical biology (Curr Opin Chem biol.) (5 (2001) 368-374). The human antibodies can be produced in a transgenic animal (e.g., a mouse) that is capable of producing all or selected human antibodies in the absence of endogenous immunoglobulins upon immunization. Transfer of an array of human germline immunoglobulin genes 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, annual immunology (Year immunol.)) 7 (1993) 3340). Human antibodies can also be generated in phage display libraries (Hoogenboom, H.R. and Winter, G., "J.Mol.biol.)" 227 (1992) 381 (-388); marks, J.D. et al, J.J. 222 (1991) 581-597). The techniques of Cole et al and Boerner et al may also be used to prepare human monoclonal antibodies (Cole et al, monoclonal antibodies and cancer therapies (monoclone a)l Antibodies and Cancer Therapy), allen R rism (Alan R.List), page 77 (1985); and Boerner et al, J.Immunol.) (147 (1991) 86-95). Human monoclonal antibodies can be prepared by using modified EBV-B cell immortalization, such as Traggiai E, becker S, suberao K, kolesnikova L, uematsu Y, gismondo MR, murphy BR, rappuoli R, lanzavecchia a. (2004): an effective method for preparing human monoclonal antibodies from memory B cells comprises the following steps: strong neutralization of SARS coronavirus (An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus) Nature medicine (Nat Med.) (10 (8): 871-5). As used herein, the term "human antibody" also includes such antibodies that are modified, for example, in the variable regions to produce the properties of antibodies and antibody fragments according to the present disclosure. As used herein, the term "variable region" (light chain variable region (V _L ) Heavy chain variable region (V) _H ) A) represents each of the light chain and heavy chain pairs directly involved in binding an antibody to an antigen.

As used herein, the term "variable region" (e.g., a light chain variable region (V _L ) Heavy chain variable region (V) _H ) Refers to the variable region of an antibody light chain or antibody heavy chain that is directly involved in binding an antibody to an antigen. In other words, the term "V _L "or" VL "and" V _H "or" VH "refers to the variable binding regions from the antibody light and heavy chains, respectively.

The variable binding region consists of discrete, well-defined sub-regions called "complementarity determining regions" (CDRs) and "framework regions" (FR). The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous amino acid sequences within the variable region of an antibody, which generally confer antigen specificity and/or binding affinity. Typically, there are three CDRs in each variable region of an antibody; the VH and VL regions together comprise six CDRs HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to herein as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, respectively. CDRs on the heavy and/or light chain can be separated in the primary amino acid sequence by framework regions, where Framework Regions (FR) are regions of less variability in the variable domain (i.e., from one antibody to another (e.g., from one antibody to another encoded by the same allele or alleles)) as compared to CDRs. For example, one chain (or each chain, respectively) may be made up of four framework regions separated by three CDRs. In certain embodiments, the antibody VH comprises four FRs and three CDRs, arranged as follows: FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4; and the antibody VL includes four FRs and three CDRs as follows: FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4. Typically, VH and VL together form an antigen binding site through their respective CDRs, although it should be appreciated that in some cases the binding site may be formed by or comprise one, two, three, four or five of the CDRs.

As used herein, a "variant" of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions, deletions, or combinations thereof. Immunoglobulin sequences may be aligned with numbering schemes (e.g., kabat, EU, international immunogenetic information systems (International Immunogenetics Information System, IMGT) and Aho) that allow equivalent residue positions to be noted and allow comparison of different molecules using antigen receptor numbering and receptor classification (Antigen receptor Numbering And Receptor Classification, ANARCI) software tools (2016, bioinformatics) 15:298-300). It is understood that in certain embodiments, an antibody or antigen binding fragment of the present disclosure may comprise all or part of a Heavy Chain (HC), a Light Chain (LC), or both. For example, full length intact IgG antibody monomers typically include VH, CH1, CH2, CH3, VL, and CL. The Fc component is further described herein.

In the present disclosure, the positions of CDR amino acids are defined according to the IMGT numbering system (IMGT: www.imgt.org/; cf. Lefranc, M.—P..et al (2009) nucleic acids research 37, D1006-D1012).

Table 1 shows amino acid sequences of heavy chain variable regions (VH), light chain variable regions (VL), CDRs, heavy Chains (HC), and Light Chains (LC) of certain exemplary antibodies according to the disclosure.

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Fragments of the antibodies described herein may be obtained from the antibodies by methods that include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of antibodies may be obtained by cloning and expressing a portion of the heavy or light chain sequence. Antibody "fragments" include Fab, fab ', F (ab') 2 and Fv fragments. The present disclosure also encompasses single chain Fv fragments (scFv) derived from the heavy and light chains of antibodies as described herein, including, for example, scFv comprising CDRs from an antibody according to the invention, heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies, wherein the heavy and light chain variable domains are joined by a peptide linker.

In certain embodiments, an antibody or antigen binding fragment thereof according to the present disclosure comprises a purified antibody, single chain antibody, fab ', F (ab') 2, fv, or scFv.

In embodiments, antibodies and antigen binding fragments of the present disclosure can be multispecific (e.g., bispecific, trispecific, tetraspecific, etc.), and can be provided in any multispecific form as disclosed herein. In certain embodiments, the antibodies or antigen binding fragments of the present disclosure are multispecific antibodies, such as bispecific or trispecific antibodies. In, for example, spiess et al, molecular immunology (mol. Immunol.)) 67 (2): 95 (2015) and Brinkmann and Kontermann, mAb 9 (2): 182-212 (2017), which are incorporated herein by reference and include, for example, bispecific T cell adapter (BiTE), DART, pestle socket (Knobs-in-hole, KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, tandabs, triplets, trici minibodies, fab-scFv, scFv-CH-CL-scFv, F (ab') 2-scFv2, tetravalent HCab, intracellular antibodies, crossMab, double-acting Fab (DAF) (two-in-one or four-in-one), dutamab, DT-IgG, charge pairs, fab-arm exchanges, SEED bodies, triomab, LUZ-Y assemblies, fcab, kappa bodies, orthogonal Fab, DVD-IgG, igG (H) -scFv, scFv- (H) IgG, igG (L) -scFv, scFv- (L), igG (L) -35igg (H) -IgG (H) -35, igG (L) -IgG (scfj-IgG), scFv (L) -IgG (84-scFv, scFv (scFv), and scFv (scFv-4, scFv-scFv (scFv) 2. Bispecific or multispecific antibodies may comprise a combination of an HBV and/or HDV specific binding domain of the present disclosure with another HBV and/or HDV specific binding domain of the present disclosure, or with a different binding domain that specifically binds HBV and/or HDV (e.g., at the same or a different epitope), or with a binding domain that specifically binds a different antigen.

The antibody fragments of the present disclosure may confer monovalent or multivalent interactions and are comprised in a variety of structures as described above. For example, scFv molecules can be synthesized to produce trivalent "trifunctional antibodies" or tetravalent "tetrafunctional antibodies. The scFv molecule may comprise a domain of an Fc region to form a bivalent minibody. In addition, the sequences of the present disclosure may be components of a multispecific molecule, wherein the sequences of the present disclosure target the epitopes of the present 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 bifunctional antibodies (Holliger and Hudson,2005, nature Biotechnology (Nature Biotechnology) 9:1126-1136).

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

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

Variant forms of the disclosed antibodies are also contemplated herein, which are engineered to reduce known or potential immunogenicity and/or other potential possibilities.

As used herein, a "neutralizing antibody" is an antibody that can neutralize (i.e., prevent, inhibit, reduce, hinder, or interfere with) the ability of a pathogen to elicit and/or persist in a host (e.g., a host organism or host cell) to infect the host. The terms "neutralizing antibody (neutralizing antibody)" or "neutralizing antibody (an antibody that neutralizes/antibodies that neutralize)" are used interchangeably herein. These antibodies may be used alone or in combination (e.g., two or more of the presently disclosed antibodies in 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 capable of neutralizing HBV B and/or D infection), after appropriate formulation, as a prophylactic or therapeutic agent in connection with active vaccination.

As used herein, "specifically bind" or "pair..specific" refers to the association or association of a binding protein (e.g., an antibody or antigen binding fragment thereof) or binding domain with a target molecule with an affinity or Ka (i.e., equilibrium association constant in 1/M for a particular binding interaction) equal to or greater than 10 ⁵ M ^-1 (which is equal to the association rate of this association reaction [ K ] _on ]With dissociation rate [ Koff ]]Is not significantly associated or associated with any other molecules or components in the sample. Antibodies or binding domains can be classified as "high affinity" binding proteins or binding domains or "low affinity" binding proteins or binding domains. "high affinity" binding protein or binding domain means that Ka is at least 10 ⁷ M ^-1 At least 10 ⁸ M ^-1 At least 10 ⁹ M ^-1 At least 10 ¹⁰ M ^-1 At least 10 ¹¹ M ^-1 At least 10 ¹² M ^-1 Or at least 10 ¹³ M ^-1 Is a binding protein or binding domain. "Low affinity" binding protein or binding domain means that Ka is at most 10 ⁷ M ^-1 At most 10 ⁶ M ^-1 Or at most 10 ⁵ M ^-1 Is a binding protein or binding domain. Alternatively, affinity may be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction, in M (e.g., 10 ^-5 M to 10 ^-13 M). The terms "bind" and "specifically bind" and similar references do not encompass non-specific adhesion.

In certain embodiments, antibodies according to the present disclosure may bind to the antigenic loop region of HBsAg. The envelope of hepatitis b virus generally contains three "HBV envelope proteins" (also known as "HBsAg", "hepatitis b surface antigen"): s protein (representing "small", also called S-HBsAg), M protein (representing "medium", also called M-HBsAg) and L protein (representing "large", also called L-HBsAg). S-HBsAg, M-HBsAg and L-HBsAg share the same C-terminus (also known as the "S domain", 226 amino acids), which corresponds to the S protein (S-HBsAg) and is critical for viral assembly and infectivity. S-HBsAg, M-HBsAg and L-HBsAg are synthesized in the Endoplasmic Reticulum (ER), assembled and secreted in particulate form by the Golgi apparatus. The S domain comprises four predicted Transmembrane (TM) domains, whereby both the N-terminus and the C-terminus of the S domain are exposed to the lumen. Both transmembrane domains TM1 and TM2 are considered necessary for the integration of the cotranslational protein into the ER membrane, and 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, comprising a total of 226 amino acids (salise j. And Sureau c.,2009, journal of virology (Journal of Virology), 83:9321-9328). The determinant of infectivity is in the antigenic loop region of HBV envelope protein. Specifically, residues between 119 and 125 of HBsAg contain CXXC motifs which are considered important for the infectivity of HBV and HDV (Jaoude GA, sureau C, J.Virol.2005; 79:10460-6).

When referring herein to positions in the amino acid sequence of the S domain of HbsAg, such positions are obtained with reference to the amino acid sequence set forth in SEQ ID NO:3 (shown below) or a natural or artificial sequence variant thereof.

MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGN CTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

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

For example, the expression "amino acids 101-172 of the S domain" refers to amino acid residues 101-172 of the polypeptide according to SEQ ID NO. 3. However, it will be appreciated by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions, e.g. of different genotypes of HBsAg or of different HBsAg mutants as described herein) may occur naturally in the amino acid sequence of the S domain of HBsAg or be artificially introduced into the amino acid sequence of the S domain of HBsAg without affecting its biological properties. Thus, as used herein, the term "S domain of HBsAg" encompasses all such polypeptides, including for example polypeptides according to SEQ ID No. 3 and natural or artificial mutants thereof. Furthermore, 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 natural or artificial mutants thereof. For example, the phrase "amino acid residues from positions 101-172 of the S domain of HBsAg" encompasses the amino acid residues from positions 101-172 of SEQ ID NO. 3 and the corresponding fragments of mutants (natural or artificial mutants) thereof. As used herein, the phrases "corresponding sequence fragment" and "corresponding fragment" refer to fragments that are located at the same position in a sequence when the sequences are subjected to optimal alignment, i.e., the sequences are aligned to obtain the highest percent identity.

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

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

In some embodiments, the antibody or antigen binding fragment neutralizes hepatitis b virus and hepatitis d virus infection. In some embodiments, the antibody or antigen binding fragment reduces viral infectivity of hepatitis b virus and hepatitis d virus.

To study and quantify viral infectivity (or "neutralization") in the laboratory, a standard "neutralization test" may be utilized. For neutralization assays, animal viruses are typically transmitted in cells and/or cell lines. A neutralization assay may be used in which cultured cells are incubated with a fixed amount of HBV or HDV in the presence (or absence) of the antibody (or antigen binding fragment) to be tested. In such assays, the level of hepatitis b surface antigen (HBsAg) or hepatitis b e antigen (HBeAg) secreted into the cell culture supernatant may be used and/or HBcAg staining may be assessed to provide a reading. For example, delta antigen immunofluorescent staining can be assessed for HDV.

In a particular embodiment of the HBV neutralization assay, cultured cells (e.g., hepavg cells, such as differentiated hepavg cells) are incubated with a fixed amount of HBV in the presence or absence of the antibody to be tested. In such embodiments, the incubation may be performed at, for example, 37 ℃ for 16 hours. The incubation may be performed in a medium (e.g., medium supplemented with 4% peg 8000). After incubation, the cells may be washed and further cultured. To measure viral infectivity, the levels of hepatitis b surface antigen (HBsAg) and hepatitis b e antigen (HBeAg) secreted into the culture supernatant can be determined by enzyme-linked immunosorbent assay (ELISA), for example from day 7 to day 11 post infection. In addition, HBcAg staining can be assessed in immunofluorescence assays. In one example of an HDV neutralization assay, substantially the same assay as HBV can be used, except that serum from an HDV vector can be used as an HDV infection inoculum (instead of HBV) on differentiated heparog cells. For detection, delta antigen immunofluorescent staining can be used as a reading.

Embodiments of the antibodies of the disclosure have high neutralizing potency (e.g., in vitro). For example, in certain embodiments, the concentration of antibody as described herein required to 50% neutralize Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV) is, for example, about 10 μg/ml or less. In other embodiments, the concentration of antibody required to 50% neutralize HBV and HDV is about 5 μg/ml. In other embodiments, the concentration of antibody as described herein required to 50% neutralize HBV and HDV is about 1 μg/ml. In other embodiments, the concentration of antibody required to 50% neutralize HBV and HDV is about 750ng/ml. In still further embodiments, the concentration of antibody as described herein required to 50% neutralize HBV and HDV (e.g., in vitro) is 500ng/ml or less. In such embodiments, the concentration of antibody as described herein required for 50% neutralization of HBV and HDV may be selected from 450ng/ml or less, 400ng/ml or less, 350ng/ml or less, 300ng/ml or less, 250ng/ml or less, 200ng/ml or less, 175ng/ml or less, 150ng/ml or less, 125ng/ml or less, 100ng/ml or less, 90ng/ml or less, 80ng/ml or less, 70ng/ml or less, 60ng/ml or less, or 50ng/ml or less.

Antibodies or antigen binding fragments according to the present disclosure that can neutralize both HBV and HDV are useful for the prevention and treatment of hepatitis b and hepatitis d. HDV infection typically occurs simultaneously with or after HBV infection (e.g., inoculation of HDV in the absence of HBV does not cause hepatitis d, as HDV requires HBV to support its own replication), and hepatitis d is typically observed in chronic HBV carriers.

Examples of the disclosed antibodies promote clearance of HBsAg and HBV. In particular embodiments, the antibodies promote clearance of HBV and hepatitis b virus subviral particles (SVP). HBsAg or subviral particle clearance can be assessed by measuring, for example, the level of HBsAg in a blood sample (e.g., a blood sample from a hepatitis b patient). Similarly, HBV clearance can be assessed by measuring HBV levels in, for example, a blood sample (e.g., a blood sample from a hepatitis b patient).

In the serum of HBV infected patients, in addition to infectious particles (HBV), there is typically an excess (typically 1,000 to 100,000 times) of empty subviral particles (SVP) consisting of HBV envelope protein (HBsAg) alone in the form of relatively small spheres and variable length filaments. 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 case of infectivity of serum including HBV, since infectivity depends not only on the number of viruses but also on the number of SVPs (Bruns M.et al 1998 J.Virol.72 (2): 1462-1468). In addition, excess subviral particles can act as baits by absorbing neutralizing antibodies and thus delay the clearance of infection. In some cases, achieving the disappearance of hepatitis b surface antigen (HBsAg) is considered the endpoint of treatment and the closest outcome of curing Chronic Hepatitis B (CHB).

Embodiments of the antibodies of the disclosure can promote clearance of HbsAg. In certain embodiments, the antibody promotes clearance of hepatitis b virus subviral particles. In some embodiments, the antibodies (e.g., in the presently disclosed pharmaceutical compositions) can be used to treat chronic hepatitis b.

In any of the presently disclosed embodiments, the antibody or antigen binding fragment binds to an HBsAg selected from HBsAg genotypes A, B, C, D, E, F, G, H, I and J or any combination thereof.

In certain embodiments, an antibody or antigen binding fragment of the disclosure binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I and J. Examples of different HBsAg genotypes include the following: gene bank accession number J02203 (HBV-D, ayw 3); gene bank accession number FJ899792.1 (HBV-D, adw 2); genbank accession number AM282986 (HBV-Sup>A); gene bank accession number D23678 (HBV-B1 Japan); gene bank accession number AB117758 (HBV-C1 Cambodia); gene bank accession number AB205192 (HBV-E ganin); gene bank accession number X69798 (HBV-F4 brazil); gene bank accession number AF160501 (HBV-G united states); gene bank accession number AY090454 (HBV-H Niglauber melon); genbank accession number AF241409 (HBV-I vietnam); gene bank accession number AB486012 (HBV-J Barroa). Exemplary amino acid sequences of the antigenic loop regions of the S domain of HBsAg of different genotypes are described herein (e.g., SEQ ID NOs: 5-15).

In some embodiments, the antibody or antigen binding fragment binds to at least 6 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I and J. In certain embodiments, the antibody or antigen binding fragment binds to at least 8 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I and J. In some embodiments, the antibody or antigen binding fragment binds to all 10 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I and J. HBV can differentiate into several genotypes depending on the genomic sequence. To date, eight well-known genotypes (A-H) of HBV genomes have been defined. In addition, two other genotypes I and J have been identified (Sunbul M.,2014, J. Wolv. Gastroenterology (World J Gastroenterol) 20 (18): 5427-5434. Genotypes are known to affect disease progression, and differences in antiviral treatment have been determined for the different genotypes.

In some embodiments, an antibody or antigen binding fragment according to the present disclosure binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mutants of HBsAg mutants having a mutation in the antigenic loop region, wherein such mutants are selected from one or more of the following: HBsAg Y100C/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 natural mutants based on the S domain of the HBsAg genotype D, genbank accession number FJ899792 (SEQ ID NO: 4). The mutated amino acid residues in each of the mutants indicated herein are indicated by the name. SEQ ID NO. 4:

MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGN CTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI

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

The amino acid sequences of the antigenic loop regions of the S domain of the HBsAg of the different mutants are shown in SEQ ID NOS.16-33.

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

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

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

In some embodiments, the antibody or antigen binding fragment binds to an epitope comprising at least one, at least two, at least three, or at least four amino acids of the HbsAg antigen loop region. In certain embodiments, the antibody or antigen binding fragment binds to at least two amino acids selected from amino acids 115-133 of the S domain of HbsAg, amino acids 120-133 of the S domain of HbsAg, or amino acids 120-130 of the S domain of HbsAg. In certain embodiments, the antibody or antigen binding fragment binds to at least three amino acids selected from amino acids 115-133 of the S domain of HbsAg, amino acids 120-133 of the S domain of HbsAg, or amino acids 120-130 of the S domain of HbsAg. In some embodiments, the antibody or antigen binding fragment binds to at least four amino acids selected from amino acids 115-133 of the S domain of HbsAg, amino acids 120-133 of the S domain of HbsAg, or amino acids 120-130 of the S domain of HbsAg. As used herein, the positions of amino acids (e.g., 115-133, 120-130) refer 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 generally 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 an antibody or antigen binding fragment thereof binds can be linear (continuous) or conformational (discontinuous). A linear or sequential epitope is an epitope recognized by an antibody according to its linear sequence of amino acids or primary structure. Conformational epitopes can be recognized by three-dimensional shapes and protein structures. Thus, if the epitope is a linear epitope and comprises more than one amino acid located at amino acid positions 115-133 or amino acid positions 120-133 of the S domain selected from HBsAg, the amino acids comprised by the epitope may be located adjacent to the primary structure (e.g. as consecutive amino acids in the amino acid sequence). In the case of conformational epitopes (3D structures), the amino acid sequence typically forms a 3D structure as an epitope, and thus the amino acids forming the epitope may or may not be located adjacent to the primary structure (i.e., may or may not be consecutive amino acids in the amino acid sequence).

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

Amino acids that are not located in adjacent positions of the primary structure (i.e., epitope-forming amino acids) to which the presently disclosed antibodies or antigen-binding fragments bind are in some cases separated by one or more amino acids to which the antibodies or antigen-binding fragments do 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 amino acids contained in an epitope that are not located in adjacent positions.

In certain embodiments, the antibody or antigen binding fragment binds to an epitope comprising at least amino acids P120, C121, R122, and C124 of the S domain of HBsAg. In other embodiments, the antibodies or antigen binding fragments of the disclosure bind to an epitope comprising the amino acid sequence according to SEQ ID No. 88:

PCRXC

wherein X is any amino acid or is not an 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, the antibodies or antigen binding fragments of the disclosure bind to an epitope comprising the amino acid sequence according to SEQ ID No. 80:

TGPCRTC

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

In other embodiments, the antibodies or antigen binding fragments of the disclosure bind to an epitope comprising the amino acid sequence according to SEQ ID NO: 85:

STTSTGPCRTC

or an amino acid sequence sharing at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO. 85.

In certain embodiments, an antibody or antigen binding fragment of the 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:81)。

in other embodiments, the antibodies or antigen binding fragments of the present disclosure bind to an epitope comprising the amino acid sequence according to SEQ ID NO. 80 and the amino acid sequence according to SEQ ID NO. 81.

In other embodiments, the antibodies or antigen binding fragments of the present disclosure bind to an epitope comprising the amino acid sequence according to SEQ ID NO:85 and/or the amino acid sequence according to SEQ ID NO: 87.

As described above, the epitopes bound by the antibodies or antigen binding fragments of the present disclosure may be linear (continuous) or conformational (discontinuous). In some embodiments, the antibodies or antigen binding fragments of the present disclosure bind to conformational epitopes, and in certain such embodiments, conformational epitopes are present only under non-reducing conditions.

In certain embodiments, the antibodies or antigen binding fragments of the disclosure bind to a linear epitope. In certain such embodiments, the linear epitope is present under both non-reducing and reducing conditions.

In a particular embodiment, the antibody or antigen binding fragment of the disclosure binds to an epitope in the antigenic loop of HBsAg formed from the amino acid sequence according to SEQ ID NO: 1:

X ₁ X ₂ X ₃ TC X ₄ X ₅ X ₆ AX ₇ G

wherein X is ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ 、X ₆ And X ₇ Any amino acid (SEQ ID NO: 1) may be used.

In some embodiments, X ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ 、X ₆ And X ₇ Is an amino acid which is conservatively substituted compared to amino acids 120 to 130 of SEQ ID NO. 3. In some embodiments, X ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ 、X ₆ And X ₇ Is an amino acid which is conservatively substituted compared to amino acids 20 to 30 of any one of SEQ ID NOs 5 to 33.

In a specific embodiment, SEQ ID NO:1X ₁ X of (2) ₁ Is a small amino acid. As used herein, "small" amino acid refers to any amino acid selected from the group consisting of: alanine, aspartic acid, asparagine, cysteine, glycine, proline, serine, threonine, and valine. In certain such embodiments, X ₁ Is proline, serine or threonine.

In certain embodiments, SEQ ID NO:1X ₂ X of (2) ₂ Is a small amino acid. In certain embodiments, X ₂ May be selected from cysteine or threonine.

In some embodiments, X of SEQ ID NO. 1 ₃ Is a charged amino acid or an aliphatic amino acid. As used herein, "charged" amino acid refers to any amino acid selected from the group consisting of: arginine, lysine, aspartic acid, glutamic acid, and histidine. As used herein, "aliphatic" amino acid refers to any amino acid selected from the group consisting of: alanine, glycine, isoleucine, leucine and valine. In certain embodiments, X ₃ Selected from arginine, lysine, aspartic acid or isoleucine.

In some embodiments, X of SEQ ID NO. 1 ₄ Are small amino acids and/or hydrophobic amino acids. As used herein, "hydrophobic" amino acid refers to any amino acid selected from the group consisting of: alanine, isoleucine, leucine, phenylalanine, valine, tryptophan, tyrosine, methionine, proline and glycine. In certain embodiments, X ₄ Selected from methionine or threineAnd (3) acid.

In some embodiments, SEQ ID NO:1X ₅ X of (2) ₅ Are small amino acids and/or hydrophobic amino acids. In certain embodiments, X ₅ Selected from threonine, alanine or isoleucine.

In some embodiments, SEQ ID NO:1X ₆ X of (2) ₆ Are small amino acids and/or hydrophobic amino acids. In certain embodiments, X ₆ Selected from threonine, proline or leucine.

In some embodiments, X of SEQ ID NO. 1 ₇ Is a polar amino acid or an aliphatic amino acid. As used herein, "polar" amino acid refers to any amino acid selected from the group consisting of: aspartic acid, asparagine, arginine, glutamic acid, histidine, lysine, glutamine, tryptophan, tyrosine, serine, and threonine. In certain such embodiments, X ₇ 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 from the amino acid sequence according to SEQ ID No. 2:

X ₁ X ₂ X ₃ TC X ₄ X ₅ X ₆ AX ₇ G

wherein X is ₁ Is P, T or S,

X ₂ is C or S;

X ₃ is R, K, D or I, which is a compound,

X ₄ is either M or T, and is selected from the group consisting of,

X ₅ is T, A or I, which is a compound,

X ₆ t, P or L, and

X ₇ is Q, H or L

(SEQ ID NO:2)。

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

In particular embodiments, antibodies or antigen binding fragments according to the present disclosure bind to an epitope in the antigenic loop of HBsAg formed from one or more, two or more, three or more, or four or more amino acids selected from the amino acid sequences of SEQ ID NOs 5-33 shown in Table 3 below.

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

Table 3: exemplary amino acid sequences of the antigenic loop region of the S domain of HBsAg of different genotypes and mutants used in the present application (residues 101-172 of the S domain of HBsAg-except SEQ ID NO:16, which refers to residues 100-172 of the S domain of HBsAg so as to include the relevant mutation).

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Thus, in certain aspects, the present disclosure provides an isolated antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure, comprising: (i) A heavy chain variable region comprising at least 90% identity to the amino acid sequence according to SEQ ID NO 41 or 67 (V _H ) The method comprises the steps of carrying out a first treatment on the surface of the (ii)A light chain variable region (V) comprising at least 90% identity to the amino acid sequence according to any one of SEQ ID NOs 42, 59, 65, 89, 90 or 110 to 120 _L ) Provided that the amino acid at position 40 of VL according to IMGT numbering is not cysteine, wherein the antibody or antigen binding fragment thereof binds to the antigenic loop region of HBsAg and neutralizes hepatitis b virus and hepatitis delta virus infection.

In further embodiments, (i) the V _H Comprises at least 95% identity to the amino acid sequence according to SEQ ID NO. 41 or 67; and/or (ii) the V _L Comprises at least 95% identity to the amino acid sequence according to any one of SEQ ID NOs 42, 59, 65, 89, 90 and 110 to 120.

In certain embodiments, V _L The amino acid at position 40 of (2) is alanine. In other embodiments, V _L The amino acid at position 40 of (2) is serine. In still other embodiments, V _L The amino acid at position 40 of (2) is glycine.

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

In some embodiments, antibodies or antigen binding fragments V suitable for use in the pharmaceutical compositions and methods of the present disclosure _L Comprising or consisting of the amino acid sequence according to SEQ ID NO. 89. In some embodiments, antibodies or antigen binding fragments V suitable for use in the pharmaceutical compositions and methods of the present disclosure _L Comprising or consisting of the amino acid sequence according to SEQ ID NO. 90. In other embodiments, antibodies or antigen-binding fragments suitable for use in the pharmaceutical compositions and methods of the present disclosureV _L Comprising or consisting of an amino acid sequence according to any of SEQ ID NOS.109 to 120. In certain embodiments, V _H Comprising or consisting of the amino acid sequence according to SEQ ID NO. 41. In certain embodiments, V _H Comprising or consisting of the amino acid sequence according to SEQ ID NO. 67.

In a particular embodiment, V _H Comprising or consisting of the amino acid sequence according to SEQ ID NO. 41 and V _L Comprising or consisting of the amino acid sequence according to SEQ ID NO. 89. In other embodiments, V _H Comprising or consisting of the amino acid sequence according to SEQ ID NO. 41 and V _L Comprising or consisting of the amino acid sequence according to SEQ ID NO. 90. In certain embodiments, V _H Comprising or consisting of the amino acid sequence according to SEQ ID NO. 41 and V _L Comprising or consisting of an amino acid sequence according to any of SEQ ID NOS.109 to 120. In other embodiments, V _H Comprising or consisting of the amino acid sequence according to SEQ ID NO. 67 and V _L Comprising or consisting of an amino acid sequence according to any of SEQ ID NOS.109 to 120.

In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure, comprising: (i) A heavy chain variable region comprising at least 90% identity to the amino acid sequence according to SEQ ID NO 95 (V _H ) The method comprises the steps of carrying out a first treatment on the surface of the And (ii) a light chain variable region (V) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO. 96 _L ) Wherein the antibody or antigen binding fragment thereof binds to the antigenic loop region of HBsAg and neutralizes hepatitis b virus and hepatitis d virus infections.

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

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

Fc portion

In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise an Fc portion. In certain embodiments, the Fc portion may be derived from a human source, such as from human IgG1, igG2, igG3, and/or IgG4. In particular embodiments, the antibody or antigen binding fragment may comprise an Fc portion derived from human IgG 1.

As used herein, the term "Fc portion" refers to a sequence comprising or derived from a portion of an immunoglobulin heavy chain that begins at the hinge region upstream of the papain cleavage site (e.g., residue 216 in natural IgG, considering the first residue of the heavy chain constant region as 114) and ends at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc portion may be a complete Fc portion or a portion (e.g., domain) thereof. In certain embodiments, the intact Fc portion comprises a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). Additional lysine residues (K) are sometimes present at the extreme C-terminus of the Fc portion, but are typically cleaved from the mature antibody.

Amino acid positions within the Fc portion herein are numbered according to the EU numbering system of Kabat, see, e.g., kabat et al, "protein sequence of immunological interest (Sequences of Proteins of Immunological Interest)", U.S. health and human service (U.S. Dept. Health and Human Services), 1983 and 1987. The amino acid positions of the Fc portion may also be numbered according to the IMGT numbering system (including the unique numbering of the C domain and the exon numbering) and the Kabat numbering system.

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

The Fc portion of the present disclosure may be modified such that it differs in amino acid sequence from the complete Fc portion of a naturally occurring immunoglobulin molecule while retaining or enhancing at least one desired function conferred by the naturally occurring Fc portion. Such functions include, for example, fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein a binding, protein G binding, and complement binding. Portions of naturally occurring Fc portions associated with such functions have been described in the art.

For example, to activate the complement cascade, when immunoglobulin molecules are attached to an antigen target, the C1q protein complex may bind to at least two IgG1 molecules or one IgM molecule (Ward, e.s. and Ghetie, v., "immunology (ter.immunol.))" 2 (1995) 77-94). Burton, D.R. (molecular immunology) 22 (1985) 161-206) describes the involvement of the heavy chain region, including amino acid residues 318 to 337, in complement fixation. Duncan, A.R. and Winter, G. ("Nature 332 (1988) 738-740) reported that Glu318, lys320 and Lys322 form binding sites for C1q using site-directed mutagenesis. The role of Glu318, lys320 and Lys322 residues in C1q binding was demonstrated by the ability of short synthetic peptides containing these residues to inhibit complement mediated cleavage.

For example, fcR binding may be mediated through the interaction of the Fc portion (of an antibody) with an Fc receptor (FcR), which is a specialized cell surface receptor on cells including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and are shown to mediate clearance of antibody-coated pathogens via phagocytosis of immune complexes as well as lysis of both erythrocytes coated with the corresponding antibodies and various other cellular targets (e.g., tumor cells) by antibody-dependent cell-mediated cytotoxicity (ADCC; van de Winkel, j.g. and Anderson, c.l., journal of white blood cell biology (j.leukoc.biol.) 49 (1991) 511-524). FcR is defined by its specificity for immunoglobulin classes; the Fc receptor of IgG antibodies is called fcγr, the Fc receptor of IgE antibodies is called fcεr, the Fc receptor of IgA antibodies is called fcαr, etc., and the neonatal Fc receptor is called FcRn. Such as Ravetch, j.v. and Kinet, j.p. "immunological annual review (annu. Rev. Immunol.)" 9 (1991) 457-492; capel, P.J. et al, immunization methods (Immunomethods) 4 (1994) 25-34; de Haas, M.et al, (J Lab. Clin. Med.) 126 (1995) 330-341; and Gessner, J.E. et al, annual book of hematology (Ann. Hematol.) 76 (1998) 231-248.

Crosslinking of the Fc domain of natural IgG antibodies (fcγr) to the receptor triggers a variety of effector functions including phagocytosis, antibody dependent cellular cytotoxicity and inflammatory mediator release, immune complex clearance and modulation of antibody production. The Fc portion that provides crosslinking of the receptor (e.g., fcγr) is contemplated herein. In humans, three classes of fcγr have been characterized to date: (i) Fcyri (CD 64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) Fcγrii (CD 32), which binds complex IgG with medium to low affinity, is widely expressed (particularly on leukocytes), is considered a core participant in antibody-mediated immunity, and can be divided into fcγriia, fcγriib and fcγriic, functions differently in the immune system, but binds IgG-Fc with similar low affinity, and the extracellular domains of these receptors are highly homologous; and (iii) fcγriii (CD 16), which binds IgG with medium to low affinity, and has been found to take two forms: fcγriiia, which has been found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is thought to mediate ADCC; and fcγriiib highly expressed on neutrophils.

Fcγriia is present on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. Fcyriib appears to play a role in the inhibition process and is found on B cells, macrophages, as well as mast cells and eosinophils. Importantly, 75% of all fcyriib has been shown to be present in the liver (Ganesan, l.p. et al 2012: "fcyriib clearance of small immune complexes on the liver sinus endothelium (fcγ RIIb on liver sinusoidal endothelium clears small immune complexes)", journal of immunology 189:4981-4988). Fcyriib is abundantly expressed on the liver sinus endothelium (called LSEC) and in Kupffer cells in the liver, and LSEC is the major site of clearance of small immune complexes (Ganesan, l.p. et al 2012: fcyriib clearance of small immune complexes on the liver sinus endothelium, journal of immunology 189:4981-4988).

In some embodiments, the antibodies and antigen binding fragments thereof disclosed herein comprise an Fc portion, particularly an Fc region, for binding fcyriib, e.g., an IgG-type antibody. In addition, the Fc portion can be engineered to enhance FcgammaRIIB binding by introducing mutations S267E and L328F, as described in Chu, S.Y. et al, 2008, inhibiting B cell receptor-mediated activation of primary human B cells by co-ligation of CD19 and FcgammaRIIB with Fc engineered antibodies (Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies), molecular immunology 45, 3926-3933. Thus, the clearance of immune complexes can be enhanced (Chu, S.et al 2014: accelerated clearance of IgE in chimpanzees is mediated by Xmab7195 (i.e., an Fc engineered antibody), with enhanced affinity for the inhibitory receptor FcgammaRIIB (Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, an Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor Fc. Gamma. RIIB) [ J. Critical care journal of medicine (Am J Respir Crit) ], international conference abstract of the United states society of thoracic sciences ]. In some embodiments, the antibodies or antigen binding fragments thereof of the present disclosure comprise an engineered Fc portion having mutations S267E and L328F, particularly as described in Chu, S.Y. et al, 2008, inhibiting B cell receptor-mediated activation of primary human B cells by co-ligation of CD19 and FcgammaRIIB with the Fc engineered antibodies, molecular immunology 45, 3926-3933.

On B cells fcyriib appears to act to inhibit further immunoglobulin production and isotype switching to e.g. IgE class. On macrophages, fcyriib is thought to inhibit phagocytosis mediated by fcyriia. On eosinophils and mast cells, form b can help inhibit activation of these cells by IgE binding to its independent receptor.

Regarding fcyri binding, modifications in native IgG of at least one of E233-G236, P238, D265, N297, a327, and P329 reduce binding to fcyri. The substitution of the IgG2 residues at positions 233-236 to the corresponding positions IgG1 and IgG4 reduces the binding of IgG1 and IgG4 to Fcgamma by 10 ³ Doubling and eliminating the response of human monocytes to antibody-sensitized erythrocytes (Armour, K.L. Et al, J. Immunol. Eur., J. Immunol.), 29 (1999) 2613-2624).

Regarding fcyrii binding, reduced binding to fcyriia is found, for example, for IgG mutations of at least one of E233-G236, P238, D265, N297, a327, P329, D270, Q295, a327, R292, and K414.

Two allelic forms of human fcyriia are the "H131" variant that binds IgG1 Fc with high affinity and the "R131" variant that binds IgG1 Fc with low affinity. See, e.g., bruhns et al, blood (Blood) 113:3716-3725 (2009).

Regarding fcyriii binding, reduced binding to fcyriiia is found, for example for mutations in at least one of E233-G236, P238, D265, N297, a327, P329, D270, Q295, a327, S239, E269, E293, Y296, V303, a327, K338 and D376. Mapping of the binding sites for Fc receptors on human IgG1, the above-described mutation sites and methods for measuring binding to FcgammaRI and FcgammaRIIA are described in Shields, R.L. et al, J.Biol.chem.) (276 (2001) 6591-6604).

Two allelic forms of human fcyriiia are the "F158" variant that binds IgG1 Fc with low affinity and the "V158" variant that binds IgG1 Fc with high affinity. See, for example, bruhns et al, blood 113:3716-3725 (2009).

Regarding binding to fcγrii, the two regions of native IgG Fc appear to be involved in the interaction between fcγrii and IgG, i.e., (i) the lower hinge site of IgG Fc, specifically amino acid residues L, L, G, G (234-237, eu numbering); and (ii) adjacent regions of the CH2 domain of IgG Fc, in particular the loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example in the P331 region (Wines, B.D. et al, J.Immunol.2000; 164:5313-5318). Furthermore, fcγRI appears to bind to the same site on IgG Fc, while FcRn and protein A bind to different sites on IgG Fc, which appear to be located at the CH2-CH3 interface (Wines, B.D. et al, J.Immunol.2000; 164:5313-5318).

In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise an Fc portion comprising a mutation that increases the binding affinity of the Fc portion to (i.e., one or more) fcγ receptors, such as human fcγriia, human fcγriiia, or both (e.g., as compared to a reference Fc portion or an antibody comprising the same portion that does not comprise a mutation). See, e.g., delilo and Ravetch, cell 161 (5): 1035-1045 (2015) and Ahmed et al, J.Structure biol.) "194 (1): 78 (2016), the Fc mutations and techniques of which are incorporated herein by reference. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise an Fc portion comprising a mutation selected from the group consisting of: G236A; S239D; a330L; and I332E; or a combination comprising the same; such as S239D/I332E; S239D/A330L/I332E; G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as "GAALIE"); or G236A/S239D/A330L/I332E.

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

In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion comprising substitution mutation M428L/N434S. In some embodiments, the binding protein comprises an Fc portion comprising the substitution mutation G236A/A330L/I332E. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion comprising a G236A mutation, an a330L mutation, and an I332E mutation (GAALIE) and do not comprise an S239D mutation. In some embodiments, the Fc portion comprises Ser at position 239. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion comprising the following substitution mutations: M428L/N434S and G236A/A330L/I332E. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion comprising the following substitution mutations: M428L/N434S and G236A/S239D/A330L/I332E. In certain further embodiments, the Fc portion does not contain any substitution mutations other than M428L/N434S and G236A/S239D/A330L/I332E.

In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises: according to the CDRs and/or variable domains and/or heavy and/or light chains of any of the exemplary anti-HBV antibodies disclosed herein and/or in PCT publication No. WO 2017/060504 (including antibodies HBC34, HBC34v7, HBC34v23, HBC34v31, HBC34v32, HBC34v33, HBC34v34, HBC34v35 (including variants of the HBC antibodies disclosed herein that include substitution mutations at position 40 in the light chain (e.g., substitution of natural cysteine with alanine, serine, etc.)), and Fc portions that include a G236A mutation, an A330L mutation, and an I332E (GAALIE) mutation, wherein the Fc portions optionally further include an M428L/N434S (MLNS) mutation.

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

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

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise a heavy chain amino acid sequence according to SEQ ID NO 138 or 91.

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

In any presently disclosed embodiment, the binding proteins of the present disclosure include an Fc portion that includes a GAALIE mutation, and the binding proteins enhance binding to human fcyriia and/or human fcyriiia as compared to a reference polypeptide (i.e., a polypeptide that may be a binding protein that includes an Fc portion that does not include a GAALIE mutation).

In certain embodiments, the reference polypeptide comprises an Fc portion that is a wild-type Fc portion, or an Fc portion comprising one or more substitution mutations (or insertions or deletions) under conditions in which the substitution mutation is not a GAALIE. In certain embodiments, the antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise HBC34v35 antibodies having GAALIE and MLNS mutations, and the reference polypeptide is HBC34v35 (including wild-type Fc portions having the same isotype as the Fc portions of antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure). In certain embodiments, the reference polypeptide does not comprise substitution mutations known or believed to affect binding to human fcyriia and/or human fcyriiia.

Binding between polypeptides, such as between an Fc portion (or binding protein comprising the same) and a human fcγ receptor (e.g., human fcγriia, human fcγriiia, or human fcγriib) or a complement protein (e.g., C1 q) can be determined or detected using methods known in the art. For example, according to the manufacturer's instructions, can useRED96 (ForteBio Inc., forteBio, fremont, california) instruments perform a Biological Layer Interferometry (BLI) assay to determine the real-time association and dissociation between a first polypeptide of interest (e.g., HBC34V35 comprising a GAALIE mutation) and a second polypeptide of interest (e.g., fcgammaRIIA (H131), fcgammaRIIA (R131), fcgammaRIIA (F158), fcgammaRIIA (V158), or FcgammaRIIB) captured on a sensor substrate.

In certain embodiments, antibodies or their analogs suitable for use in the pharmaceutical compositions and methods of the present disclosureThe antigen binding fragment comprises an Fc portion comprising a GAALIE mutation, and the antibody or antigen binding fragment thereof enhances binding to human fcyriia (H131), human fcyriia (R131), human fcyriiia (F158), human fcyriiia (V158), or any combination thereof, as compared to a reference polypeptide comprising an Fc portion that does not comprise a GAALIE mutation. In certain embodiments, enhanced binding is determined by an increase in signal shift (e.g., one or more of: higher peak signal, higher association rate, slower dissociation rate, or larger area under the curve) relative to a reference binding protein in the BLI assay. In certain embodiments, the BLI test involves the use of Octet ^(R) RED96 (Fremont ForteBio, calif.) instrument. In further embodiments, the BLI assay comprises a labeled human fcγr captured on an anti-penta tag sensor and exposed to a binding protein. In some embodiments, the binding protein comprises an IgG Fab, and the BLI assay further comprises exposing the captured human fcγr to an antibody or antigen binding fragment in the presence of an anti-IgG Fab binding fragment to crosslink the binding protein by the Fab fragment.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion comprising a GAALIE mutation, and the antibodies or antigen-binding fragments thereof enhance binding to human fcyriia (H131), human fcyriia (R131), human fcyriiia (F158), and/or human fcyriiia (V158) compared to a reference polypeptide, wherein the enhanced binding comprises a signal shift (nm) in a BLI assay that is 1.5, 2, 2.5, 3, or more times the signal shift observed using the reference antibody.

In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion comprising a GAALIE mutation, and which antibodies or antigen-binding fragments thereof enhance binding to human fcyriia (H131), human fcyriia (R131), human fcyriiia (F158), and human fcyriiia (V158) as compared to a reference polypeptide.

In any of the presently disclosed embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure includes an Fc portion comprising a GAALIE mutation, and the antibody or antigen-binding fragment thereof reduces binding to human fcyriib as compared to a reference polypeptide. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure include an Fc portion comprising a GAALIE mutation and do not bind to human fcyriib, e.g., as determined by the absence of a statistically significant signal shift from baseline in the BLI assay.

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

In any presently disclosed embodiment, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure includes an Fc portion that includes a GAALIE mutation and activates human fcyriia, human fcyriiia, or both to a greater extent than a reference polypeptide (i.e., a polypeptide, which may be an antibody or antigen-binding fragment thereof, including an Fc portion that does not include a GAALIE mutation). In certain embodiments, the reference polypeptide comprises an Fc portion that is a wild-type Fc portion, or comprises one or more substitution mutations under conditions where the substitution mutation is not GAALIE. In certain embodiments, the antibody or antigen binding fragment thereof comprises an HBC34v35 antibody having a GAALIE mutation (and optionally other substitution mutations, such as MLNS), and the reference polypeptide is HBC34v35 having a wild-type Fc portion.

Activation of human fcγr can be determined or detected using methods known in the art. For example, a well-validated commercially available biological reporter assay involves incubating HBsAg-specific binding protein with recombinant HBsAg (Engerix B, gelanin SmithKline) in the presence of Jurkat effector cells (Promega; catalog number: G9798), stably expressing (i) Fc gamma R of interest and (ii) Fcgamma R at NFATFirefly luciferase reporter under the control of the response element. Binding of Fc to cell surface expressed fcγr drives NFAT-mediated expression of luciferase reporter. Then, according to the manufacturer's instructions, bio-Glo- ^TM The luciferase assay reagent (Promega corporation) measures luminescence with a photometer (e.g., bertonian corporation). Activation is expressed as the average of the Relative Luminescence Units (RLU) over the background by applying the following formula: (binding protein (e.g., mAb) concentration [ x]RLU under RLU-background RLU).

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

In any of the presently disclosed embodiments, the antibody or antigen binding fragment thereof includes an Fc portion comprising a GAALIE mutation that does not activate human fcyriib, as determined by the absence of a statistically significant and/or measurable RLU in a luminescent biological reporter assay as described above.

In any of the presently disclosed embodiments, the antibody or antigen binding fragment thereof comprises an Fc portion comprising a GAALIE mutation, and activates human Natural Killer (NK) cells to a greater extent than the reference polypeptide in the presence of HBsAg. In certain embodiments, NK cell activation is determined by CD107a expression (e.g., by flow cytometry). In certain embodiments, NK cells comprise cells containing the V158/V158 homozygous, F158/F158 homozygous, or V158/F158 heterozygous FcgammaRIIIa genotype.

It is understood that any antibody or antigen binding fragment thereof comprising an Fc portion comprising a GAALIE mutation according to the present disclosure may perform or possess any one or more of the features described herein; for example, enhanced binding to human fcyriia and/or human fcyriiia compared to a reference polypeptide; reduced binding to (and/or no binding to) human fcyriib compared to a reference polypeptide; reduced binding to human C1q (and/or no binding to human C1 q) compared to the reference polypeptide; activating fcyriia, human fcyriiia, or both to a greater extent than the reference polypeptide; non-activating human fcyriib; and/or activate human Natural Killer (NK) cells to a greater extent in the presence of HBsAg than a reference polypeptide (e.g., an antibody that is specific for HBsAg and that includes an Fc portion that does not contain a GAALIE mutation).

Alternatively or additionally, the Fc portion of an antibody or antigen binding fragment thereof of the present disclosure may include at least a portion known in the art to be required for protein a binding; and/or the Fc portion of the antibodies of the present disclosure includes at least a portion of an Fc molecule known in the art to be required for protein G binding. In some embodiments, the function retained comprises clearance of HBsAg and HBVg. Thus, in certain embodiments, the Fc portion comprises at least a portion of that required for fcγr binding as known in the art. As described above, the Fc portion may thus comprise at least: (i) The lower hinge site of native IgG Fc, specifically amino acid residue L, L, G, G (234-237, eu numbering); and (ii) adjacent regions of the CH2 domain of native IgG Fc, in particular the loops and chains of the upper CH2 domain adjacent to the lower hinge region, e.g. in the P331 region, e.g. in the upper CH2 domain of native IgG Fc around P331, a region of at least 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids, e.g. between amino acids 320 and 340 (EU numbering) of native IgG Fc.

In some embodiments, an antibody or antigen binding fragment thereof according to the present disclosure comprises an Fc region. As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed from two or more Fc portions of an antibody heavy chain. For example, the Fc region may be a monomeric or "single chain" Fc region (i.e., scFc region). The single chain Fc region comprises an Fc portion linked (e.g., encoded in a single contiguous nucleic acid sequence) within a single polypeptide chain. An exemplary scFc region is disclosed in WO 2008/143954 A2, and is incorporated herein by reference. The Fc region may be or comprise a dimeric Fc region. "dimeric Fc region" or "dcFc" refers to a dimer formed from the Fc portions of two separate immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc portions (e.g., an Fc region of a naturally occurring immunoglobulin) or a heterodimer of two different Fc portions (e.g., one Fc monomer of a dimeric Fc region comprises at least one amino acid modification (e.g., substitution, deletion, insertion, or chemical modification) that is not present in the other Fc monomer, or one Fc monomer may be truncated compared to the other).

Specific embodiments include those antibodies and antigen-binding fragments having a heavy chain according to SEQ ID NO. 91 or SEQ ID NO. 92 (e.g., VH-hinge-CH 1-CH2-CH 3), and those antibodies and antigen-binding fragments having a light chain according to SEQ ID NO. 93 or SEQ ID NO. 94 (i.e., VL-CL). In certain embodiments, the antibody or antigen binding fragment comprises a heavy chain according to SEQ ID NO. 91 and a light chain according to SEQ ID NO. 93. In other embodiments, the antibody or antigen binding fragment comprises a heavy chain according to SEQ ID NO. 92 and a light chain according to SEQ ID NO. 94. In other embodiments, the antibody or antigen binding fragment comprises a heavy chain according to SEQ ID NO. 91 and a light chain according to SEQ ID NO. 94. In other embodiments, the antibody or antigen binding fragment comprises a heavy chain according to SEQ ID NO. 92 and a light chain according to SEQ ID NO. 93. In some embodiments, the antibody or antigen binding fragment comprises or consists of a heavy chain according to SEQ ID NO. 129. In some embodiments, the antibody or antigen binding fragment comprises or consists of a heavy chain according to SEQ ID NO. 138. These sequences are listed in the sequence listing.

The Fc portion of the present disclosure may comprise Fc sequences or regions of the same or different classes and/or subclasses. For example, the Fc portion may be derived from an immunoglobulin (e.g., a human immunoglobulin) of the IgG1, igG2, igG3, or IgG4 subclass, or any combination thereof. In certain embodiments, the Fc portions of the Fc regions are of the same class and subclass. However, the Fc region (or one or more Fc portions of the Fc region) may also be chimeric, whereby the chimeric Fc region may comprise Fc portions derived from different immunoglobulin classes and/or subclasses. For example, at least two of the Fc portions of the dimeric or single-chain Fc region may be from different immunoglobulin classes and/or subclasses. In certain embodiments, the dimeric Fc region may include sequences from two or more different isoforms or subclasses; for example, SEED bodies ("Strand exchange engineering domains (strand-exchange engineered domains)"), see Davis et al, protein engineering and selection (Protein Eng. Des. Sel.) 23 (4): 195 (2010).

Additionally or alternatively, the chimeric Fc region may comprise one or more chimeric Fc portions. For example, a chimeric Fc region or portion may comprise one or more portions of an immunoglobulin derived from a first subclass (e.g., an IgG1, igG2, or IgG3 subclass), while the remainder of the Fc region or portion is a different subclass. For example, an Fc region or portion of an Fc polypeptide may comprise CH2 and/or CH3 domains derived from an immunoglobulin of a first subclass (e.g., an IgG1, igG2, or IgG4 subclass) and a hinge region of an immunoglobulin of a second subclass (e.g., an IgG3 subclass). For example, the Fc region or portion may comprise a hinge and/or CH2 domain derived from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and a CH3 domain derived from an immunoglobulin of a second subclass (e.g., an IgG1, igG2, or IgG3 subclass). For example, a chimeric Fc region may comprise an Fc portion (e.g., an intact Fc portion) of an immunoglobulin from a first subclass (e.g., an IgG4 subclass) and an Fc portion of an immunoglobulin from a second subclass (e.g., an IgG1, igG2, or IgG3 subclass). For example, the Fc region or portion may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or portion may comprise a CH1 domain and a CH2 domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. For example, the Fc region or portion may comprise a portion of a CH2 domain from a particular antibody subclass, e.g., EU positions 292-340 of the CH2 domain. For example, the Fc region or portion may comprise amino acids derived from CH2 of the IgG4 portion at positions 292-340 and the remainder of CH2 derived from the IgG1 portion (alternatively, 292-340 of CH2 may be derived from the IgG1 portion and the remainder of CH2 is derived from the IgG4 portion).

It is also understood that any antibody, antigen binding fragment, or Fc region or portion of the disclosure may belong to any allotype and/or haplotype. For example, human immunoglobulin G allotypes include those disclosed in Jefferis and LeFranc, mAb 1 (4): 1-7 (2009), including G1m (1 (a); 2 (x); 3 (f); and 17 (z)); G2m (23 (n)); G3m (21 (G1); 28 (G5); 11 (b 0); 5 (b 2); 13 (b 3); 14 (b 4); 10 (b 5); 15(s); 16 (t); 6 (c 3); 24 (c 5); 26 (u); and 27 (v)); A2m (1 & 2); and Km (1; and 3) and haplotypes and amino acid sequences produced and combinations thereof are incorporated herein by reference in certain embodiments, the antibodies, antigen binding fragments, or Fc regions or portions of the disclosure comprise IgG1 allotypes G1m17, k1.

Furthermore, the Fc region or portion may (additionally or alternatively) comprise, for example, a chimeric hinge region. For example, chimeric hinges can be derived, e.g., partially from an IgG1, igG2, or IgG4 molecule (e.g., upper and lower middle hinge sequences) and partially from an IgG3 molecule (e.g., middle hinge sequence). In another example, the Fc region or portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule. In another example, the chimeric hinge may comprise upper and lower hinge domains from an IgG4 molecule and an intermediate hinge domain from an IgG1 molecule. Such chimeric hinges can be obtained, for example, by introducing a proline substitution (Ser 228 Pro) at EU position 228 in the intermediate hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge can comprise amino acids at EU positions 233-236, from an IgG2 antibody and/or Ser228Pro mutation, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g., chimeric hinge of sequence ESKYGPPCPPCPAPPVAGP). Additional chimeric hinges that may be used in the Fc portion of antibodies according to the present disclosure are described in US2005/0163783 A1.

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

Nucleic acid molecules

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

Table 4 shows an exemplary V according to the present disclosure _H 、V _L CH, CL, HC, and LC coding nucleotide sequences:

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because of the redundancy of the genetic code, the present disclosure also encompasses sequence variants of these nucleic acid sequences and in particular such sequence variants encoding the same amino acid sequence.

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

In a specific embodiment, a nucleic acid molecule according to the present disclosure comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOS: 103-110 and 130-136.

In certain embodiments, the polynucleotide comprises V according to SEQ ID NO. 103 _H Coding nucleotide sequence and V according to SEQ ID NO. 105 _L A coding nucleotide sequence. In other embodiments, the polynucleotide comprises V according to SEQ ID NO. 103 _H Coding nucleotide sequence and V according to SEQ ID NO 104 _L A coding nucleotide sequence. In other embodiments, the polynucleotide comprises V according to SEQ ID NO. 108 _H Coding nucleotide sequence and V according to SEQ ID NO. 109 _L A coding nucleotide sequence.

Also provided herein are polynucleotides encoding antibodies or antigen binding fragments, wherein the polynucleotides comprise V according to SEQ ID NO 103 _H Coding nucleotide sequence and V according to SEQ ID NO. 110 _L A coding nucleotide sequence or consisting of, wherein the encoded antibody or antigen binding fragment binds to the antigenic loop region of HBsAg and neutralizes hepatitis b virus and hepatitis delta virus infections.

In any of the presently disclosed embodiments, the polynucleotide can comprise a CH 1-hinge-CH 2-CH3 encoding nucleotide sequence according to SEQ ID NO. 130 and/or comprise a HC (VH-CH 1-hinge-CH 3-CH 3) encoding nucleotide sequence according to SEQ ID NO. 131. In some embodiments, the polynucleotide comprises a CL coding nucleotide sequence according to SEQ ID NO. 132 and/or comprises an LC (VL-CL) coding nucleotide sequence according to SEQ ID NO. 133. In other embodiments, the polynucleotide comprises a CL coding nucleotide sequence according to SEQ ID NO. 134 and/or comprises an LC (VL-CL) coding nucleotide sequence according to SEQ ID NO. 135 or SEQ ID NO. 136.

Carrier body

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

The term "vector" refers to a construct comprising a nucleic acid molecule. Vectors in the context of the present disclosure are suitable for incorporation into or carrying the desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors and the like. A storage vector is a vector that allows for convenient storage of nucleic acid molecules. Thus, the vector may comprise sequences corresponding to, for example, a desired antibody or antibody fragment thereof according to the present description.

As used herein, an "expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include promoters that affect transcription (e.g., heterologous promoters), optional operator sequences that control such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that control transcription and translation termination. Any element of the expression vector that contributes to transcription of the nucleic acid molecule of interest may be heterologous to the vector. The vector may be a plasmid, phage particle, 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 cases, integrate into the genome itself. In this specification, "plasmid," "expression plasmid," "virus," and "vector" are generally used interchangeably.

Cloning vectors are typically vectors that contain cloning sites that can be used to incorporate a nucleic acid sequence into the vector. The cloning vector may be, for example, a plasmid vector or a phage vector.

The transfer vector may be a vector suitable for transferring a nucleic acid molecule into a cell or organism, such as a viral vector. In the context of the present disclosure, the vector may be, for example, an RNA vector or a DNA vector. The vector may be a DNA molecule. For example, vectors within the meaning of the application comprise cloning sites, selection markers (e.g., antibiotic resistance factors) and sequences suitable for vector multiplication (e.g., origins of replication). In some embodiments, the vector in the context of the present application is a plasmid vector. In certain such embodiments, the vector comprises a lentiviral vector or a retroviral vector.

Cells

In further aspects, the disclosure also provides cells (also referred to as "host cells") expressing antibodies, antigen binding fragments, or fusion proteins according to the disclosure; or a cell comprising a vector or polynucleotide according to 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 (E.coli). In some embodiments, the cell is a mammalian cell. In certain such embodiments, the cells are mammalian cell lines, such as CHO cells (e.g., DHFR-CHO cells (Urlaub et al, proc. Natl. Acad. Sci. USA (PNAS) 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, sp2/0 cells, NS0 cells, human liver cells, e.g., hepa RG cells, myeloma cells, or hybridoma cells, other examples of mammalian host cell lines include mouse support cells (e.g., TM4 cells), SV40 (COS-7) transformed monkey kidney CV1 lines, small hamster kidney cells (BHK), african green monkey kidney cells (VERO-76), monkey kidney cells (CV 1), human cervical cancer cells (HELA), human lung cells (W138), human liver cells (Hep G2), canine kidney cells (MDCK, buffalo rat liver cells (BRL 3A), mouse mammary tumors (MMT 060), MRC5 cells, and human mouse host cells (e.g., human tumor cells) and human host cell lines (e.g., human tumor cells) in the French 4, such as those described in Louser's, vol. 35, totsin the further examples include the methods of producing antibodies by Louser's host cell lines, such as Louser's systems, vol. 35, totso's, vol. 35, and Totso's cell lines.

In certain embodiments, the host cell is a prokaryotic cell, such as E.coli. Expression of peptides in prokaryotic cells, such as E.coli, is well established (see e.g.Pluckthun, A. (Bio/Technology) 9:545-551 (1991.) for example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. 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 for expressing the antibodies or antigen binding fragments thereof of the present disclosure are known in the art and include, for example, spodoptera frugiperda (Spodoptera frugipera) Sf9 cells, spodoptera frugiperda (Trichoplusia ni) BTI-TN5B1-4 cells, and Spodoptera frugiperda SfSWT01"Mimic ^TM "cells". See, for example, palmberger et al, J.Biotechnology journal (J.Biotechnol.) 153 (3-4): 160-166 (2011). A number of baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells.

Eukaryotic microorganisms (such as filamentous fungi or yeasts) are also suitable hosts for cloning or expressing the protein-encoding vectors and include fungal and yeast strains having a "humanized" glycosylation pathway, thereby producing antibodies having a partially or fully human glycosylation pattern. See Gerngross, nature Biotech 22:1409-1414 (2004); li et al, nature Biotechnology 24:210-215 (2006).

Plant cells may also be used as hosts for expression of the antibodies or antigen-binding fragments thereof of the present disclosure. For example, PLANTIBODIES ^TM Techniques (described, for example, in U.S. Pat. nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429) employ transgenic plants to produce antibodies.

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

In particular embodiments, cells may be transfected with vectors and expression vectors according to the present description. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, such as a eukaryotic cell. In the context of the present specification, the term "transfection" encompasses any method known to the skilled person for introducing a nucleic acid molecule into a cell, such as a eukaryotic cell, including 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.

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

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

Thus, the present disclosure also provides recombinant host cells that heterologously express the antibodies or antigen-binding fragments thereof of the present disclosure. For example, the cells may belong to a different species than the species from which the antibodies were obtained in whole or in part (e.g., CHO cells expressing human antibodies or engineered human antibodies). In some embodiments, the cell type of the host cell does not express the antibody or antigen binding fragment in nature. Furthermore, the host cell may confer post-translational modifications (PTM; e.g., glycosylation or fucosylation) to an antibody or antigen-binding fragment that is not present in the native state of the antibody or antigen-binding fragment (or in the native state of the parent antibody from which the antibody or antigen-binding fragment was engineered or derived). Such PTMs may cause functional differences (e.g., reduced immunogenicity). Thus, an antibody or antigen-binding fragment of the present disclosure produced by a host cell as disclosed herein may include one or more post-translational modifications that are different from the antibody (or parent antibody) in its native state (e.g., a human antibody produced by CHO cells may comprise more post-translational modifications that are different from antibodies isolated from humans and/or produced by native human B cells or plasma cells).

Optional additional features of antibodies or antigen binding fragments

Antibodies and antigen binding fragments of the present disclosure can be conjugated to, for example, a drug for delivery to a treatment site or to a detectable label to facilitate imaging of a site comprising a cell of interest. Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods of imaging using detectable labels. The labeled antibodies can be used in a variety of assays employing a variety of labels. Detection of antibody-antigen complex formation between an antibody (or antigen binding fragment or fusion protein) of the present disclosure and an epitope of interest on HBsAg, in particular on the antigenic loop region of HBsAg, can be facilitated by linking a detectable substance to the antibody. Suitable detection means include the use of labels such as radionuclides, enzymes, coenzymes, fluorescers, chemiluminescent agents, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic groups complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine (rhodomine), dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol (luminol); examples of suitable bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 125I, 131I, 35S or 3H. Such labeled reagents can be used in a variety of well known assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), fluorescent immunoassays, and the like. Labeled antibodies and antigen binding fragments according to the present disclosure may thus be used, for example, as in US 3,766,162; US 3,791,932; US 3,817,837; and such assays as described in US 4,233,402.

An antibody or antigen binding fragment thereof according to the present disclosure may be conjugated to a therapeutic moiety, such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope. Examples of radioisotopes include, but are not limited to, I-131, I-123, I-125, Y-90, re-188, re-186, at-211, cu-67, bi-212, bi-213, pd-109, tc-99, in-111, and the like. Such antibody conjugates can be used to modify a given biological response; the drug moiety should not be construed as limited to typical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moieties to antibodies are well known. See, for example, arnon et al (1985), "monoclonal antibodies for immune targeting drugs in cancer therapy (Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy)", "monoclonal antibodies and cancer therapy (Monoclonal Antibodies And Cancer Therapy)," Reisfeld et al (editorial) (Alan R.Lists, inc.), "pages 243-256; hellstrom et al (1987) "antibody for drug delivery (Antibodies For Drug Delivery)", "controlled drug delivery (Controlled Drug Delivery)", robinson et al (edit) (2 nd edition; marcel Dekker, inc.), "pages 623-653; antibody vector for cytotoxic agent in Thorpe (1985) "cancer therapy: review (Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review) "," monoclonal antibody 84: biological and clinical applications (Monoclonal Antibodies'84:Biological and Clinical Applications), picchera et al (editorial), pages 475-506 (Milan editrica Kurtis, 1985); "Analysis, results and future observations of therapeutic use of radiolabeled antibodies in cancer therapy (Analysis, results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy)", "monoclonal antibodies for cancer detection and therapy (Monoclonal Antibodies For Cancer Detection And Therapy), baldwin et al (editorial) pages 303-316; thorpe et al (1982), review of immunology (Immunol. Rev.) 62:119-158.

Alternatively, the antibody or antigen binding fragment thereof may be conjugated to a second antibody or antibody fragment thereof (or a second fusion protein) to form a heterologous conjugate as described in US 4,676,980. Furthermore, a linker may be used between the label and the described antibody, for example as described in US 4,831,175. Antibodies, antigen binding fragments, and fusion proteins may be directly labeled with radioiodine, indium, yttrium, or other radioactive particles known in the art, for example as described in US 5,595,721. The treatment may consist of a combination of treatments with conjugated and unconjugated antibodies and/or antigen-binding fragments administered simultaneously or subsequently, e.g. WO00/52031; described in WO 00/52473.

Antibodies and antigen binding fragments as described herein may also be attached to a solid support. In addition, the antibodies of the present disclosure, or functional antibody fragments thereof, may be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Examples of polymers and methods for attaching them to peptides are shown in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. In some embodiments, the polymer may be selected from polyoxyethylated polyols and polyethylene glycols (PEG). PEG is readily soluble in water at room temperature and has the general formula: r (O-CH) ₂ -CH ₂ ) _n O-R, wherein R may be hydrogen or a protecting group, such as an alkyl or alkanol group. In certain embodiments, the protecting group may have from 1 to 8 carbons. For example, the protecting group may be methyl. The symbol n is a positive integer. In one embodiment, n is between 1 and 1,000. In another embodiment, n is between 2 and 500. In some embodiments, the average molecular weight of the PEG is selected from between 1,000 and 40,000, between 2,000 and 20,000, and between 3,000 and 12,000. Furthermore, PEG may have at least one hydroxyl group, for example the PEG may have a terminal hydroxyl group. For example, it is a terminal hydroxyl group that is activated to react with a free amino group on the inhibitor. However, it is understood that the type and amount of reactive groups can be varied to achieve the covalently conjugated PEG/antibodies of the present description.

Water-soluble polyoxyethylated polyols may also be used in the context of antibodies and antigen-binding fragments described herein. It includes polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. In one embodiment, POGs are used. Without being bound by any theory, this branching is not necessarily considered a foreign agent in the body, because the glycerol backbone of polyoxyethylated glycerol is the same backbone in mono-, di-and triglycerides naturally occurring in e.g. animals and humans. The molecular weight of POG may be in the same range as that of PEG. Another drug delivery system that may be used to increase circulatory half-life is a liposome. Methods of preparing liposome delivery systems are known to those skilled in the art. Other drug delivery systems are known in the art and are described in, for example, the references Poznansky et al (1980) and Poznansky (1984).

Typically, the antibody or antigen binding fragment will be present in a composition that is substantially free of other polypeptides, e.g., wherein less than 90% (by weight), typically less than 60%, and more typically less than 50% of the composition is made up of other polypeptides.

The antibodies or antigen binding fragments of the disclosure may be immunogenic in a non-human (or heterologous) host (e.g., a mouse). In particular, the antibody, antigen binding fragment or fusion protein may have unique positions that are immunogenic in a non-human host but not in a human host. In particular, such molecules of the present disclosure for human use include molecules that cannot be readily isolated from a host (e.g., mouse, goat, rabbit, rat, non-primate mammal, etc.) and are generally not obtainable by humanization or from xenogeneic mice.

Production of antibodies, antigen binding fragments and fusion proteins

Antibodies and antigen binding fragments according to the present disclosure may be prepared by any method known in the art. For example, general methods for preparing monoclonal antibodies using hybridoma technology are well known (Kohler, G. And Milstein, C.,1975; kozbar et al, 1983). In one embodiment, an alternative EBV immortalization method described in WO2004/076677 is used.

In one embodiment, the antibodies are produced using the method described in WO 2004/076677. In such methods, antibody-producing B cells are transformed with EBV and polyclonal B cell activators. Additional stimulators of cell growth and differentiation may optionally be added during the transformation step to further enhance efficiency. These stimulators may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further increase the efficiency of immortalization, but its use is not required. 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 methods, the plasma cells are cultured in limited numbers or as single plasma cells in microwell plates. Antibodies can be isolated from plasma cell cultures. In addition, RNA may be extracted from plasma cell cultures and PCR may 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 expression vectors, which are then transfected into HEK293T cells or other host cells. Cloning of the nucleic acid in the expression vector, transfection of the host cell, culture of the transfected host cell, and isolation of the produced antibody may be performed using any method known to those of skill in the art.

If desired, the antibodies can be further purified using filtration, centrifugation, and various chromatographic methods (e.g., HPLC or affinity chromatography). Techniques for purifying antibodies (e.g., monoclonal antibodies), including techniques for producing pharmaceutical grade antibodies, are well known in the art.

Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibodies, antibody fragments or fusion proteins of the present specification. The desired DNA sequence may be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and Polymerase Chain Reaction (PCR) techniques may be suitably employed.

Any suitable host cell/vector system may be used to express the DNA sequences encoding the antibody or fusion protein molecules of the present disclosure, or fragments thereof. Bacteria (e.g., E.coli) and other microbial systems can be used in part to express antibody fragments, such as Fab and F (ab') 2 fragments, and in particular Fv fragments and single chain antibody fragments, e.g., single chain Fv. Eukaryotic (e.g., mammalian) host cell expression systems can be used to produce larger antibody molecules, including intact antibody molecules. Suitable mammalian host cells include, but are not limited to CHO, HEK293T, per.c6, NS0, myeloma or hybridoma cells.

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

An antibody molecule or antibody fragment may comprise only a heavy or light chain polypeptide, in which case only the heavy or light chain polypeptide coding sequence need be used to transfect a host cell. To produce a product 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, which comprises sequences encoding both the light chain polypeptide and the heavy chain polypeptide.

Alternatively, antibodies and antigen binding fragments according to the present disclosure may be produced by: (i) Expressing a nucleic acid sequence according to the present disclosure in a host cell, for example, by using a vector according to the present specification; and (ii) isolating the expressed desired product. Furthermore, the method may comprise (iii) purifying the isolated antibody or antigen binding fragment. Transformed B cells and cultured plasma cells can be screened for those that produce antibodies and antigen binding fragments of the desired specificity or function.

Screening may be performed by any immunoassay (e.g., ELISA), by staining of tissues or cells (including transfected cells), by neutralization assays, or by one of a variety of other methods known in the art for identifying the desired specificity or function. The assay may be selected based on simple recognition of one or more antigens, or may be selected based on a desired function, such as selecting neutralizing antibodies instead of just antigen binding antibodies, selecting antibodies that can alter the characteristics of the targeted cell (e.g., its signaling cascade, its shape, its growth rate, its ability to affect other cells, its response to other cells or other agents or changes in conditions, its differentiation state, etc.).

Individual transformed B cell clones can then be generated from the positive transformed B cell cultures. The cloning step for isolating individual clones from the positive cell mixture may be performed using limiting dilution, micromanipulation, single cell deposition by cell sorting, or another method known in the art.

Nucleic acids from 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 transfected host cells described herein may be used in a variety of ways, for example as a source of monoclonal antibodies, as a source of nucleic acids (DNA or mRNA) encoding monoclonal antibodies of interest, for research, and the like.

Pharmaceutical composition

The present disclosure provides pharmaceutical compositions comprising antibodies that neutralize hepatitis b virus and pharmaceutically acceptable aqueous vehicles. A vehicle is generally understood to be a material suitable for storing, transporting, formulating and/or administering a compound, such as a pharmaceutically active compound, in particular an antibody according to the present disclosure. For example, the vehicle may be a physiologically acceptable liquid suitable for storing, transporting and/or administering the pharmaceutically active compound, in particular an antibody according to the present disclosure.

The pharmaceutical compositions described herein are prepared for injection or infusion into a patient. In some embodiments, the composition may be prepared for intravenous ("IV" or "i.v."), intra-arterial, or intra-ventricular infusion. In other embodiments, the composition may be prepared for intravenous, intra-arterial, intraventricular, intramedullary, intraperitoneal, intrathecal, intraventricular, or injection. In particular embodiments, the compositions are prepared for subcutaneous ("SC" or "s.c.") injection. In particular embodiments, the compositions described herein are pharmaceutically acceptable sterile aqueous solutions that exhibit suitable pH, isotonicity and stability for administration to human subjects. Suitable aqueous vehicles for formulating the compositions described herein include water (e.g., sterile water, USP water for Injection) as well as isotonic vehicles, such as sodium chloride Injection, ringer's Injection, lactated Ringer's Injection.

The pharmaceutical composition according to the present description comprises an antibody selected from HBV neutralizing antibodies according to the present description. For example, in some embodiments, a pharmaceutical composition according to the present description comprises an (isolated) antibody comprising: (i) A heavy chain variable region (VH) comprising at least 90% identity to the amino acid sequence according to SEQ ID No. 41; and (ii) a light chain variable region (VL) comprising at least 90% identity to the amino acid sequence according to any one of SEQ ID NOs 59, 89 or 90, provided that the amino acid at position 40 of VL according to IMGT numbering is not a cysteine, wherein the antibody or antigen binding fragment thereof binds to the antigenic loop region of HBsAg and neutralizes hepatitis b and hepatitis d virus infections.

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

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

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

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

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

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

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

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

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

In certain embodiments, the antibody is a multispecific antibody. In certain embodiments, the antibody is a bispecific antibody.

In certain embodiments, the antibody comprises an Fc portion.

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

In certain embodiments, the Fc portion comprises a mutation that enhances binding to (e.g., human) fcγr (e.g., fcγriia, fcγriiia, or both) as compared to a reference Fc portion that does not comprise the mutation.

In certain embodiments, the Fc portion is or is derived from an IgG isotype.

In certain embodiments, the mutation that enhances binding to FcRn comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof.

In certain embodiments, the mutation that enhances binding to FcRn comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; or (viii) any combination of (i) to (vii).

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

In certain embodiments, the mutation that enhances binding to fcγr comprises S239D; I332E; a330L; G236A; or any combination thereof.

In certain embodiments, the mutation that enhances binding to fcγr comprises: (I) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E.

In certain embodiments, the mutation that enhances binding to fcγr comprises or consists of G236A/a330L/I332E. In some embodiments, the mutation that enhances binding to fcγr does not comprise S239D. In some embodiments, the Fc portion comprises a native Ser (S) at position 239.

In certain embodiments, the Fc portion comprises the following amino acid substitution mutations: M428L; N434S; G236A; a330L; and I332E. In certain additional embodiments, the Fc portion comprises no further mutations.

In certain embodiments, the antibody comprises a Heavy Chain (HC) amino acid sequence according to SEQ ID NO. 91.

In certain embodiments, the antibody comprises a Heavy Chain (HC) amino acid sequence according to SEQ ID NO. 92.

In certain embodiments, the antibody comprises a Light Chain (LC) amino acid sequence according to SEQ ID No. 93.

In certain embodiments, the antibody comprises a Light Chain (LC) amino acid sequence according to SEQ ID No. 94.

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

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

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

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

In some embodiments, a pharmaceutical composition according to the present description comprises an (isolated) antibody comprising: (i) A Heavy Chain (HC) comprising an amino acid sequence according to SEQ ID NO. 91; and (ii) a Light Chain (LC) comprising an amino acid sequence according to SEQ ID NO. 93, wherein the antibody binds to the antigenic loop region of HBsAg and neutralizes hepatitis B virus and hepatitis D virus infection.

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

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

In some embodiments, a pharmaceutical composition according to the present description comprises an antibody comprising: comprising a CDRH1 amino acid sequence according to SEQ ID NO. 34, a CDRH2 amino acid sequence according to SEQ ID NO. 35 or 66, a heavy chain variable region (V _H ) The method comprises the steps of carrying out a first treatment on the surface of the And a light chain variable region (V) comprising a CDRL1 amino acid sequence according to SEQ ID NO. 37, a CDRL2 amino acid sequence according to SEQ ID NO. 38 or 39 and a CDRL3 amino acid sequence according to SEQ ID NO. 58 or 40 _L ) The method comprises the steps of carrying out a first treatment on the surface of the And an Fc portion, wherein the Fc portion comprises G236A/A330L/I332E.

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

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

In certain embodiments, the V _H Comprising or consisting of an amino acid sequence according to any one of SEQ ID NOs 41 or 67, and said V _L Comprising the sequences according to SEQ ID NOS 42, 59, 65, 89, 90 and 111-120The amino acid sequence of any one of (c) or consists thereof.

In other embodiments, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof comprising: (i) Comprising a CDRH1 amino acid sequence according to SEQ ID NO. 97, a CDRH2 amino acid sequence according to SEQ ID NO. 98, a heavy chain variable region (V _H ) The method comprises the steps of carrying out a first treatment on the surface of the (ii) Comprising a CDRL1 amino acid sequence according to SEQ ID NO. 100, a CDRL2 amino acid sequence according to SEQ ID NO. 100 and a light chain variable region (V _L ) The method comprises the steps of carrying out a first treatment on the surface of the And (iii) an Fc portion, wherein the Fc portion comprises G236A/A330L/I332E.

In particular such embodiments of antibodies to pharmaceutical compositions, V _H Comprising or consisting of the amino acid sequence according to SEQ ID NO 95 and wherein V _L Comprising or consisting of the amino acid sequence according to SEQ ID NO. 96.

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

In some embodiments: the antibodies of the pharmaceutical composition enhance binding to human fcyriia, human fcyriiia, or both, wherein human fcyriia is optionally H131 or R131 and/or human fcyriiia is optionally F158 or V158, compared to a reference polypeptide comprising an Fc portion that does not comprise G236A/a 330L/I332E; antibodies of the pharmaceutical composition have reduced binding to human fcyriib compared to a reference polypeptide comprising an Fc portion that does not comprise G236A/a 330L/I332E; the antibodies of the pharmaceutical composition do not bind to human fcyriib; the antibody of the pharmaceutical composition has reduced binding to human C1q compared to a reference polypeptide comprising an Fc portion that does not comprise G236A/a 330L/I332E; the antibody of the pharmaceutical composition does not bind to human C1 q; the antibody of the pharmaceutical composition activates fcyriia, human fcyriiia, or both to a greater extent than a reference polypeptide comprising an Fc portion that does not comprise G236A/a330L/I332E, wherein the human fcyriia is optionally H131 or R131 and/or the human fcyriiia is optionally F158 or V158; the antibodies of the pharmaceutical composition do not activate human fcyriib; and/or in the presence of HBsAg, the antibody of the pharmaceutical composition activates human Natural Killer (NK) cells to a greater extent than a reference polypeptide comprising an Fc portion that does not comprise G236A/a 330L/I332E.

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

The composition according to the present description may comprise one or more of the following: buffers, surfactants or triblock copolymers, salts (e.g., sodium chloride), and stabilizers such as sugar alcohols, disaccharide or polysaccharide stabilizers and/or stabilizing amino acids (e.g., arginine and/or glycine). Furthermore, where necessary or desired, the compositions described herein may be formulated to additionally include one or more antioxidants (e.g., ascorbic acid, methionine, ethylenediamine tetraacetic acid (EDTA)).

The pharmaceutical compositions of the present disclosure exhibit and maintain a pH that maintains antibody viability while also being suitable for injection or infusion. The pH of the compositions described herein typically ranges from about 5.5 to about 8.5. In certain embodiments, the pH of the pharmaceutical composition ranges from about 5.5 to about 6.5, such as in the range of 5.5 to 6.5. In some embodiments, the pH of the pharmaceutical composition ranges from 5.8 to 6.2, for example about 6.0. In certain embodiments, the pH may be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the pH of the composition ranges from 6 to 8, for example about 7. In certain such embodiments, the pH may be about 6, such as 6.

The composition may include a buffer to achieve and maintain a desired pH. Buffers suitable for use in the compositions described herein include, for example, acetate, citrate, histidine, succinate, phosphate and hydroxymethyl aminomethane (Tris) buffers. In a particular embodiment, the composition comprises a buffer selected from the group consisting of histidine buffer and phosphate buffer. In particular embodiments, the composition exhibits a pH of 6 and comprises a histidine buffer. In such embodiments, histidine may be included in the composition at a concentration ranging from 10mM to 40mM (e.g., 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, or 40 mM). For example, in particular embodiments, compositions according to the present disclosure exhibit a pH of 6 and include histidine at a concentration selected from the group consisting of 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, and 40 mM.

The pharmaceutical compositions described herein may also include a surfactant or a triblock copolymer. Surfactants, sometimes referred to as "detergents," may serve one or more functions. For example, in aqueous antibody solutions, surfactants are used to maintain antibody function, aid in solubilization of antibodies or other excipients, and/or to control microbial growth. Surfactants that may be used in the compositions described herein include, for example, polysorbate 80 (tween 80), polysorbate 20 (tween 20). Additionally or alternatively, triblock copolymers such as poloxamer 188 may be used. In some embodiments, the composition comprises surfactant at a concentration ranging from 0.01% to 0.05% (w/v). In such embodiments, the surfactant may be selected from polysorbate 80 (tween 80), polysorbate 20 (tween 20), and poloxamer 188. In specific embodiments, the pharmaceutical compositions of the present description comprise polysorbate 80 (tween 80) at a concentration ranging from 0.01% to 0.05% (w/v). In other embodiments, the pharmaceutical compositions of the present description include polysorbate 80 (tween 80) at a concentration of 0.02% (w/v).

Where the composition according to the present disclosure includes a sugar alcohol, disaccharide or polysaccharide stabilizer, the stabilizer may be selected from, for example, mannitol, sorbitol, sucrose, trehalose and dextran 40. In a particular embodiment, the stabilizer is a disaccharide. In certain embodiments, the pharmaceutical composition comprises a disaccharide at a concentration selected from 4.0% to 10% (w/v). In certain such embodiments, the disaccharide is sucrose. In other embodiments, the pharmaceutical composition comprises a concentration selected from 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.9.9%, 9.0%, 7.1%, 9.9.0%, 7.9.1%, 7.9%, 7.9.9%, 7.9.2%, 7.9.9% and/or any two of these values within any two% of the ranges of these values (or any two w/w% or values). In other embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 7%, such as 7% (w/v).

In some embodiments, the composition is suitable for administration to a mammal, such as a human subject. In such embodiments, the composition is sterile and may be specially prepared to be pyrogen-free. Furthermore, the composition may be isotonic with respect to humans.

The compositions described herein may be prepared for direct administration to a subject (i.e., without reconstitution or mixing steps), or they may be prepared as lyophilized materials for reconstitution in an aqueous vehicle prior to injection or infusion into a patient. Pharmaceutical compositions according to the present disclosure may be provided for administration directly to a subject, for example in a pre-filled syringe, or in a vial, such as a glass vial. In some embodiments, the pharmaceutical compositions of the present disclosure are supplied in hermetically sealed containers. In some embodiments, the composition may be in the form of a kit designed such that the combined composition is reconstituted just prior to administration to a subject. For example, the lyophilized antibodies can be provided in kit form with sterile water or sterile buffer.

Administration of pharmaceutical compositions according to the present disclosure in methods and uses according to the present disclosure may be administered alone or in combination with adjuvants (also referred to herein as "additional active ingredients") that may be useful in the prevention and/or treatment of hepatitis b virus infection.

The present disclosure encompasses administration of a pharmaceutical composition according to the present disclosure, wherein the pharmaceutical composition is administered to a subject prior to, concurrently with, or after an adjuvant or another treatment regimen useful for treating and/or preventing a hepatitis b virus infection. The pharmaceutical compositions administered in combination with the adjuvant may be administered as the same or different compositions and by the same or different routes of administration. As used herein, expressions such as "combination therapy," "combination administration (combined administration)", "combination administration (administered in combination)", and the like refer to the combined action of a drug (which would be "combination" administration). For this purpose, the combined drugs are usually present at the site of action simultaneously and/or within overlapping time windows. It is also possible that the effect of one drug is sustained (even though the drug itself may no longer be present in a detectable manner) upon administration of another drug, so that the effects of the two drugs may interact. However, one drug is administered long before another drug (e.g., more than one month, two months, three months or more, or one year) so that when the other drug is administered it is no longer present at a detectable level (or its effect is not sustained), and is not generally considered to be "combined" administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a PD-1 inhibitor, e.g., a PD-1 specific antibody or binding fragment thereof, such as pidotizumab (pidilizumab), nivolumab (nivolumab), pembrolizumab (pembrolizumab), MEDI0680 (previously referred to as AMP-514), AMP-224, BMS-936558, or any combination thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, divaruzumab (MEDI 4736), atezolizumab (RG 7446), aviuzumab (avelumab, MSB 0010718C), MPDL3280A, or any combination thereof.

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

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with CTLA4 inhibitors. In particular embodiments, the pharmaceutical compositions of the present disclosure are used in combination with CTLA 4-specific antibodies or binding fragments thereof, such as ipilimumab (ipilimumab), tremelimumab (tremelimumab), CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoxazumab (MGA 271), 376.96, or both. The B7-H3 antibody binding fragment may be an scFv or fusion protein thereof, such as those described, for example, in Dangaj et al, cancer research (Cancer Res.) 73:4820,2013 and U.S. Pat. No. 9,574,000 and PCT patent publication Nos. WO/201640724A1 and WO 2013/025779A 1.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a CD244 inhibitor.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. anti-CD-160 antibodies are described, for example, in PCT publication No. WO 2010/084158.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a TIM3 inhibitor.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a Gal9 inhibitor.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an adenosine signaling inhibitor (e.g., decoy adenosine receptor).

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

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a KIR inhibitor, such as Li Lushan anti (BMS-986015).

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

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with IDO inhibitors, such as l-1-methyltryptophan, ai Kaduo stat (epacoadostat) (INCB 024360; liu et al, blood 115:3520-30,2010), ebselen (ebselen) (Terentis et al, biochemistry (biochem.)) 49:591-600,2010, indomethacin (Indoximod), NLG919 (Mautino et al, american cancer research institute 2013, 104th meeting (American Association for Cancer Research 104th Annual Meeting 2013), 4 months, 6 days-10 days), 1-methyl-tryptophan (1-MT) -tirapazamine, or any combination thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an arginase inhibitor, such as N (ω) -nitro-L-arginine methyl ester (L-NAME), N- ω -hydroxy-nor-L-arginine (nor-NOHA), L-NOHA, 2 (S) -amino-6-dihydroxyboronecaproic Acid (ABH), S- (2-dihydroxyboronoethyl) -L-cysteine (BEC), or any combination thereof.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a VISTA inhibitor, such as CA-170 (curie, lexington, mass.).

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with TIGIT inhibitors (e.g., COM902 (Compugen, toronto, ontario Canada)), CD155 inhibitors (e.g., COM701 (Compugen)), or both.

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of PVRIG, PVRL2, or both. anti-PVRIG antibodies are described, for example, in PCT publication No. WO 2016/134333. anti-PVRL 2 antibodies are described, for example, in PCT publication No. WO 2017/021526.

In certain embodiments, the compositions of the present disclosure are used in combination with an LAIR1 inhibitor.

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

In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an agent (i.e., agonist) that increases the activity of a stimulatory immune checkpoint molecule. For example, the compositions of the present disclosure may be used in combination with a CD137 (4-1 BB) agonist (e.g., wu Ruilu mAb (urelumab)), a CD134 (OX-40) agonist (e.g., MEDI6469, MEDI6383, or MEDI 0562), lenalidomide (lenalidomide), pomalidomide (pomalidomide), CD27 agonist (e.g., CDX-1127), a CD28 agonist (e.g., TGN1412, CD80, or CD 86), a CD40 agonist (e.g., CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (e.g., IL-2), a GITR agonist (e.g., humanized monoclonal antibody described in PCT patent publication No. WO 2016/054638), an ICOS (CD 278) agonist (e.g., GSK3359609, mAb 88.2, JTX-2011, ICOS 145-8, ICOS 314-8, or any combination thereof). In any of the embodiments disclosed herein, the method can comprise administering the pharmaceutical composition of the present disclosure alone or in any combination with one or more agonists, including any of the above, of the stimulatory immune checkpoint molecule.

In some embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI), an interferon (e.g., ifnα, ifnβ, or both), or any combination thereof. In some embodiments, the NRTI comprises one or more of the following: tenofovir (tenofovir); tenofovir disoproxil (tenofovir disoproxil) (e.g., tenofovir disoproxil fumarate (tenofovir disproxil fumarate)); tenofovir alafenamide (tenofovir alafenamide); entecavir (entetavir); lamivudine (Lamivudine); adefovir (Adefovir); adefovir dipivoxil (adefovir dipivoxil).

Medical treatment and use

In another aspect, the present disclosure provides the use of a pharmaceutical composition according to the present disclosure in the treatment of hepatitis b virus infection. In a particular embodiment, the present disclosure provides a method for treating a hepatitis b virus infection, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the present disclosure.

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

In certain embodiments, the subject is an adult. In certain embodiments, the subject has an age ranging from 18 years to 65 years. In certain embodiments, the subject has a weight of 40kg to 125kg. In certain embodiments, the subject has a Body Mass Index (BMI) of 18 to 35kg/m ² 。

In certain embodiments, the subject administered the pharmaceutical composition of the present disclosure suffers from chronic HBV infection; the HBV infection is for example defined by positive serum HBsAg, HBV DNA and/or HBeAg 2 times separated by at least 6 months.

In certain embodiments, the subject to whom the pharmaceutical composition of the present disclosure is administered does not suffer from cirrhosis. The absence of cirrhosis is determined by: fibrincan assessment (e.g., within 6 months prior to administration of the single dose of the pharmaceutical composition); or liver biopsy (e.g., within 12 months prior to administration of the single dose of the pharmaceutical composition), wherein preferably the absence of cirrhosis is determined by absence of Metavir F3 fibrosis or absence of F4 cirrhosis.

In certain embodiments, a subject administered a pharmaceutical composition of the present disclosure receives a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI) within, optionally, 120 days, further, optionally, 60 days, prior to administration of a single dose of the pharmaceutical composition. In other words, the subject has previously received NRTI, such as within 120 days or within 60 days of administration of the pharmaceutical composition.

In certain embodiments, the NRTI comprises one or more of the following: tenofovir; tenofovir disoproxil (e.g., tenofovir disoproxil fumarate); tenofovir alafenamide; entecavir; lamivudine; adefovir; adefovir dipivoxil.

In certain embodiments, the serum HBV DNA concentration of a subject administered a pharmaceutical composition of the present disclosure is less than 100IU/mL (e.g., 99, 98, 97, 96, 95, 90, 80, 70, 60, etc.) no more than 28 days prior to administration of a single dose.

In certain embodiments, the serum HBsAg concentration of a subject administered the pharmaceutical composition of the present disclosure is less than 3,000iu/mL prior to administration of a single dose. In certain embodiments, the serum HBsAg concentration of a subject administered the pharmaceutical composition of the present disclosure is less than 1,000iu/mL prior to administration of a single dose.

In certain embodiments, the serum HB surface antigen (HBsAg) concentration of a subject administered the pharmaceutical composition of the present disclosure is greater than or equal to 3,000iu/mL no more than 28 days prior to administration of the single dose. In certain embodiments, the serum HB surface antigen (HBsAg) concentration of a subject administered the pharmaceutical composition of the present disclosure is greater than or equal to 1,000iu/mL no more than 28 days prior to administration of the single dose. HBsAg concentrations can be determined using, for example, the yabach corporation (Abbott) ARCHITECT assay.

In certain embodiments, the subject administered the pharmaceutical composition of the present disclosure is negative for HB e-antigen (HBeAg) no more than 28 days prior to administration of the single dose.

In certain embodiments, the subject is negative for anti-HB antibodies no more than 28 days prior to administration of the single dose.

In certain embodiments: (i) The subject administered the pharmaceutical composition of the present disclosure does not suffer from fibrosis and/or does not suffer from cirrhosis; and/or (ii) alanine Aminotransferase (ALT) <2 x upper normal limit (ULN) in the (serum) of a subject administered the pharmaceutical composition of the present disclosure.

In certain embodiments, the methods comprise administering a single dose of the pharmaceutical compositions of the present disclosure.

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

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

In certain embodiments, the single dose of the pharmaceutical composition comprises 6mg, up to 10mg, up to 15mg, up to 18mg, up to 25mg, up to 30mg, up to 35mg, up to 40mg, up to 45mg, up to 50mg, up to 55mg, up to 60mg, up to 75mg, up to 90mg, up to 300mg, up to 900mg, or up to 3000mg of the antibody.

In certain embodiments, the single dose of the pharmaceutical composition comprises an amount of the antibody ranging from 1mg to 3000mg, or ranging from 5mg to 3000mg, or ranging from 6mg to 3000mg, or ranging from 10mg to 3000mg, or ranging from 25mg to 3000mg, or ranging from 30mg to 3000mg, or ranging from 50mg to 3000mg, or ranging from 60mg to 3000mg, or ranging from 75mg to 3000mg, or ranging from 90mg to 3000mg, or ranging from 100mg to 3000mg, or ranging from 150mg to 3000mg, or ranging from 200mg to 3000mg, or ranging from 300mg to 3000mg, or ranging from 400mg to 3000mg, or ranging from 500mg to 3000mg, or ranging from 600mg to 3000mg, or ranging from 750mg to 3000mg, or ranging from 900mg to 3000mg, or ranging from 1000mg to 3000mg, or ranging from 1500mg to 3000mg, or ranging from 2000mg to 3000 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1mg to 900mg, or ranging from 5mg to 900mg, or ranging from 6mg to 900mg, or ranging from 10mg to 900mg, or ranging from 25mg to 900mg, or ranging from 30mg to 900mg, or ranging from 50mg to 900mg, or ranging from 60mg to 900mg, or ranging from 75mg to 900mg, or ranging from 90mg to 900mg, or ranging from 100mg to 900mg, or ranging from 150mg to 900mg, or ranging from 200mg to 900mg, or ranging from 300mg to 900mg, or ranging from 500mg to 900mg, or ranging from 750mg to 900 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1mg to 500mg, or ranging from 5mg to 500mg, or ranging from 6mg to 500mg, or ranging from 10mg to 500mg, or ranging from 25mg to 500mg, or ranging from 30mg to 500mg, or ranging from 50mg to 500mg, or ranging from 60mg to 500mg, or ranging from 75mg to 500mg, or ranging from 90mg to 500mg, or ranging from 100mg to 500mg, or ranging from 150mg to 500mg, or ranging from 200mg to 500mg, or ranging from 300mg to 500 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1mg to 300mg, or ranging from 5mg to 300mg, or ranging from 6mg to 300mg, or ranging from 10mg to 300mg, or ranging from 25mg to 300mg, or ranging from 30mg to 300mg, or ranging from 50mg to 300mg, or ranging from 60mg to 300mg, or ranging from 75mg to 300mg, or ranging from 90mg to 300mg, or ranging from 100mg to 300mg, or ranging from 150mg to 300mg, or ranging from 200mg to 300 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1mg to 200mg, or ranging from 5mg to 200mg, or ranging from 6mg to 200mg, or ranging from 10mg to 200mg, or ranging from 25mg to 200mg, or ranging from 30mg to 200mg, or ranging from 50mg to 200mg, or ranging from 60mg to 200mg, or ranging from 75mg to 200mg, or ranging from 90mg to 200mg, or ranging from 100mg to 200mg, or ranging from 150mg to 200 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1mg to 100mg, or ranging from 5mg to 100mg, or ranging from 6mg to 100mg, or ranging from 10mg to 100mg, or ranging from 25mg to 100mg, or ranging from 30mg to 100mg, or ranging from 60mg to 100mg, or ranging from 75mg to 100mg, or ranging from 90mg to 100 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1mg to 25mg, or ranging from 5mg to 25mg, or ranging from 6mg to 25mg, or ranging from 10mg to 25mg, or ranging from 15mg to 25mg, or ranging from 20mg to 25 mg.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 1mg to 15mg, or ranging from 5mg to 15mg, or ranging from 6mg to 15mg, or ranging from 10mg to 15 mg.

In some embodiments of the present invention, in some embodiments, 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, 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, 910, 915, 920, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 995, or more mg of the antibody.

In certain embodiments, the single dose of the pharmaceutical composition comprises the antibody in an amount of less than 3000mg, less than 2500mg, less than 2000mg, less than 1500mg, less than 1000mg, less than 900mg, less than 500mg, less than 300mg, less than 200mg, less than 100mg, less than 90mg, less than 75mg, less than 50mg, less than 25mg, or less than 10mg, but greater than or equal to 1mg, greater than or equal to 2mg, greater than or equal to 3mg, greater than or equal to 4mg, greater than or equal to 5mg, or greater than or equal to 6 mg.

In a particular embodiment, the single dose of the pharmaceutical composition comprises about 75mg of the antibody. In other embodiments, the single dose of the pharmaceutical composition comprises about 90mg of the antibody. In still other embodiments, the single dose of the pharmaceutical composition comprises up to 300mg of the antibody. In yet other embodiments, the single dose of the pharmaceutical composition comprises up to 900mg of the antibody. In yet other embodiments, the single dose of the pharmaceutical composition comprises up to 3,000mg of the antibody.

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

In any of the methods described herein for treating a hepatitis b virus infection, the pharmaceutical composition may be administered by injection or infusion. When administered by infusion, the pharmaceutical composition may be administered by intravenous, intra-arterial, or intra-ventricular infusion, for example. When administered by injection, the pharmaceutical composition may be administered by, for example, intravenous, intra-arterial, intraventricular, intramedullary, intraperitoneal, intrathecal, intraventricular, or subcutaneous injection. In particular embodiments of the methods described herein, the pharmaceutical composition is administered by subcutaneous ("SC") injection or by intravenous ("IV") injection.

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

In certain embodiments, wherein the serum HBsAg of the subject is reduced by a factor >2 about 56 days after administration of the single dose compared to the serum HBsAg of the subject (e.g., the concentration of HBsAg in serum, e.g., as determined using the yaban ARCHITECT assay) from 0 to 28 days prior to administration of the single dose.

In certain embodiments, after administration of the single dose of the pharmaceutical composition (e.g., 56 days after administration of the single dose): (i) The subject has reduced or less severe intrahepatic transmission of HBV compared to a reference subject (e.g., a subject having a similar severity of HBV infection and the same gender, age, weight, and/or general health status as a subject receiving the pharmaceutical composition) receiving placebo or no HBV therapy within the same period of time; and/or (ii) the subject has an adaptive immune response against HBV, e.g., comprising a T cell response specific for HBV.

In some embodiments, the subject's serum HBsAg is reduced by 1.0log compared to (pre-administration) baseline after administration of the single dose ¹⁰ IU/mL、1.5log ¹⁰ IU/mL or more, wherein optionally, after administration of the single dose, the decrease lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or more. In some embodiments, the single dose comprises 6mg of antibody or more. In some embodiments, the subject's serum HBsAg is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 days after administration of the single dose, as compared to (pre) baseline Decrease in 53 days, 54 days, 55 days, 56 days or more.

In certain embodiments, the subject's serum HBsAg is reduced for at least 8 days, at least 15 days, at least 22 days, or at least 29 days after administration of the single dose, as compared to (pre-administration) baseline.

In certain embodiments, at least 100ng/mL of antibody remains unbound to serum HBsAg for at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after administration of the single dose. In certain embodiments, at least 100ng/mL of antibody remains unbound to serum HBsAg for at least 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, or more after administration of the single dose. In certain embodiments, at least 100ng/mL of antibody remains unbound to serum HBsAg for at least 8 days after administration of the single dose.

In certain embodiments, at least 1000ng/mL of antibody remains unbound to serum HBsAg for at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more after administration of the single dose. In certain embodiments, at least 1000ng/mL of antibody remains unbound to serum HBsAg for at least 14 days after administration of the single dose.

In certain embodiments, after administration of the single dose, the antibody concentration in the subject is C _max Between 300ng/mL and 6,000 ng/mL. In some embodiments, the C of the antibody in the subject _max Is at least 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, 1000ng/mL, 1100ng/mL, 1200ng/mL, 1300ng/mL, 1400ng/mL, 1500ng/mL, 1600ng/mL, 1700ng/mL, 1800ng/mL, 1900ng/mL, 2000ng/mL, 2100 ng/mLng/mL、2200ng/mL、2300ng/mL、2400ng/mL、2500ng/mL、2600ng/mL、2700ng/mL、2800ng/mL、2900ng/mL、3000ng/mL、3100ng/mL、3200ng/mL、3300ng/mL、3400ng/mL、3500ng/mL、3600ng/mL、3700ng/mL、3800ng/mL、3900ng/mL、4000ng/mL、4100ng/mL、4200ng/mL、4300ng/mL、4400ng/mL、4500ng/mL、4600ng/mL、4700ng/mL、4800ng/mL、4900ng/mL、5000ng/mL、5100ng/mL、5200ng/mL、5300ng/mL、5400ng/mL、5500ng/mL、5600ng/mL、5700ng/mL、5800ng/mL、5900ng/mL、6000ng/mL。

The present disclosure also includes the following exemplary embodiments.

Example 1A method of treating Hepatitis B Virus (HBV) infection in a subject, the method comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody comprises the heavy chain amino acid sequence of SEQ ID NO:91 and the light chain amino acid sequence of SEQ ID NO:93, and

(a) Said single dose of said pharmaceutical composition comprises at least 6mg of said antibody; and is also provided with

(b) After administration of the single dose, the subject's serum HBsAg is reduced by at least 1.0log from baseline ¹⁰ IU/mL、1.5log ¹⁰ IU/mL or more; and is also provided with

(c) The reduction in serum HBsAg of the subject after administration of the single dose lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or more.

Example 2A method of treating Hepatitis B Virus (HBV) infection in a subject, the method comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody comprises the heavy chain amino acid sequence of SEQ ID NO:91 and the light chain amino acid sequence of SEQ ID NO:93, and

(a) Said single dose of said pharmaceutical composition comprises at least 75mg of said antibody; and is also provided with

(b) After administration of the single dose, the subject's serum HBsAg is reduced by at least 1.0log from baseline ¹⁰ IU/mL, at least 1.5log ¹⁰ IU/mL or more; and is also provided with

(c) The reduction in serum HBsAg in the subject after administration of the single dose lasts at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days or more.

Example 3. A method of treating Hepatitis B Virus (HBV) infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody, wherein:

(a) The antibody comprises a heavy chain amino acid sequence of SEQ ID NO. 91 and a light chain amino acid sequence of SEQ ID NO. 93,

(b) At least 1000ng/mL of antibody remains unbound to serum HBsAg for at least 14 days after administration of a single dose; and is also provided with

(c) The subject has a baseline serum HBsAg level of less than 3000IU/mL.

Embodiment 4. The method of any one of embodiments 1 to 3, wherein the serum HBsAg of the subject is reduced by at least 1.0log compared to baseline within 8 days of administration of a single dose ¹⁰ IU/mL。

Embodiment 5 the method of any one of embodiments 1-4, wherein the single dose of the pharmaceutical composition comprises at least 75mg of the antibody, and within 8 days of administration of the single dose, the subject's serum HBsAg is reduced by at least 1.5log from baseline ¹⁰ IU/mL。

Embodiment 6 the method of any one of embodiments 1 to 5, wherein the serum HBsAg of the subject is reduced by at least 0.5log from baseline 56 days after administration of the single dose ¹⁰ IU/mL。

Embodiment 7. The method of any of embodiments 1 to 6, wherein the subject has a C of the antibody between 300ng/mL and 6,000ng/mL _max 。

Embodiment 8. The method of embodiment 7 wherein the C of the antibody in the subject _max At least 300ng/mL, 400ngPer mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, 1000ng/mL, 1100ng/mL, 1200ng/mL, 1300ng/mL, 1400ng/mL, 1500ng/mL, 1600ng/mL, 1700ng/mL, 1800ng/mL, 1900ng/mL, 2000ng/mL, 2100ng/mL, 2200ng/mL, 2300ng/mL, 2400ng/mL, 2500ng/mL, 2600ng/mL, 2700ng/mL, 2800ng/mL, 2900ng/mL, 3000ng/mL, 3100ng/mL, 3200ng/mL 3300ng/mL, 3400ng/mL, 3500ng/mL, 3600ng/mL, 3700ng/mL, 3800ng/mL, 3900ng/mL, 4000ng/mL, 4100ng/mL, 4200ng/mL, 4300ng/mL, 4400ng/mL, 4500ng/mL, 4600ng/mL, 4700ng/mL, 4800ng/mL, 4900ng/mL, 5000ng/mL, 5100ng/mL, 5200ng/mL, 5300ng/mL, 5400ng/mL, 5500ng/mL, 5600ng/mL, 5700ng/mL, 5800ng/mL or 5900ng/mL.

Embodiment 9. The method of any of embodiments 1 to 8, wherein the single dose of the pharmaceutical composition comprises at most 10mg, at most 15mg, at most 18mg, at most 25mg, at most 30mg, at most 35mg, at most 40mg, at most 45mg, at most 50mg, at most 55mg, at most 60mg, at most 75mg, at most 90mg, at most 300mg, at most 900mg, or at most 3000mg of the antibody,

Or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 3000mg, or ranging from 10mg to 3000mg, or ranging from 25mg to 3000mg, or ranging from 30mg to 3000mg, or ranging from 50mg to 3000mg, or ranging from 60mg to 3000mg, or ranging from 75mg to 3000mg, or ranging from 90mg to 3000mg, or ranging from 100mg to 3000mg, or ranging from 150mg to 3000mg, or ranging from 200mg to 3000mg, or ranging from 300mg to 3000mg, or ranging from 500mg to 3000mg, or ranging from 750mg to 3000mg, or ranging from 900mg to 3000mg, or ranging from 1500mg to 3000mg, or ranging from 2000mg to 3000mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 900mg, or ranging from 10mg to 900mg, or ranging from 25mg to 900mg, or ranging from 30mg to 900mg, or ranging from 50mg to 900mg, or ranging from 60mg to 900mg, or ranging from 75mg to 900mg, or ranging from 90mg to 900mg, or ranging from 100mg to 900mg, or ranging from 150mg to 900mg, or ranging from 200mg to 900mg, or ranging from 300mg to 900mg, or ranging from 500mg to 900mg, or ranging from 750mg to 900mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 500mg, or ranging from 10mg to 500mg, or ranging from 25mg to 500mg, or ranging from 30mg to 500mg, or ranging from 50mg to 500mg, or ranging from 60mg to 500mg, or ranging from 75mg to 500mg, or ranging from 90mg to 500mg, or ranging from 100mg to 500mg, or ranging from 150mg to 500mg, or ranging from 200mg to 500mg, or ranging from 300mg to 500mg, or ranging from 400mg to 500mg,

Or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 300mg, or ranging from 10mg to 300mg, or ranging from 25mg to 300mg, or ranging from 30mg to 300mg, or ranging from 50mg to 300mg, or ranging from 60mg to 300mg, or ranging from 75mg to 300mg, or ranging from 90mg to 300mg, or ranging from 100mg to 300mg, or ranging from 150mg to 300mg, or ranging from 200mg to 300mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 200mg, or ranging from 10mg to 200mg, or ranging from 25mg to 200mg, or ranging from 30mg to 200mg, or ranging from 50mg to 200mg, or ranging from 60mg to 200mg, or ranging from 75mg to 200mg, or ranging from 90mg to 200mg, or ranging from 100mg to 200mg, or ranging from 150mg to 200mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 100mg, or ranging from 10mg to 100mg, or ranging from 25mg to 100mg, or ranging from 30mg to 100mg, or ranging from 50mg to 100mg, or ranging from 60mg to 100mg, or ranging from 75mg to 100mg, or ranging from 90mg to 100mg,

Or wherein said single dose of said pharmaceutical composition comprises said antibody in an amount ranging from 6mg to 25mg, or ranging from 10mg to 25mg, or ranging from 15mg to 25mg, or ranging from 20mg to 25mg,

or wherein said single dose of said pharmaceutical composition comprises said antibody in an amount ranging from 6mg to 50mg, or ranging from 6mg to 25mg, or ranging from 6mg to 50mg, or ranging from 10mg to 25mg, or ranging from 6mg to 15mg, or ranging from 10mg to 15mg, or wherein the single dose of the pharmaceutical composition comprises 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 985mg, 990mg, 995mg or 1000mg or more of said antibody,

Or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount of less than 3000mg, less than 2500mg, less than 2000mg, less than 1500mg, less than 1000mg, less than 900mg, less than 500mg, less than 300mg, less than 200mg, less than 100mg, less than 90mg, less than 75mg, less than 50mg, less than 25mg, or less than 10 mg.

Embodiment 10. The method according to any one of embodiments 1 to 9, wherein the single dose of the pharmaceutical composition comprises the antibody in a concentration in the range of 100mg/mL to 200mg/mL, such as 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL or 200mg/mL, preferably 150 mg/mL.

Embodiment 11. The method of any one of embodiments 1 to 10, wherein the single dose of the pharmaceutical composition comprises about 75mg of the antibody.

Embodiment 12. The method of any one of embodiments 1 to 10, wherein the single dose of the pharmaceutical composition comprises about 90mg of the antibody.

Embodiment 13. The method of any one of embodiments 1 to 10, wherein the single dose of the pharmaceutical composition comprises at most 300mg of the antibody.

Embodiment 14. The method of any one of embodiments 1 to 10, wherein the single dose of the pharmaceutical composition comprises at most 900mg of the antibody.

Embodiment 15. The method of any one of embodiments 1 to 10, wherein the single dose of the pharmaceutical composition comprises at most 3,000mg of the antibody.

Embodiment 16. The method of any one of embodiments 1 to 15, wherein the method comprises administering the single dose by subcutaneous injection, optionally wherein the single dose comprises, or consists of, 6mg of the antibody, 18mg of the antibody, or 75mg of the antibody.

Embodiment 17. The method of any one of embodiments 1 to 16, wherein the method comprises administering the single dose by intravenous injection.

Embodiment 18 the method of any one of embodiments 1 to 17, wherein the pharmaceutical composition further comprises water, optionally USP water.

Embodiment 19. The method of any of embodiments 1 to 18, wherein the pharmaceutical composition further comprises histidine, optionally in the pharmaceutical composition at a histidine concentration in the range of 10mM to 40mM, such as 20mM.

Embodiment 20. The method of any of embodiments 1 to 19, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally 5%, 6%, 7%, 8% or 9%, preferably about 7% (w/v).

Embodiment 21. The method of any of embodiments 1 to 20, wherein the pharmaceutical composition further comprises a surfactant or triblock copolymer, optionally polysorbate or poloxamer-188, preferably polysorbate 80 (PS 80), wherein optionally the polysorbate or poloxamer-188 is present in a range of 0.01% to 0.05% (w/v), preferably 0.02% (w/v).

Embodiment 22. The method of any of embodiments 1 to 21, wherein the pH of the pharmaceutical composition is in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or is 5.8, 5.9, 6.0, 6.1, or 6.2.

Embodiment 23. The method of embodiment 22, wherein the pharmaceutical composition comprises:

(i) 150mg/mL of the antibody;

(ii) USP water;

(iii) 20mM histidine;

(iv) 7% sucrose; and

(v)0.02％PS80，

wherein the pharmaceutical composition has a pH of 6.

Embodiment 24. The method of any one of embodiments 1 to 23, wherein the subject is an adult.

Embodiment 25. The method of embodiment 24 wherein the subject has an age ranging from 18 years to 65 years.

Embodiment 26 the method of any one of embodiments 1 to 25, wherein the subject has a body weight of 40kg to 125kg and/or the subject has a Body Mass Index (BMI) of 18 to 35kg/m ² 。

Embodiment 27. The method of any one of embodiments 1 to 26, wherein the subject has chronic HBV infection; for example, defined by 2 serum HBsAg, HBV DNA, and/or HBeAg positives, wherein the 2 separations are at least 6 months apart.

Embodiment 28. The method of any one of embodiments 1 to 27, wherein the subject does not have liver cirrhosis.

Embodiment 29. The method of embodiment 28, wherein the absence of cirrhosis is determined by:

fibrincan assessment (e.g., within 6 months prior to administration of the single dose of the pharmaceutical composition); or (b)

Liver biopsy (e.g., within 12 months prior to administration of the single dose of the pharmaceutical composition),

wherein preferably the absence of cirrhosis is determined by absence of Metavir F3 fibrosis or absence of F4 cirrhosis.

Embodiment 30. The method of any one of embodiments 1 to 29, wherein the subject has received a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI) optionally within 120 days, further optionally within 60 days, prior to administration of the single dose.

Embodiment 31. The method of embodiment 30, wherein the NRTI comprises one or more of the following: tenofovir; tenofovir disoproxil (e.g., tenofovir disoproxil fumarate); tenofovir alafenamide; entecavir; lamivudine; adefovir; adefovir dipivoxil.

Embodiment 32. The method of any one of embodiments 1 to 31, wherein the subject has a serum HBV DNA concentration of less than 100IU/mL no more than 28 days prior to administration of the single dose.

Embodiment 33. The method of any of embodiments 1 to 32, wherein the subject's serum HBsAg concentration is less than 3,000iu/mL prior to administration of the single dose, and optionally, the subject's serum HBsAg concentration is less than 1,000iu/mL prior to administration of the single dose.

Embodiment 34 the method of any one of embodiments 1 to 32, wherein the subject has a serum HBsAg concentration greater than or equal to 3,000iu/mL no more than 28 days prior to administration of the single dose, and optionally, the subject has a serum HBsAg concentration greater than or equal to 1,000iu/mL no more than 28 days prior to administration of the single dose.

Embodiment 35 the method of any one of embodiments 1-34, wherein the subject is negative for HB e antigen (HBeAg) no more than 28 days prior to administration of the single dose.

Embodiment 36. The method of any one of embodiments 1-35, wherein the subject is negative for anti-HB antibodies no more than 28 days prior to administration of the single dose.

Embodiment 37 the method of any one of embodiments 1 to 36, wherein prior to administering the single dose:

(i) The subject is not suffering from fibrosis and/or is not suffering from cirrhosis; and/or

(ii) Alanine Aminotransferase (ALT) <2x upper normal limit (ULN) for the subject.

Embodiment 38 the method of any one of embodiments 1-37, wherein the serum HBsAg of the subject is reduced by > 2-fold 56 days after administration of the single dose, as compared to the serum HBsAg of the subject (e.g., the concentration of HBsAg in serum, e.g., as determined using the yaban ARCHITECT assay) 0 to 28 days prior to administration of the single dose.

Embodiment 39. The method of any one of embodiments 1 to 38, wherein after administration of the single dose (e.g., 56 days after administration of the single dose),

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

(ii) The subject has an adaptive immune response against HBV.

Embodiment 40. The method of any one of embodiments 1 to 39, wherein the subject is a male.

Embodiment 41. The method of any one of embodiments 1 to 39, wherein the subject is female.

Example 42. A pharmaceutical composition comprising an antibody, wherein the antibody comprises the heavy chain amino acid sequence of SEQ ID No. 91 and the light chain amino acid sequence of SEQ ID No. 93, wherein the pharmaceutical composition comprises the antibody in a concentration ranging from 100mg/mL to 200mg/mL, such as 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL or 200mg/mL, preferably 150mg/mL, and wherein at least 1000ng/mL of the antibody remains unbound to serum HBsAg for at least 14 days after administration of a single dose following administration of the composition to a subject in need thereof. And the subject's baseline serum HBsAg level is less than 3000IU/mL.

Embodiment 43. The pharmaceutical composition of embodiment 42, wherein the pharmaceutical composition comprises at most 6mg, at most 18mg, at most 75mg, at most 90mg, at most 300mg, at most 900mg, or at most 3000mg of the antibody.

Embodiment 44. The pharmaceutical composition of embodiment 42 or 43, wherein the pharmaceutical composition comprises about 75mg of the antibody.

Embodiment 45. The pharmaceutical composition of embodiment 42 or 43, wherein the pharmaceutical composition comprises about 90mg of the antibody.

Embodiment 46. The pharmaceutical composition of embodiment 42 or 43, wherein the pharmaceutical composition comprises about 300mg of the antibody.

Embodiment 47. The pharmaceutical composition of embodiment 42 or 43, wherein the pharmaceutical composition comprises about 900mg of the antibody.

Embodiment 48. The pharmaceutical composition of embodiment 42 or 43, wherein the pharmaceutical composition comprises about 3,000mg of the antibody.

Embodiment 49 the pharmaceutical composition of any one of embodiments 42 to 48, wherein the pharmaceutical composition further comprises water, optionally USP water.

Embodiment 50. The pharmaceutical composition according to any of embodiments 42 to 49, wherein the pharmaceutical composition further comprises histidine, optionally in the pharmaceutical composition at a histidine concentration of 10mM to 40mM, such as 20mM.

Embodiment 51. The pharmaceutical composition according to any of embodiments 42 to 50, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally 5%, 6%, 7%, 8% or 9%, preferably about 7% (w/v).

Embodiment 52 the pharmaceutical composition according to any of embodiments 42 to 51, wherein the pharmaceutical composition further comprises a surfactant, optionally a polysorbate, preferably polysorbate 80 (PS 80), wherein optionally the polysorbate is present in the range of 0.01% to 0.05% (w/v), preferably 0.02% (w/v).

Embodiment 53. The pharmaceutical composition of any of embodiments 42 to 52, wherein the pH of the pharmaceutical composition is in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or is 5.8, 5.9, 6.0, 6.1, or 6.2.

Embodiment 54 the pharmaceutical composition of any one of embodiments 42 to 53, wherein the pharmaceutical composition comprises:

(i) 150mg/mL of the antibody;

(ii) USP water;

(iii) 20mM histidine;

(iv) 7% sucrose; and

(v)0.02％PS80，

wherein the pharmaceutical composition has a pH of 6.

Embodiment 55. The method of any one of embodiments 1-41, wherein the subject is negative for HBeAg or positive for HBeAg.

Examples

Hereinafter, specific examples illustrating embodiments and aspects of the present disclosure are presented. However, the scope of the present disclosure is not limited by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and practice the present disclosure. However, the scope of the present disclosure is not limited by the illustrated embodiments. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description, accompanying drawings, and the following examples. All such modifications are intended to be within the scope of the appended claims.

Example 1: production and testing of engineered antibodies

Analysis of some HBC34 antibody variants from PCT publication No. WO 2017/060504 revealed that the cysteine amino acids at position 40 (IMGT numbering) in the light chain variable region were unpaired and represent a potential propensity. Without wishing to be bound by theory, unpaired cysteine residues are potentially reactive and may potentially trigger aggregation through intramolecular disorder or intermolecular disulfide formation. Variants of HBC34-V7 (WO 2017/060504) were engineered in which the cysteine amino acid at position 40 was substituted with serine (thereby yielding "HBC 34-V34") or with alanine (thereby yielding "HBC 34-V35"). The nucleotide sequences encoding these additional variant antibodies were codon optimized and the antibodies were found in expiocho ^TM IgG1 (g 1m17,1 allotype) was expressed in cells (ThermoFisher, siemens). Codon-optimized nucleotide sequences encoding the VH and VL domains of HBC34-V35 are provided in SEQ ID NOs 103 and 104, respectively.

The ability of HBC34-V34 and HBC34-V35 to bind antigen was studied using a direct antigen binding ELISA. HBC34-V7 was used as a comparator. As shown in FIG. 1, both HBC34-V34 and HBC34-V35 bind efficiently to both recombinant HBsAg antigens ("adw", upper panel; "adr", lower panel), and HBC34-V35 has very similar binding to the parent HBC 34-V7.

Variant antibodies were examined for binding to all known HBsAg genotypes ((a) - (J)). Briefly, human epithelial cells (Hep 2 cells) were transfected with plasmids expressing each of the 10 HBV HBsAg genotypes A, B, C, D, E, F, G, H, I and J. All antibodies were tested at various concentrations to stain transiently transfected permeabilized cells. Two days after transfection, hep2 cells were collected, fixed and permeabilized with saponin for immunostaining with HBC34 and five selected variants. HBC34-V7 was included as a comparator. Binding of antibodies to transfected cells was analyzed using a bacton Dickinson company (Becton Dickinson) FACSCanto2 (BD Biosciences) with FlowJo software (treesar). As shown in FIGS. 2A-2J, HBC34-V34 and HBC34-V35 recognize all 10 HBV HBsAg genotypes. HBC34-V35 showed a slightly stronger staining than HBC 34-V34.

These data show that antibody variants HBC34-V34 and HBC34-V35 widely recognize and bind HBsAG at levels comparable to HBC 34-V7.

Example 2: HBC antibodies having modified Fc regions efficiently bind antigen

Modifications in the Fc region may provide advantages for therapeutic antibodies. HBC34-V35 is expressed as IgG1 with wild-type Fc, or with Fc containing the "MLNS" mutation (M428L/N434S) or with MLNS in combination with "GAALIE" (G239A/A330L/I332E). Binding of each construct to recombinant HBsAg (adw) was tested in two separate antigen binding ELISA experiments. Three (3) batches of HBC34-v35 (wild-type Fc) were tested. Two (2) batches of HBCs 34-V35-MLNS and two (2) batches of HBCs 34-V35-MLNS-GAALIE were tested. HBC34v7 (batch) was tested as a comparator.

As shown in fig. 3A and 3B, the introduced Fc mutation did not affect the antigen binding activity of HBC 34-V35. EC50 values varied slightly between the various constructs and the two experiments, but were generally lower.

Example 3: other functional studies

In vitro and in vivo neutralization studies were performed using HBC34-V35, HBC34-V35-MLNS and HBC 34-V35-MLNS-GAALIE. In one study, neutralizing activity of antibodies was tested using HBV infected mouse PXB cells. In another study, antibodies were tested using human hepatocytes infected with genotype C HBV.

For both studies, hebsbumin (human hepatitis b immunoglobulin) was used as a positive control. The following data were captured at various time points: quantifying HBV DNA; quantitative HBsAg; quantification of HBeAg; and hAlb quantification.

Example 4: identification and characterization of the human monoclonal antibody HBC24

Human monoclonal antibodies were isolated from human patients in a manner similar to that described in Traggiai E. Et al, 2004, nature medical science 10 (8): 871-5. General purpose medicineThe nucleotide and amino acid sequences of the antibody variable regions and Complementarity Determining Regions (CDRs) therein were determined to characterize the antibody and were referred to as "HBC24". Thus, HBC24 is a polypeptide having the CDR, V as shown in Table 3 above _H And V _L Fully human monoclonal antibodies of the IgG1 type of the sequence. The V encoding HBC24 is provided in Table 4 _H And V _L Is a sequence of nucleotides.

Example 5: removal of HB antigen and viral entry inhibition in mouse model

Immunodeficient mice with transplanted human hepatocytes were used to test the effectiveness of the anti-HBV antibodies of the present disclosure in clearing HBsAg. Briefly, primary human hepatocytes were transplanted into SCID mice whose mouse hepatocytes had been previously enzymatically destroyed. Mice are T cell and B cell deficient. This model can be used to study HBV infection, including entry, transmission, cccDNA modulation, hepatocyte innate immune response, and viral integration into the host genome.

On day 28, mice were vaccinated with D-rAAV 8-1.3HBV strain ayw by tail vein injection, 1.0X10 each ⁷ And the viral genome. Treatment was performed on day 0. AAV/HBV infected mice (n=4 per treatment group) were administered PBS (control) or HBC34-v35 (1, 5 or 15mg/kg intraperitoneally, 2 times/week). The antibodies were murine (murinized) except for the antigen binding Fab region.

Plasma and serum samples, viral load, HBV DNA (by PCR) and HB Ag (HBsAg, HBeAg, HBcrAg) were collected periodically throughout the study. Mice were sacrificed at week 6. As shown in fig. 4-7, treatment with the highest dose of HBC34-v35 reduced viral load and viral entry into hepatocytes.

Example 6: production and functional testing of HBC24 germline variants

HBC24 was analyzed for the presence of somatic mutations in the variable region relative to germline sequences. The identified somatic mutations were inverted to germline sequences to produce HBC24 variants. HBC24 and variants were tested for binding (in vitro) and neutralization (in vitro; in vivo) of HBV and HBD serotypes using the assays described in examples 1 and 3.

Example 7: introduction of Fc modification into HBC24 and variants

Other HBC24 variants containing MLNS and GAALIE mutations in both Fc monomers were generated. The HC amino acid sequences of the selected variants are provided in SEQ ID NOs 120 and 121. The following assays for variants were used to examine: (1) in vitro binding of antigen; (2) neutralization of HBV serotypes in vitro.

Example 8: in vitro effector function studies

An in vitro study was performed to examine the following capabilities of HBC34 antibodies with modified Fc: (1) binding to human fcγr and complement; (2) activating fcyriia, fcyriib, and fcyriiia; and (3) promote ADCC and activate human Natural Killer (NK) cells. The test articles, cell lines and reagents used are described in tables 5-7 below. The following abbreviations are used in this example: GLP = good laboratory specification; ADCC = antibody dependent cellular cytotoxicity; ADCP = antibody dependent cellular phagocytosis; fc = crystallizable fragment; HBsAg = hepatitis b surface antigen; mAb = monoclonal antibody; PBS = phosphate buffered saline; UHPL-SEC = ultra-high performance liquid size exclusion chromatography; ATCC = american type culture collection; fcγr=fcγreceptor; CHO cells = chinese hamster ovary cells; RLU = relative light emitting unit; BLI = biological layer interferometry.

TABLE 5 test article

Test article	HBC34v35-MLNS
		● Isotype type	●IgG1k
● Relative molecular weight	●≈150kDa
		● Concentration of	●3.47mg/ml
● Source(s)	● Inside part
		● Treatment and storage conditions	● Short term storage at-80 ℃ below zero for 4 DEG C
● Formulation buffer	●PBS，pH 7.2
		Test article	HBC34-V35-MLNS-GAALIE
● Isotype type	●IgG1k
		● Relative molecular weight	●≈150kDa
● Concentration of	●2.1mg/ml/0.86mg/ml
		● Source(s)	● Inside part
● Treatment and storage conditions	● Short term storage at-80 ℃ below zero for 4 DEG C
		● Formulation buffer	●PBS，pH 7.2
Test article	HBC34v35-LALA
		● Isotype type	●IgG1k
● Relative molecular weight	●≈150kDa
		● Concentration of	●1.2mg/ml
● Source(s)	● Inside part
		● Treatment and storage conditions	● Short term storage at-80 ℃ below zero for 4 DEG C
● Formulation buffer	●PBS，pH 7.2
		Test article	mAb 17.1.41
● Isotype type	●IgG1k
		● Relative molecular weight	●≈150kDa
● Concentration of	●4.4mg/ml
		● Source(s)	● Inside part
● Treatment and storage conditions	● Short term storage at-80 ℃ below zero for 4 DEG C
		● Formulation buffer	●PBS，pH 7.2

TABLE 6 cell lines

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TABLE 7 other reagents

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Experimental procedure

Measurement of binding to human Fcgamma-receptor

The binding of HBC34V35-MLNS and HBC34-V35-MLNS-GAALIE to human FcgammaR was measured on an Octet instrument (BLI, biological layer interferometry). Briefly, 2 μg/ml of His-tagged human fcγr (fcγriia allele H131, fcγriia allele R131, fcγriiaa allele F158, fcγriiia allele V158, and fcγriib) was captured onto an anti-penta-His sensor for 6 minutes. Then, in the presence of 1 μg/ml of an afiniband F (ab ') 2 fragment goat anti-human IgG, i.e. a F (ab') 2 fragment specific for cross-linking a human mAb by Fab fragment, the fcγr loaded sensor was exposed to kinetic buffer (ph 7.1) containing 2 μg/ml of each mAb for 4 minutes followed by a dissociation step in the same buffer for another 4 minutes (right part of the figure). Association and dissociation curves as a function of interference pattern were measured in real time using Octet RED96 (fortbio).

Measurement of binding to human complement protein C1q

The binding of HBC34V35-MLNS and HBC34-V35-MLNS-GAALIE to human complement was measured on an Octet instrument (BLI, biological layer interferometry). Briefly, intact IgG1 of HBC34V35 MLNS and HBC34-V35-MLNS-GAALIE mAb was captured at 10 μg/ml by Fab fragment using an anti-human Fab (CH 1 specific) sensor for 10 min. The IgG-loaded sensor was then exposed to kinetic buffer containing 3 μg/ml purified human C1q (pH 7.1) for 4 minutes (left part of the figure), followed by a dissociation step in the same buffer for another 4 minutes (right part of the figure). Association and dissociation curves as a function of interference pattern were measured in real time using Octet RED96 (fortbio).

Preparation of human NK cells from Whole blood

According to the manufacturer's instructions, useNK cells are freshly isolated from EDTA whole blood by the NK isolation kit. Briefly, anticoagulated blood was mixed with 15ml NK separation mix in a 50ml tube and incubated at room temperature using a rotator at about 12 revolutions per minute for 5 minutes. The tube was then placed +.>The separator was kept in the magnetic field for 15 minutes. Magnetically labeled cells adhere to the vessel wall, while aggregated erythrocytes settle to the bottom . Then still in the tubeTarget NK cells were collected from the supernatant while in the separator. NK cells were centrifuged, treated with distilled water to remove residual erythrocytes, centrifuged again and finally resuspended in AIM-V medium.

Determination of antibody-dependent NK cell killing

MAbs were serially diluted from 100. Mu.g/ml to 0.001. Mu.g/ml in AIM-V medium. Target cells (PLC/PRF/5; macNab et al, J.England cancer (British Journal of Cancer), 34 (5), 1976) were 7.5X10 in 23. Mu.l ³ Individual cells/well were added to round bottom 384 well plates, then serial dilutions of antibody were added to each well (23 μl per well), and the antibody/cell mixture was incubated at room temperature for 10 minutes. After incubation, human NK cells were grown at 7.5X10 in 23. Mu.l ⁴ Cell density of wells was added with an effector to target ratio of 10:1. Control wells for measuring maximum lysis (containing target cells with 23 μl of 3% triton x-100) and spontaneous lysis (containing target cells and effector cells but no antibody) were also included. The plates were incubated with 5% CO at 37 ℃C ₂ Incubation was continued for 4 hours. Cell death was determined by measuring Lactate Dehydrogenase (LDH) release using an LDH detection kit according to the manufacturer's instructions. Briefly, the plates were centrifuged at 400x g for 4 minutes and 35 μl of supernatant was transferred to 384 well plates. LDH reagent was prepared and 35 μl was added per well. Absorbance at 490nm and 650nm was measured every 2 minutes for 8 minutes using a kinetic protocol. The percent specific lysis was determined by applying the following formula: (specific release-spontaneous release)/(maximum release-spontaneous release) ×100.

Determination of antibody-dependent NK cell activation

Activation of primary NK cells was tested using freshly isolated cells from two donors that had previously been genotyped to express homozygous high (V158 allele) or low (F158 allele) affinity fcγriiia. Serial dilutions of mAb (10-fold serial dilutions from 100 μg/ml to 0.0001 μg/ml in AIM-V medium) were incubated with NK cells for 4 hours. NK cell activation was measured by flow cytometry by staining NK cells with anti-CD 107a mAb (anti-CD 107 PE, BAOJIAN, 1/35 dilution used) as a functional marker of NK cell activity.

Determination of antibody-dependent activation of human fcyriiia

HBC34V35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 4-fold from 5. Mu.g/ml to 0.076. Mu.g/ml in ADCC assay buffer. Target antigen (HBsAg from Engerix B, glaring smith) was added to a white flat bottom 96-well plate at 0.6 μg/ml in 25 μl, then serial dilutions of antibody were added to each well (25 μl per well) and the antibody/cell mixture was incubated at room temperature for 10 minutes. The effector cells of the ADCC bioassay were thawed and used at 7.5X10 ⁴ Cell density per well 25. Mu.l (final HBsAg concentration 0.2. Mu.g/ml) was added. Control wells for measuring antibody independent activation (wells containing HBsAg and effector cells but no antibody) and spontaneous luminescence of the plate (wells containing ADCC assay buffer only) were also included. The plates were incubated with 5% CO at 37 ℃C ₂ Incubation was continued for 24 hours. Activation of human fcyriiia (V158 or F158 variant) in this biological assay results in NFAT-mediated luciferase reporter expression. According to the manufacturer's instructions, bio-Glo- ^TM The luciferase assay reagent measures luminescence with a photometer. The data (i.e., specific fcγriiia activation) are expressed as the average of Relative Luminescence Units (RLUs) over background by applying the following formula: (RLU at mAb concentration x-RLU against background).

Determination of antibody-dependent activation of human fcyriia

HBC34V35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 5-fold from 50. Mu.g/ml to 0.00013. Mu.g/ml in ADCP assay buffer. Target antigen (HBsAg from Engerix B) was added to a white flat bottom 96-well plate at 0.6 or 6 μg/ml in 25 μl, then serial dilutions of antibody were added to each well (25 μl per well) and the antigen/antibody incubation was continued for 25 min at room temperature. Thawing effector cells from fcyriia-activated bioassay at 50.0X10 ⁴ Cell density per well 25. Mu.l (final HBsAg concentration 0.2 or 2. Mu.g/ml, respectively) was added. Also included are methods for measuring antibody non-linearitiesControl wells with dependent activation (wells containing HBsAg and effector cells but no antibody) and spontaneous luminescence of the plate (wells containing ADCP assay buffer only). The plates were incubated with 5% CO at 37 ℃C ₂ Incubation was continued for 23 hours. Activation of human fcyriia (H131 variant) in this biological assay results in NFAT-mediated luciferase reporter expression. According to the manufacturer's instructions, bio-Glo- ^TM The luciferase assay reagent measures luminescence with a photometer. The data (i.e., specific fcyriia activation) are expressed as the average of Relative Luminescence Units (RLU) over background by applying the following formula: (mAb concentration [ x ]]RLU under RLU-background RLU).

Assay for antibody-dependent activation of human fcyriib

HBC34V35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 5-fold from 100. Mu.g/ml to 0.00026. Mu.g/ml in ADCP assay buffer. Target antigen (HBsAg from Engerix B) was added to a white flat bottom 96-well plate at 3 μg/ml in 25 μl, then serial dilutions of antibody were added to each well (25 μl per well), and the antigen/antibody incubated at room temperature for 15 minutes. Thawing effector cells from fcyriib-activated bioassay and thawing the same at 75.0x10 ⁴ Cell density per well 25. Mu.l (final HBsAg concentration 1. Mu.g/ml) was added. Control wells for measuring antibody independent activation (wells containing HBsAg and effector cells but no antibody) and spontaneous luminescence of the plates (wells containing ADCP assay buffer only) were also included. The plates were incubated with 5% CO at 37 ℃C ₂ Incubation was continued for 20 hours. Activation of human fcyriib in this biological assay results in NFAT-mediated luciferase reporter gene expression. According to the manufacturer's instructions, bio-Glo- ^TM The luciferase assay reagent measures luminescence with a photometer. The data (i.e., specific fcyriib activation) are expressed as the average of Relative Luminescence Units (RLU) over background by applying the following formula: (mAb concentration [ x ]]RLU under RLU-background RLU).

Determination of binding of antibodies to human hepatoma cell line PLC/PRF/5

PLC/PRF/5 cells were trypsinized at 37℃for 5 min, transferred to 7ml of growth medium, centrifuged at 400Xg for 4 min at 4℃and washed well in PBS at 4 ℃. Some cells were fixed with 4% formaldehyde (at 4 ℃C20 minutes); other cells were fixed and then permeabilized with permeabilization buffer (20 min at 4 ℃). Cell pellets were resuspended in 2.64ml of wash buffer (fixed cells) or permeabilization buffer (fixed and permeabilized cells) (Table 7) and distributed at 200 μl/well into 96-well round bottom plates (corresponding to 100,000 cells/well). The plates were centrifuged at 400g for 4 min at 4 ℃. Test antibodies were added to wells containing cells in serial dilutions at 1:5 points at a final concentration of 10 μg/ml and incubated on ice for 30 minutes. After washing 2 times at 4℃for 4 minutes in washing buffer (fixed cells) or permeabilization buffer (fixed and permeabilized cells) at 400x g, 50. Mu.l/well of Alexa were used 647 labeled secondary antibodies (table 7) were added to the cells and incubated on ice for 20 minutes. Cells were further washed 2 times with wash buffer (fixed cells) or permeabilization buffer (fixed and permeabilized cells), resuspended in 200 μl/well of wash buffer (fixed cells) or permeabilization buffer (fixed and permeabilized cells), and the signal (MFI, mean fluorescence intensity) was quantified with a cytofluorometer (BD FACSCanto II).

Results

The direct antiviral mechanism is important for in vivo neutralization of HBV. The indirect, fc-dependent mechanisms mediated by the interaction of the Fc region with fcγ receptors (fcγr) on immune cells may also contribute significantly to in vivo efficacy and to mediate endogenous immune responses. By measuring binding to fcγr and antibody dependent activation of human fcγr, fcγr dependent mechanisms can be assessed in vitro (Hsieh, y. -t et al, journal of immunology (Journal of Immunological Methods), 441 (C), 56-66.doi.org/10.1016/j.jim.2016.12.002).

In this study, the ability of HBC34V35-MLNS and HBC34-V35-MLNS-GAALIE to bind to the full set of human FcgammaR (FcgammaRIIIa V158 and F158 alleles, fcgammaRIIa H131 and R131 alleles and FcgammaRIIb) was compared in parallel using the biological layer interferometry (BLioctet system, forteBio Inc.). As shown in fig. 8A-8E, fcs carrying MLNS-GAALIE mutations altered interactions with fcγr; in particular, fcs carrying these mutations enhance binding to fcγriiia and fcγriia, while reducing binding to fcγriib. Notably, the binding of HBC34-V35-MLNS-GAALIE to C1q was eliminated as measured by biolayer interferometry (fig. 9).

The ability of HBC34-V35-MLNS and HBC34-V35-MLNS-GAALIE to activate human FcgammaRIIIa and FcgammaRIIA was also tested using a cell-based reporter gene bioassay. These assays reflect activation of human fcγr using Jurkat cells engineered with NFAT-mediated luciferase reporter genes. Although HBC34V35-MLNS activated poorly or not in the presence of HBsAg, HBC34-V35-MLNS-GAALIE showed dose-dependent activation of all tested FcgammaR (FIGS. 10A, 10B, 11A and 11B). In contrast, HBC34-V35-MLNS-GAALIE did not activate FcgammaRIIB even when tested at 100 μg/ml (FIG. 12).

ADCC activity was also measured using natural killer cells (NK) isolated from human peripheral blood mononuclear cells of a donor that had previously been genotyped to express heterozygous high (V158) and low (F158) affinity fcγriiia (F/V). Isolated NK cells were used to measure killing of the hepatoma cell line PLC/PR/5 after exposure to: HBC34v35; HBC34v35-MLNS; HBC34-V35-MLNS-GAALIE; or another mAb (17.1.41, targeting another epitope on the antigen loop of HBsAg; see Eren, R.et al, (Hepatology) doi.org/10.1053/jhep.2000.9632; galun, E.et al, (Hepatology) doi.org/10.1053/jhep.2002.31867). Killing in the presence of HBsAg specific mAbs HBC34V35, HBC34V35-MLNS, HBC34-V35-MLNS-GAALIE and 17.1.41 was not observed (FIG. 13A). The observed lack of antibody-dependent killing of PLC/PR/5 cells may be associated with low expression of HBsAg on the surface of these cells (fig. 13B), which may not be sufficient to trigger killing of NK cells without wishing to be bound by theory. In contrast, when PLC/PR/5 cells were fixed and permeabilized, high levels of HBsAg were detected with HBCs 34v35 and 17.1.41, indicating that most HBsAg was present either intracellularly or in secreted form (i.e., subviral particles) (fig. 13B).

Activation of primary human NK cells (V/F) in the presence of HBC34V35-MLNS or HBC34-V35-MLNS-GAALIE and HBsAg was also examined using anti-CD 107a mAb. The data is shown in fig. 14A and 14B.

These in vitro data show that HBV-specific binding proteins of the present disclosure carrying GAALIE Fc mutations bind and activate low affinity activated fcyriia and fcyriiia more effectively than non-GAALIE Fc parent antibodies. The GAALIE-carrying binding protein also did not bind to or activate the low affinity inhibitory FcgammaRIIB. The GAALIE-carrying binding protein also did not bind to C1q. Furthermore, the GAALIE-carrying binding protein does not promote ADCC to liver cancer cells, but activates human NK cells in the presence of soluble HBsAg.

Example 9: phase 1 clinical study of HBC34-v35-MLNS-GAALIE

A multicenter phase 1, randomized, placebo controlled study was performed to evaluate the safety, tolerability, pharmacokinetic and antiviral activity of HBC34-v35-MLNS-GAALIE (comprising the heavy chain amino acid sequence shown in SEQ ID NO:91 and the light chain amino acid sequence shown in SEQ ID NO: 93). Study sites were as follows: part A (single center) and part B/C (multi-center).

In part A (up to 40 subjects), the main objective was to assess the safety and tolerability of HBC34-v35-MLNS-GAALIE in healthy adult subjects. The secondary objective was to characterize serum Pharmacokinetics (PK) of HBC34-v35-MLNS-GAALIE in healthy adult subjects and to evaluate immunogenicity (induction of anti-drug antibodies [ ADA ]) of HBC34-v35-MLNS-GAALIE in healthy adult subjects,

In part B (up to 56 subjects) and part C (up to 24 subjects), the main objective was to assess the safety and tolerability of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis. The secondary objectives are: characterization of serum PK of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis; assessing antiviral activity of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis; and the immunogenicity of HBC34-v35-MLNS-GAALIE (ADA-induced) in adult subjects with chronic HBV infection without cirrhosis was evaluated. The exploration targets include: assessing the effect of HBC34-v35-MLNS-GAALIE on additional viral parameters; assessing the effect of HBC34-v35-MLNS-GAALIE on the immune response (or exploratory biomarker) of an adult subject with chronic HBV infection without cirrhosis; and assessing the effect of host polymorphism (or exploratory biomarker) on HBC34-v35-MLNS-GAALIE response in adult subjects with chronic HBV infection without cirrhosis.

Evaluation of Standard details

For part a, the main endpoints of the study are as follows:

incidence of adverse events (TEAE) occurring in treatment

Clinical assessment, including but not limited to laboratory test results

The secondary objectives of this study are as follows:

HBC34-v35-MLNS-GAALIE serum-free PK parameters, for example: c (C) _max 、Clast、T _max 、T _last 、AUC _inf 、AUC _last 、AUC _exp ％、t _1/2 、λ _z 、V _z (IV only), CL (IV only), V _z /F (SC only) and CL/F (SC only)

Incidence and titre of ADA on HBC34v-35-MLNS-GAALIE (if applicable)

For the B/C part, the main endpoints of the study are as follows:

incidence of TEAE

Clinical assessment, including but not limited to laboratory test results

The secondary objectives of this study are as follows:

HBC34-v35-MLNS-GAALIE serum-free and total PK parameters, for example: c (C) _max 、C _last 、T _max 、T _last 、AUC _inf 、AUC _last 、AUC _exp ％、t _1/2 、λz、V _z F and CL/F.

Incidence and titre of ADA on HBC34-v35-MLNS-GAALIE (if applicable) serum HBsAg was maximally reduced from baseline (day 1 before dosing)

Exploratory endpoints of the study may include:

assessment of additional viral parameters (e.g. HBV RNA and HBcrAg)

Host immune response analysis

Host factor analysis as determined by RNA sequencing

Fcgamma receptor (Fcgamma R) polymorphism as determined by genotyping

● IgG allotypes as determined by genotyping

Number of subjects planned

Part A: up to 40 healthy adult subjects.

Part B: up to 56 adult subjects with chronic HBV infection without cirrhosis who received nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI) therapy, were negative for HBeAg and had HBsAg <1000 IU/mL.

Part C: up to 24 adult subjects with chronic HBV infection without cirrhosis who received NRTI therapy, HBsAg. Gtoreq.1000 IU/mL.

Incorporated diagnostic and primary criteria

Part a inclusion criteria include:

healthy adult subjects aged 18 years (or legal promised ages, whichever is older) to 55 years with weights greater than or equal to 40kg to less than or equal to 125 kg. Patient health, determined from medical history (chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, asthma, anxiety and depression must be well controlled), and physical examination has no clinical significance findings, 12-lead ECG, important markers and laboratory values. Female subjects must have a negative pregnancy test or confirm postmenopausal status. Postmenopausal status is defined as no menses and no alternative medical cause for 12 months. Women with fertility (WOCBP) must be tested negative for blood pregnancy at screening and negative for urine pregnancy at day 1, cannot breast feed, and must be willing to use a high-efficiency contraceptive method as disclosed herein from 14 days prior to study drug administration to 40 weeks after study drug administration.

Male subjects and fertility female partners must agree to meet one of the following contraceptive requirements from study drug administration time to 40 weeks after study drug administration: vas deferens ligature and record azoospermia, or use male condoms plus partners often use high-efficiency contraceptives. Male subjects must also agree not to donate sperm from the study drug administration time to 40 weeks after study drug administration. Patients agreed not to donate blood during the study.

The patient would like to follow the study requirements and be able to provide written informed consent.

The B/C part inclusion criteria include:

1.18 years (or legal promise age, whichever is older) to 65 years

2. Weight of 40kg or more to 125kg or less with chronic HBV infection (defined as positive for serum HBsAg, HBV DNA or HBeAg 2 times at least 6 months apart based on previous or current laboratory files (any combination of these tests at 6 months apart is acceptable))

3. No liver cirrhosis

4. NRTI therapy was performed at least 2 months at the time of screening, negative for HBeAg. Examples of NRTI therapies include, but are not limited to: tenofovir disoproxil/tenofovir alafenamide; entecavir; lamivudine; adefovir/adefovir dipivoxil.

5. HBV DNA <100IU/mL at screening

HBsAg > lower detection limit

7. HBsAg <3000IU/mL at screening (fraction B only); queue 1b of participants who included HBsAg <1000IU/mL (6 mg SC)

8. HBsAg is larger than or equal to 1000IU/mL (only part C) during screening

9. HBeAg negative in screening (part B only)

10. Negative anti-HB at screening

11. In addition to chronic HBV infection, the patient must be physically healthy, the patient's physical health is determined from medical history (e.g., chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, asthma, anxiety, and depression must be well controlled), and physical examination has no clinical significance findings, 12-lead ECG, important markers, and laboratory values.

12. Female subjects must have a negative pregnancy test or confirm postmenopausal status. Postmenopausal status is defined as no menses and no alternative medical cause for 12 months. Women with fertility must be tested negative for blood pregnancy at screening and negative for urine pregnancy at day 1, cannot breast feed, and must be willing to use a high-efficiency contraceptive method from 14 days before study drug administration to 40 weeks after study drug administration.

13. Male subjects and fertility female partners must agree to meet one of the following contraceptive requirements from study drug administration time to 40 weeks after study drug administration: vasectomy and recording azoospermia, or use of male condoms plus one of the listed contraceptive options for WOCBP contraception by chaperones (see herein). Male subjects must also agree not to donate sperm from the time of first study drug administration to 40 weeks after study drug administration.

14. Is willing to comply with the study requirements and can provide written informed consent.

Methods of birth control that are considered very effective include:

oral, intravaginal or transdermal hormonal contraception methods determined to be used in combination with ovulation inhibition (estrogen and progestin containing) or oral, injectable or implantable hormonal contraception methods determined to be used with progestin only in combination with ovulation inhibition. It is currently unclear whether HBC34-v35-mlns_gaalie will affect the effectiveness of hormonal contraceptive methods; thus, another contraceptive regimen (i.e., barrier method) was suggested for the entire study period and within 40 weeks after study drug administration.

Placement of intrauterine device

Placement of an intrauterine hormone releasing system

Surgical sterilization of male mate (with appropriate files of absence of sperm in ejaculation after vasectomy; for female subjects under study, vasally ligated male mate should be the only mate of the subject)

True sexual abstinence without contact with the specificity when conforming to the preferred and usual lifestyle of the subject. Periodic abstinence (e.g., calendar, ovulation, symptomatic body temperature, post-ovulation methods) and in vitro ejaculation are unacceptable contraceptive methods. If subjects with no desire to develop an originating relationship (based on the longer) during the study and up to 40 weeks after administration of the study medication or during follow-up of the subjects in the study, the subject with no desire to agree to use one of the above-described contraceptive methods.

Barrier methods used in combination with hormonal contraceptives as described above

Postmenopausal status is defined as no menses and no alternative medical cause for 12 months.

Male subjects and fertility female partners must agree to meet one of the following contraceptive requirements within 40 weeks from study treatment administration time to study drug administration.

Vasectomy and azoospermia was recorded

Male condom plus partner one of the contraceptive options listed above (hormonal contraception, intrauterine contraceptive device) using WOCBP contraception

Male subjects must also agree not to donate sperm for 40 weeks after the last study drug administration.

Duration of participation in the study

Part A: the duration of study drug treatment was a single dose. The estimated total study time (including screening and follow-up) for each subject was up to 28 weeks.

B/C part: the duration of study drug treatment was a single dose. The estimated total study time (including screening and follow-up) for each subject was up to 44 weeks.

Duration of follow-up

Part A: all subjects were followed 24 weeks after study drug administration.

B/C part: all subjects were followed 8 weeks after study drug administration. Subjects with > 2-fold reduction in HBsAg at week 8 received prolonged follow-up for a total of up to 40 weeks, or until < 2-fold reduction in HBsAg at 2 consecutive collections relative to baseline, whichever occurs first. Based on the newly emerging data, the extended follow-up may cease.

Study design

The Safety Review Committee (SRC) conducted a continuous review of safety, tolerability and antiviral activity data (only parts B and C) at specified time points based on available data collected throughout the study. Although the SRC ranks the primary data of the dose escalation and optional queue registration of the scrutiny throughout the regimen, the SRC also scrutinizes other relevant data of other queues as indicated to provide decisions.

The study was performed in 3 parts:

● Part A: randomization of HBC34-v35-MLNS-GAALIE, double blind, placebo-controlled, single increment dose (SAD) study, which administered HBC34-v35-MLNS-GAALIE to healthy adult subjects by Subcutaneous (SC) injection or Intravenous (IV) infusion.

● Part B: randomization of HBC34-v35-MLNS-GAALIE, double-blind, placebo-controlled, SAD study, which administered HBC34-v35-MLNS-GAALIE by SC injection to adult subjects with chronic HBV infection without cirrhosis who were receiving NRTI therapy, were negative for HBeAg and had HBsAg <1000 IU/mL.

● Part C: an optional, randomized, double-blind, placebo-controlled, SAD study of HBC34v35-MLNS-GAALIE, which study administered HBC34v35-MLNS-GAALIE by SC injection to adult subjects with chronic HBV infection without cirrhosis who are receiving NRTI therapy and have an HBsAg of ≡1000 IU/mL.

Overall risk/benefit assessment

The potential risk for healthy adult subjects is based on the common safety risk observed with mAb-like therapeutics, and for HBC34-v35-MLNS-GAALIE: allergic reactions and other serious allergic reactions and injection/infusion-related reactions are not specific. The risk of developing such conditions following administration with HBC34v35-MLNS-GAALIE is specifically unknown.

Part a of the study collected information on the safety and tolerability of HBC34v35-MLNS-GAALIE and related data on PK profile and anti-drug antibody (ADA) production. HBC34-v35-MLNS-GAALIE was not expected to provide benefits to healthy subjects who participated in part A of the study. The subject will be monitored for significant potential risk and will be subjected to routine medical alerts and risk minimization activities.

The potential benefits of HBC34-v35-MLNS-GAALIE in subjects with chronic HBV infection compared to current standards of care are:

● Reduce serum HBsAg, inhibit intrahepatic transmission of HBV, eliminate infected hepatocytes, and stimulate an adaptive immune response against HBV.

● Flood genotype therapy for HBV infection is well tolerated and SC is administered for a limited duration

In addition to allergic reactions, other severe allergic reactions, and injection/infusion-related reactions, the potential risks associated with the administration of HBC34-v35-MLNS-GAALIE to subjects with chronic HBV infection include immune complex diseases and hepatotoxicity due to the elimination of infected hepatocytes by ADCC/ADCP and/or cytotoxic T cells induced by vaccine effects. The study design of section B/C includes several elements that mitigate these risks:

● Fraction B was included in subjects with serum HBsAg <1000IU/mL to reduce the risk of immune complex disease and hepatotoxicity. In addition, SRC will review safety data for part B prior to incorporating subjects with potentially higher baseline HBsAg values into optional part C of the study.

● Parts B and C were included in subjects receiving NRTI and screened for HBV DNA <100IU/mL and had good liver reserves and low levels of liver inflammation at baseline, as determined by the following attributes: ALT or AST is less than or equal to 2 XULN, has no history of liver decompensation, and has no significant fibrosis and cirrhosis.

● Two sentinel subjects were randomly assigned to HBC34-v35-MLNS-GAALIE or placebo at 1:1 and dosed. These whistle subjects were monitored for at least 72 hours after dosing, and if the investigator did not find a safety issue, the remaining 6 subjects in the same cohort (5 active agents and 1 placebo) were dosed.

● The dose escalation occurs after up to 4 weeks of SRC review of available safety data following dose administration to account for potential immune complex disease and the expected time of hepatotoxicity due to elimination of infected hepatocytes by ADCC/ADCP and/or cytotoxic T cells induced by vaccine effects.

● Safety monitoring, including liver function testing, urinalysis, renal function, vital signs and physical examination results, is aimed at detecting evidence of HBC34-v35-MLNS-GAALIE related immune adverse events.

Part A

Three consecutive cohorts of part a evaluate 90g, up to 300mg and up to 900mg administered by SC injection. The SRC reviews available clinical and laboratory safety data up to 2 weeks after dosing for all available subjects in the cohort prior to dose escalation. Two optional cohorts in part a can be added to evaluate up to 900mg and 3000mg administered by IV infusion. Inclusion of these optional cohorts may be performed after the available week 2 data for all available subjects in SRC review cohort 3a (up to 900mg SC).

While all SC queues in part a (queues 1a, 2a and 3 a) are brought in order, the queues may be brought in parallel if the dose level that the additional queues are checking is at or below the dose level that was previously found to have acceptable safety and tolerability characteristics in the queues of part a.

In each cohort, 2 sentinel subjects were randomly assigned at 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo. Dosing and monitoring the subjects in a hospitalized environment for at least 24 hours; if the investigator did not find a safety issue, the remaining subjects in the same cohort were dosed. The remaining subjects were randomized at 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo.

The maximum dose escalation factor for part a does not exceed 5 times.

Part B

The first cohort in part B (cohort 1B) was included after the available week 2 data for all available subjects in SRC review cohort 1a (90 mg SC).

Part B was scheduled to evaluate five cohorts, each of 6mg (cohort 1B), 18mg (cohort 2B), up to 75mg (cohort 3B), up to 300mg (cohort 4B), and up to 900mg (cohort 5B) administered by SC injection. The SRC reviews available clinical and laboratory safety data and antiviral activity data for all available subjects in the previous cohort prior to dose escalation until 4 weeks post-dosing.

Two optional queues in section B may be added according to the same dosing regimen. The optional cohort is administered at a lower, equivalent or intermediate dose level relative to the dose level explored in the planned part B cohort or after cohort 5B at a dose level of no more than 900 mg. The maximum dose level of the optional cohort in part B does not exceed the highest single dose found in part a to have acceptable safety and tolerability characteristics. The optional queue is included at any time within the part B scheduled queue based on approval of the SRC.

While all of the queues in part B will be brought in order, the queues may be brought in parallel if the dose level that additional queues are checking is at or below the dose level that was previously found to have acceptable safety and tolerability characteristics in the queues of part a and part B.

In each cohort, 2 sentinel subjects were randomly assigned at 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection. These subjects were dosed and monitored for at least 72 hours post dosing (including hospitalization monitoring for at least the first 24 hours); if the investigator did not find a safety issue, the remaining subjects in the same cohort were dosed. The remaining subjects were randomized at 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection.

The maximum dose escalation factor for part B does not exceed 5 times.

Part C

Part C is optional and can be based on acceptable safety and tolerability characteristics of HBC34-v35-MLNS-GAALIE in HBeAg negative subjects with HBsAg levels <1000IU/mL in part B. The available data for all subjects in part a and part B is reviewed by the SRC until the first cohort in part C is entered after the 4 th week visit to the cohort of subjects in part B who are receiving a match or higher dose relative to the recommended starting dose level in part C.

Three optional queues may be included in section C. Each cohort can be assessed for up to 900mg administered by SC injection, and the dose used in the part C cohort does not exceed the highest dose level in part B found by SRC to have acceptable safety and tolerability characteristics. Queues may be brought in parallel.

In each cohort, 2 sentinel subjects were randomly assigned at 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection. These subjects were dosed and monitored for at least 72 hours post dosing (including hospitalization monitoring for at least the first 24 hours); if the investigator did not find a safety issue, the remaining subjects in the same cohort were dosed. The remaining subjects were randomized at 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo by SC injection.

Study procedure

Part A

Screening

● Healthy adult subjects will be included in 1 of the 5 cohorts in part a (3 in plan, optionally 2). Screening was performed no more than 4 weeks prior to day 1 visit and included written informed consent, qualification, demographic data collection and history, physical examination, vital signs, laboratory tests, 12-lead Electrocardiography (ECG), and other evaluations according to the evaluation schedule (SoA).

● Eligible subjects entered the clinical study site on day-1 or day 1. On day 1, qualification criteria related to vital signs, pregnancy tests, drugs of abuse, blood donation, the presence of any clinically significant acute pathology, and the use of prescription, OTC, herbal or study medication are evaluated to ensure continued qualification of the study. Any changes in the medical history will also be assessed and recorded. The eligible subjects in each cohort were randomly assigned to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to study drug administration. Subjects received a single dose of study drug (HBC 34-v35-MLNS-GAALIE or placebo) on day 1.

● Collecting Adverse Events (AEs) associated with the screening activity from the date of consent; any other events that occur during the screening period are reported as a medical history. All Serious Adverse Events (SAE) were collected from the date of consent.

● The serological parameters for the screening viruses were as follows: active infection with HIV, HCV and HBV

Day of administration (day 1)

● Eligible subjects were randomly assigned to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to study drug administration on day 1.

● The eligible subjects received a single dose of study drug and were evaluated for suitability on day 1.

● At the beginning of each cohort, 2 sentinel subjects were randomly assigned to either HBC34v-35-MLNS-GAALIE or placebo at 1:1. These subjects were dosed and monitored in a hospitalized environment for at least 24 hours. Vital signs, ECG, symptom-oriented physical examinations and AEs were reviewed by researchers; if the investigator did not find a safety issue, the remaining subjects in the same cohort were dosed. The remaining subjects in the cohort were randomly assigned at 5:1 to receive a single dose of HBC34-v35-MLNS-GAALIE or placebo. All subjects were monitored closely after dose administration.

Follow-up period

● After all study evaluations on day 2, subjects were discharged. All subsequent study visits were outpatient.

● Subjects returned to the clinical study site for on-site assessment according to SoA, including but not limited to physical examination, vital signs, laboratory tests, PK assessment, and review of AE and concomitant medications, until week 24.

B/C part

Screening

● Screening was performed no more than 4 weeks prior to day 1 visit and included written informed consent, qualification, demographic data collection and history, physical examination, vital signs, laboratory tests, 12-lead ECG, and other assessment according to SoA. Collecting adverse events associated with screening activities from the date of consent; any other events that occur during the screening period are reported as a medical history. All SAEs were collected from the date of consent.

● Adult subjects with HBeAg negative chronic HBV infection without cirrhosis and HBsAg <3000IU/mL and receiving NRTI therapy for > 2 months were included in part B (cohort 1B (6 mg SC), 1 cohort of 7 cohorts (5 of the programs, optionally 2) of participants with HBsAg <1,000 IU/mL). Subject screening occurred no more than 4 weeks prior to day 1 visit. Subjects entered the clinical study site on day-1 or day 1. On day 1, qualification criteria related to NRTI compliance, vital signs, pregnancy testing, any clinically significant acute pathology, liver function decompensation, and use of prescription, OTC, herbal or study drugs will be assessed to ensure continued qualification. Any changes in the medical history will also be assessed and recorded. Eligible subjects in each cohort were randomly assigned to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to study drug administration on day 1.

● To rule out the presence of cirrhosis, subjects in parts B and C were subjected to a fibriscan evaluation. If the subject underwent a fibriscan within 6 months prior to screening or a liver biopsy within the first year of screening, confirming that there was no Metavir F3 fibrosis or F4 cirrhosis, this procedure need not be performed.

● The serological parameters for the screening viruses were as follows: active infections with HIV, HCV and hepatitis d virus. Subjects with positive HCV serological results can be subjected to HCV-RT PCR reflex testing to determine eligibility.

● Chronic HBV infection will be determined at screening and defined as follows: based on previous or current laboratory documentation, 2 serum HBsAg, HBV DNA or HBeAg separated by at least 6 months are positive (any combination of these tests separated by 6 months is acceptable).

Day of administration (day 1)

● Eligible subjects were randomly assigned to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to study drug administration on day 1.

● Subjects entered the clinical study site on day 1.

● The eligible subjects received a single dose of study drug and will be evaluated for suitability on day 1.

● At the beginning of each cohort, 2 sentinel subjects were randomly assigned to either HBC34-v35-MLNS-GAALIE or placebo at 1:1. These subjects were dosed and monitored for at least 72 hours post dosing (including hospitalization monitoring for at least the first 24 hours); if the investigator did not find a safety issue, the remaining subjects in the same cohort were dosed. Vital signs, symptom-oriented physical examinations and AEs were reviewed by a researcher prior to administration to any additional subjects. The remaining subjects in the cohort were randomly assigned at 5:1 to receive a single dose of antibody composition or placebo. All subjects were monitored closely after dose administration.

Follow-up period

● After all study evaluations on day 2, subjects were discharged. All subsequent study visits were outpatient.

● Subjects returned to the clinical study site for evaluation according to SoA, including but not limited to physical examination, vital signs, laboratory tests, PK evaluation, efficacy evaluation, and review of AE and concomitant medications, until week 8.

Prolonging the follow-up period

Subjects with > 2-fold decrease in HBsAg at week 8 returned to the clinical study site within week 40 for on-site assessment according to SoA, or until HBsAg decreased < 2-fold from baseline at 2 consecutive collections, subject to first occurrence. Based on the newly emerging data, the extended follow-up may cease.

Product, dose and mode of administration

HBC34v35-MLNS-GAALIE is supplied in lyophilized solid form for reconstitution using sterile water for injection (USP) at a concentration of 150mg/mL and administration by SC injection or IV infusion. Unit doses are based on volume and method of administration. After reconstitution with sterile water for injection (USP) to 150mg/mL, the drug product at the time of administration contained 20mM histidine, 7% sucrose, 0.02% PS80, and pH 6. Placebo is a sterile, preservative-free physiological saline 0.9% solution for IV infusion or SC injection.

● Queue 1a: HBC34v35-MLNS-GAALIE, single dose 90mg, was administered by SC injection

● Queue 2a: HBC34v35-MLNS-GAALIE, single dose up to 300mg, administered by SC injection

● Queue 3a: HBC34v35-MLNS-GAALIE, single dose up to 900mg, administered by SC injection

● Queue 4a (optional): HBC34v35-MLNS-GAALIE, administered by IV infusion in a single dose of up to 900mg

● Queue 5a (optional): HBC34v35-MLNS-GAALIE, administered by IV infusion in a single dose of up to 3000mg

● Queue 1b: HBC34v35-MLNS-GAALIE, single dose 6mg, was administered by SC injection

● Queue 2b: HBC34v35-MLNS-GAALIE, single dose of 18mg, was administered by SC injection

● Queue 3b: HBC34v35-MLNS-GAALIE, single dose up to 75mg, administered by SC injection

● Queue 4b: HBC34v35-MLNS-GAALIE, single dose up to 300mg, administered by SC injection

● Queue 5b: HBC34v35-MLNS-GAALIE, single dose up to 900mg, administered by SC injection

● Queue 6b (optional): HBC34v35-MLNS-GAALIE, single dose up to 900mg, administered by SC injection

● Queue 7b (optional): HBC34v35-MLNS-GAALIE, single dose up to 900mg, administered by SC injection

● Queue 1c (optional): HBC34v35-MLNS-GAALIE, single dose up to 900mg, administered by SC injection

● Queue 2c (optional): HBC34v35-MLNS-GAALIE, single dose up to 900mg, administered by SC injection

● Queue 3c (optional): HBC34v35-MLNS-GAALIE, single dose up to 900mg, administered by SC injection

Table 8: part a dose escalation planning

IV = intravenous; sc=subcutaneous.

Part B: single escalation dose study of subjects with chronic HBV infection

In part B, subjects with chronic HBV infection received a single dose of study drug. The presence of HBC34-v35-MLNS-GAALIE therapeutic targets (i.e., HBsAg) in subjects with chronic HBV infection alters the potential risk of HBC34-v35-MLNS-GAALIE administration. Potential risks include immune complex diseases due to antigen-antibody complex formation and hepatotoxicity due to elimination of infected hepatocytes by ADCC/ADCP and/or "vaccine effect". To minimize the risk for the subject, part B will be performed in subjects receiving NRTI and screening for HBV DNA <100IU/mL and good liver reserves and low liver inflammation levels, as determined by the lack of fibrosis/cirrhosis and ALT <2 x ULN.

Five dose level cohorts were used for part B. The dose was stepped up approximately 3 to 4 times to reach a maximum planned dose of 900mg administered by SC injection:

Two optional cohorts were included to reach a maximum dose of 900mg administered by SC injection. The queue 7b may be included for, but is not limited to, collection and assessment of immune response samples and liver fine aspiration samples at times and places available at selected places when available. These dose levels are based on preclinical animal models and transformed PK/PD modeling, predicting a significant decrease in HBsAg in the 2 to 15mg/kg dose range. Details of the dose escalation plan for section B can be found in table 9.

Table 9: part B dose escalation planning

Sc=subcutaneous

Optional part C: single escalation dose study of subjects with chronic HBV infection

To assess the safety, tolerability and antiviral activity of HBC34-v35-MLNS-GAALIE in subjects with baseline HBsAg levels of > 1000IU/mL, an optional C-fraction was performed after the safety, tolerability and antiviral activity of HBC34-v35-MLNS-GAALIE had been established in HBeAg negative subjects with HBsAg <1000IU/mL in fraction B. Part C consisted of three optional dose level cohorts, with each cohort evaluating doses administered up to 900mg by SC injection. Table 10 one or more of the optional queues in section C may be incorporated for, but are not limited to, collecting and evaluating immune response samples and liver fine aspiration samples at selected locations when available.

Table 10: part C queue overview

Research section	Queues	Pathway	Dosage level	Active material: placebo
					C	1c (optional)	SC	At most 900mg	6:2
C	2c (optional)	SC	At most 900mg	6:2
					C	3c (optional)	SC	At most 900mg	6:2

Reference therapy, dose and mode of administration:

subjects randomly assigned to placebo were administered a sterile, preservative-free 0.9% physiological saline solution by SC injection (A, B and part C) or IV infusion (part a only).

Local tolerance

For all study sections, local tolerability assessment was performed according to the assessment schedule (figures for subjects receiving study drug by SC injection). The injection site will be marked and drawn for later viewing and should be recorded. Pain/tenderness, swelling, redness, bruising and itching at the injection site should be monitored.

Fig. 15A to 15C show the local tolerance evaluation time of the a portion. FIGS. 16A-16E show the local tolerance evaluation time of the B/C portion.

At the discretion of the investigator, an unplanned visit is allowed as needed to track any unresolved local tolerogenic symptoms.

Screening for drugs of abuse

Urine was collected for drug abuse screening for parts A, B and C of the study. The panel included amphetamine, cocaine, methadone, and opioids.

Pharmacokinetic assessment

Blood samples will be collected to assess the concentration of HBC34-v 35-MLNS-GAALIE. Provided herein are sample collection time points for HBC34-v35-MLNS-GAALIE PK analysis of study part a. Provided herein are sample collection time points for HBC34-v35-MLNS-GAALIE PK analysis for study of parts B and C.

Pharmacokinetic analysis

Part A

The free PK parameters of HBC34-v35-MLNS-GAALIE were calculated using standard non-compartmental methods. Parameters include, but are not limited to, serum C _max 、C _last 、T _max 、T _last 、AUC _inf 、AUC _last 、AUC _exp ％、 _t1/2 、λ _z 、V _z (IV only), CL (IV only), V _z F (SC only) and CL/F (SC only). Other parameters are calculated as needed.

B/C part

Calculation of free and Total PK parameters for HBC34-v35-MLNS-GAALIE using standard non-compartmental methodsA number. Parameters include, but are not limited to, serum C _max 、C _last 、T _max 、T _last 、AUC _inf 、AUC _last 、AUC _exp ％、t _1/2 、λz、V _z F and CL/F. Other parameters are calculated as needed.

PK/pharmacodynamic analysis was performed to explore exposure-response relationships between PK parameters and selected antiviral variables.

Antiviral Activity assay

For parts B and C, selected data relating to antiviral activity of HBC34-v35-MLNS-GAALIE, such as HBsAg, anti-HB, HBeAg, anti-HBe, HBV RNA, HBcrAg and HBV DNA levels (n, average, SD, median, Q1, Q3, minimum and maximum) are summarized in cohorts and study visits and corresponding changes from baseline. Summary of HBsAg loss (number and percentage of subjects) is provided by cohort and study visit (defined as undetectable HBsAg determined at 2 separate, consecutive times separated by at least 2 weeks).

Immunogenicity of

Blood samples were collected to analyze the immunogenic response to determine the presence/absence and titer (as applicable) of the drug-resistant antibodies (ADA) according to time points defined in the evaluation schedule (fig. 15A-16E). The HBC34-v35-MLNS-GAALIE (NAb) neutralization potential of the samples was characterized as appropriate.

Evaluation of screening viral parameters, antiviral Activity and drug resistance monitoring

In parts B and C, the evaluation of screening virus parameters includes: HBsAg, anti-HBs, HBeAg (qualitative) and HBV DNA.

Evaluation of antiviral activity following screening included: HBsAg, anti-HB, HBeAg (qualitative; should be collected only for part C subjects positive qualitatively for HBeAg at screening), HBeAg (quantitative; should be collected only for part C subjects positive qualitatively for HBeAg at screening), anti-HBe, HBV RNA, hepatitis b nuclear related antigen (HBcrAg) and HBV DNA.

All subjects receiving study drug were monitored for resistance to monitor potential development of resistance to NRTI or HBC34-v 35-MLNS-GAALIE. HBV genomic sequencing was attempted on subjects confirming HBV DNA breakthrough (as defined by HBV DNA.gtoreq.500 IU/mL measured at 2 consecutive study visits) or subjects terminating the study prematurely in the case of HBV DNA.gtoreq.500 IU/mL. Since it is not known at visit whether the subject has a virologic breakthrough, samples for resistance monitoring were collected in all study visits noted in SOA. Samples collected for resistance monitoring may be used for additional viral analysis, including viral sequencing.

Immune response assessment

To examine potential biomarkers of host immune response and infection, subjects may agree with an optional sub-study in which peripheral immune samples with or without liver immune samples (by fine needle aspiration) will be collected at the time points outlined in fig. 15A-16E. These optional sub-studies and related evaluations are performed at times and locations available at the selected location.

Fcgamma receptor (fcγr) genotyping and immunoglobulin allotypic forms

Blood samples for fcγr genotyping and immunoglobulin allotypes were collected at baseline for all subjects in parts B and C to assess the likely correlation between fcγreceptor polymorphism or immunoglobulin allotype and antiviral activity of HBC34-v 35-MLNS-GAALIE.

Statistical method

Statistical analysis is mainly descriptive. All study data are presented by a subject data list. For all study parts, the summary table presented the results of HBC34-v35-MLNS-GAALIE and placebo on a cohort basis, with placebo subjects combined across dose cohorts on a per part route of administration.

The study was conducted according to ethical principles derived from the declaration of helsinki (Declaration of Helsinki) and met the requirements of Good Clinical Practice (GCP) and applicable regulatory requirements, including archiving of important documents.

Abbreviations and term definition lists in this example

ADA drug-resistant antibodies

AE adverse events

ALT alanine aminotransferase

ANC absolute neutrophil count

AP alkaline phosphatase

AST aspartate aminotransferase

Area under AUC curve

BLQ below quantitative limit

BMI body mass index

BUN blood urea nitrogen

CLcr creatinine clearance rate

CRF case report forms

General adverse event terminology standard for CTCAE

DNA deoxyribonucleic acid

ECG electrocardiogram

eCRF electronic case report form

EF follow-up end

ET treatment end

FDA U.S. food and drug administration (Food and Drug Administration)

Good clinical practice of GCP

GGT gamma glutamyl transferase

GLP good laboratory specification (Good Laboratory Practice)

GNA ethylene glycol nucleic acid

HBcrAg hepatitis B nuclear related antigen

HBeAg hepatitis B e antigen

HBIG hepatitis B immunoglobulin

HBsAg hepatitis B surface antigen

HBV hepatitis B virus

HCC hepatocellular carcinoma

HED human equivalent dose

Hgb hemoglobin

ICF informed consent

ICH International conference of coordination (International Conference on Harmonisation)

IgG immunoglobulin G

IgM immunoglobulin M

IEC independent ethics committee

INR International normalized ratio

IRB institutional review board

IV intravenous

IWRS interactive network response system

LDH lactate dehydrogenase

LLN lower normal limit

LLOQ quantitative lower limit

LLT low-level terminology

mAb monoclonal antibodies

Meddra medical dictionary for medical matter management

Nab neutralizing antibodies

No side effect levels were observed with NOAEL

OTC over-the-counter medicine

PK pharmacokinetics

PT preferred terminology

Q1 first quartile

Q3 third quartile

RBC red blood cells (count)

RNA ribonucleic acid

SAD single increment dose

SAE serious adverse events

SC subcutaneous

Standard deviation of SD

SoA evaluation schedule

SOC system organ

SRC Security review Committee

SUSAR suspected unexpected serious adverse reactions

TCR tissue cross-reactivity

Adverse events occurring in TEAE treatment

US united states

ULN upper normal limit

WBC white blood cells (count)

WHO world health organization (World Health Organization)

WOCBP fertility female

Example 10: HBsAg activates dendritic cells: HBC34-v35 antibody immunocomplexes

Activation of monocyte-derived (mo) DCs in the presence of Immune Complexes (IC) (vendor: bioIVT) formed by HBC34-V35-MLNS_GAALIE (HC SEQ ID NO:91,LC SEQ ID NO:93) or HBC34-V35-MLNS (HC SEQ ID NO:92,LC SEQ ID NO:93) and HBsAg in HBV+ patient serum was tested.

Materials and methods:

cd14+ monocytes were isolated from human PBMCs of healthy donors (n=2) and cultured in RPMI 1640, 10% fbs (Hyclone), 1% non-essential amino acids, 1% glutamine, 1% pen/Strep, 1% sodium pyruvate, 50 μΜ β -mercaptoethanol, 50ng/mL GM-CSF (meitian) and 1000U/mL IL-4 (R & D) for 6 days. Differentiated immature monocyte-derived DCs (moDCs) were then stimulated with HBsAg alone (diluted to final 250IU/ml in serum of both patients at 1890 and 4460 IU/ml), with HBsAg and HBC34-v35-MLNS or IC of HBC34-v35-MLNS_GAALIE (mAb at 20-100. Mu.g/ml) or mAb alone for 22 hours. The reagent was tested to be endotoxin free. The surface expression of the co-stimulatory markers CD83 and CD86 and HLA-DR was measured by flow cytometry. The levels of ten (10) human pro-inflammatory cytokines (IFN, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF. Alpha.) were measured using the Meso Scale Diagnostics (MSD) V-PLEX pro-inflammatory panel 1 human kit. The medium was used as a negative control. LPS (Sigma, 100 ng/ml) was used as positive control.

The data is shown in fig. 20-24B. Immune Complexes (IC) of HBsAg and HBC34-v35-MLNS-GAALIE can induce upregulation of the moDC surface costimulatory markers CD83 and CD86 and HLA-DR. In addition, the IC of HBsAg with HBC34-v35-MLNS-GAALIE induces moDC to secrete cytokines TNFa, IL-6 and IL-10.

Example 11: reduction of HBsAg in human subjects

The phase 1 clinical study described in example 9 included SAD a and B part cohorts as shown in fig. 25. Extrapolated from preclinical data, a computer simulated model was generated and predicted to reduce HBsAg by about 1.5log10 IU/mL after 60mg HBC34-v35-MLNS-GAALIE x 4 doses (FIG. 26).

Figure 27 summarizes certain demographics and baseline characteristics of subjects administered HBC34-v35-MLNS-GAALIE at 6mg (cohort 1 b), 18mg (cohort 2 b), or 75mg (cohort 3 b).

In the part B SAD cohort shown in FIG. 25 (HsAg.ltoreq.3000 IU/mL of subjects with chronic HBV), cohort 1B (lowest dose cohort, 6mg S.C.), cohort 2B (18 mg S.C.), and cohort 3B (75 mg S.C.) each included two of eight subjects receiving placebo, while the remaining six subjects received HBC34-v35-MLNS-GAALIE.

The subject was well tolerated by the HBC34-v35-MLNS-GAALIE antibody (FIG. 28). No clinically significant laboratory abnormalities or changes were observed in liver function tests. No injection site reaction was observed. No subjects developed clinical or laboratory signs of immune complex disease. Two subjects in cohort 1b reported a total of two adverse events of grade 1, which were allergic rhinitis and palpitations but no objective tachycardia. Four subjects in cohort 2b reported a total of four adverse events, which were chest discomfort (non-cardiac) grade 1 adverse events; scratch and dizziness of the right knee of the subject; seasonal allergic grade 2 adverse reactions. Four subjects in cohort 3b reported a total of eight adverse events, three grade 1 adverse events, namely headache, musculoskeletal stiffness, and nasopharyngitis, which were considered to be related to study drug; four adverse events grade 1, namely ear infection, fatigue, acne and dry pharynx; and a grade 2 adverse event, namely headache. Although in cohorts 1B and 2B no shift in the level of alanine Aminotransferase (ALT) or a value above the Upper Limit of Normal (ULN) was observed at 29 days post-dosing (fig. 29A-29B), one participant in cohort 1B showed an elevation of level 1 (ALT) 24 weeks post-dosing.

Sixteen of the subjects receiving HBC34-v35-MLNS-GAALIE antibodies rapidly achieved >1log10 IU/mL HBsAg decline within about 1 week after dosing in part B SAD cohorts (FIG. 30A). No detectable or lower than expected free PK of HBC34-v35-MLNS-GAALIE antibodies was reported in both participants in cohort 2B, with a decrease in HbsAg of approximately <0.5log10 IU/mL (fig. 30A, 30B, 32 and 33B).

Surprisingly, for blind cohort 1B, the serum hepatitis B virus surface antigen (HBsAg) cut-off for the eighth balance was reduced by 1.3log10 IU/mL for all six subjects, while most subjects reached the lowest point the day (fig. 30A, 30B and 31). Notably, these results using 6mg x 1 exceeded predictions made using the computer simulation model of 60mg x 4 (fig. 26).

Similarly, serum HBsAg for four subjects decreased by more than 1.5log10iu/mL on day eight for blind cohort 2B (fig. 30A, 30B, 31 and 32). Notably, these results using 18mg x 1 exceeded predictions made using the computer simulation model of 60mg x 4 (see fig. 26). No HBC34-v35-MLNS-GAALIE antibodies were detected in samples of two subjects receiving a dose of 18mg but not achieving a HbsAg reduction of 1.5log10 IU/mL.

Within the range of baseline HBsAg values, all participants with a decrease of ≡1log10 IU/mL compared to baseline achieved absolute levels below 30IU/mL at the nadir (FIGS. 30A, 30B, 31 and 32). Five of the six subjects in cohort 3B achieved absolute levels below 10IU/mL at the nadir (fig. 30A and 30B). Furthermore, the subjects in cohort 3b had a maximum decrease in HBsAg>2log ₁₀ IU/mL), and the duration is the longest. Average 1.96log10 IU/mL at nadir and 1.5log10 IU/mL at day 29 (FIG. 31).

Example 12: reduced dose-dependent durability of hepatitis B surface antigen following a single dose of HBC34-v35-MLNS-GAALIE

This example provides data from an ongoing phase 1 study that evaluates the safety, tolerability and antiviral activity of HBC34-v35-MLNS-GAALIE in participants with chronic HBV infection.

The method comprises the following steps: this randomized, double-blind, placebo-controlled phase 1 single escalation dose study included adults with hepatitis b e antigen (HBeAg) negative chronic HBV infection without cirrhosis. The participants were screened for HBsAg <3,000IU/mL and received nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI) therapy for > 2 months. Eight participants in each cohort were randomly allocated at 6:2 to receive 6mg, 18mg, 75mg or 300mg of HBC34-v35-MLNS-GAALIE or placebo in a single subcutaneous dose. Preliminary data are presented over 8 weeks of follow-up; dose escalation and follow-up are ongoing.

Results: twenty four participants were included. Most participants had an HBsAg decrease of 1log or more compared to baseline within 1-3 days of dosing ₁₀ IU/mL (FIGS. 30A, 30B, 31 and 32). The greatest and most durable HBsAg reduction was observed in the 75mg cohort (figures 30A, 30B and 31). HBsAg reduction in each queue>0.2log ₁₀ The average decrease at nadir for 6mg, 18mg and 75mg groups among 6 participants in IU/mL was 1.30, 1.27 and 1.96log, respectively ₁₀ IU/mL, and at week 8 are 0.17, 0.20 and 0.82log, respectively ₁₀ IU/mL (FIG. 31). Ten adverse events were reported and the severity was either level 1 or level 2 (fig. 28). No clinically significant laboratory abnormalities or signs of immune complex disease were observed. Data for participants receiving a single dose of 300mg HBC34-v35-MLNS-GAALIE are presented in FIGS. 30A and 30B.

Conclusion: a single dose of 6mg, 18mg or 75mg HBC34-v35-MLNS-GAALIE showed a rapid decrease in HBsAg. HBC34-v35-MLNS-GAALIE is generally safe and well tolerated. These data support the use of HBC34-v35-MLNS-GAALIE for functionally curing patients suffering from chronic HBV infection.

Example 13: measurement of PK in human subjects

The purpose of this study was to characterize serum Pharmacokinetics (PK) in subjects with HBC34-v35-MLNS-GAALIE in SADB cohorts, SADC cohort 1C.

The concentration of HBC34-v35-MLNS-GAALIE in serum was determined using a validated electrochemiluminescence method on a Meso Scale Discovery (Rockwell, malyland) platform, where LLOQ was 10ng/mL. UsingThe PK parameters were estimated by standard non-compartmental methods in V8.2 (Certara l.p., princeton, NJ) and summarized using descriptive statistics. By equation ba=auc _sc /AUC _iv X 100 calculation of bioavailability (F%) with AUC _iv And AUC _sc The area under the curve after SC administration is indicated.

In cohort 3b, all 6 active participants were in agreement with a corresponding >1log HBsAg decline in free and total HBC34-v35-MLNS-GAALIE after a single 75mg dose, which lasted for at least 28 days (fig. 33C and 35C). The total PK profile of the cohorts 1B and 3B methods projected PK profiles based on data of healthy subjects (figures 35A, 35B, 36, 39 and 40). In cohorts 2b and 1c, unexpected free HBC34-v35-MLNS-GAALIE PK was observed for 7 of the 12 active subjects following the 18mg dose. Furthermore, low antiviral activity (< 0.5log HBsAg) was observed in 4 out of 7 subjects exhibiting unexpected PK profile. The total PK was consistent with the free PK in 8/24HBV participants who had unexpected free PK properties at 6mg (B n =1/6; fig. 33A and 35A) and 18mg (B n =3/6; 1cn=4/6; fig. 33B and 35B).

Example 14: measurement of pharmacokinetics in human subjects and health safety in human subjects

Forty-one healthy human subject (each cohort n=8, one of which was replaced for non-safety related reasons) was enrolled and accepted HBC34-v35-MLNS-GAALIE or placebo (fig. 37). The cohort received a single dose of 90mg (subcutaneous), 300mg (subcutaneous), 900mg (intravenous), or 3,000mg (intravenous). After subcutaneous injection, HBC34-v35-MLNS-GAALIE is absorbed, wherein the median T _max Seven days. Exposure (C) _max And AUC) was increased in dose-scale fashion in the subcutaneous range of 90-900mg (fig. 39). Inter-subject variability within each cohort is generally low (CV 35%). The PK profile of HBC34-v35-MLNS-GAALIE was consistent with that of a typical IgG, with a half-life of about 28 days. HBC34-v35-MLNS-GAALIE in the range of 90-3,000mThe g dose range was well tolerated (figure 38). Subjects totaling 24/41 (59%) experienced adverse events, which were predominantly grade 1 (FIG. 38). No serious adverse events were reported and no clinically significant effect on laboratory parameters or electrocardiography was observed. These results demonstrate that HBC34-v35-MLNS-GAALIE is safe and well tolerated in healthy subjects after a single dose of up to 3,000mg and exhibits favorable PK properties supporting subcutaneous administration.

Example 15: measurement of pharmacokinetics and health safety in human subjects

The purpose of this study was to assess the safety and tolerability of HBC34-v35-MLNS-GAALIE in healthy subjects and to characterize its serum Pharmacokinetics (PK).

Part A is a randomized, blind, placebo-controlled study of HBC34-v35-MLNS-GAALIE in healthy subjects aged 18-55 years, wherein creatinine clearance is <90 ml/min. FIG. 37 summarizes certain demographic characteristics of the subjects and the dose of HBC34-v35-MLNS-GAALIE administered to the subjects. Eight subjects per cohort were randomly allocated at 6:2 to receive a single dose of HBC34-v35-MLNS-GAALIE or placebo by Subcutaneous (SC) or Intravenous (IV) injection. Serum PK samples were collected at 24 hours (day 1) and 3 days, day 7, day 14, and week 4, week 8, week 12, week 18, week 24.

The concentration of HBC34-v35-MLNS-GAALIE in serum was determined using a validated electrochemiluminescence method on a Meso Scale Discovery (Rockwell, malyland) platform, where LLOQ was 10ng/mL. UsingThe PK parameters were estimated by standard non-compartmental methods in V8.2 (l.p.) and summarized using descriptive statistics. By equation ba=auc _sc /AUC _iv X 100 calculation of bioavailability (F%) with AUC _iv And AUC _sc The area under the curve after IV and SC administration is shown, respectively. Adverse Event (AE) monitoring, clinical laboratory and physical examination, and electrocardiographic assessment were performed throughout the study. At the position ofInjection site tolerance assessment was performed about 30 minutes, 2 hours, 12 hours, 24 hours and 48 hours, and 1 week after administration.

Adverse events are summarized in fig. 38. The AE most commonly reported in all groups was headache, which was observed in 10/41 (24.4%) participants. 6/41 (14.6%) participants reported injection site responses and all participants were of grade 1 severity, except grade 2 AE of injection site erythema resolved without intervention. No grade 3/4 AE, SAE or AE that led to study discontinuation were observed. No clinically significant laboratory abnormalities were observed.

After SC injection, HBC34-v35-MLNS-GAALIE is absorbed, wherein the median T _max For 3-7 days, and preliminary half-life t _1/2 For about 25 days. C was observed in the SC dose range of 90-900mg _max And AUC _inf Is increased in the dosage ratio. Inter-subject variability within each cohort was generally low (CV of about 35%) for all PK parameters. After SC administration, the bioavailability of HBC34-v35-MLNS-GAALIE was about 76%. PK data are summarized in figures 39 and 40.

Thus, HBC34-v35-MLNS-GAALIE was well tolerated in healthy volunteers after a single dose of up to 3000 mg. Adverse events were usually mild and no adverse events led to study discontinuation. Systemic exposure to HBC34-v35-MLNS-GAALIE was maintained for 24 weeks within the dose range assessed. It was estimated that the bioavailability and half-life of SC-administered HBC34-v35-MLNS-GAALIE were 76% and 25 days, respectively.

Example 16: phase 1 clinical study, part D

Part D is a randomized, double-blind, placebo-controlled, single-increment dose study of HBC34-v35-MLNS-GAALIE administered to adult subjects with chronic HBV infection without cirrhosis, who did not receive nucleotide (acid) reverse transcriptase inhibitor therapy, were HBV DNA ≡1,000iu/mL, were negative for HBeAg or positive for HBeAg and had any HBsAg level. Part D contains three optional cohorts, each cohort consisting of 8 participants who will be randomly allocated at 6:2 to receive HBC34-v35-MLNS-GAALIE or placebo on study day 1. Each cohort can be assessed for doses of up to 900mg of HBC34-v35-MLNS-GAALIE administered by subcutaneous injection. Subjects will return to the clinical study site for evaluation, including but not limited to physical examination, vital signs, laboratory tests, pharmacokinetic assessment, efficacy assessment, and review of adverse events and concomitant medications, until week 8. Subjects meeting certain criteria at week 8 visit may be followed up to week 40.

Sequence and SEQ ID numbering table (sequence Listing):

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all U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patent applications, and non-patent publications cited in this specification or the accompanying application data sheets are incorporated herein by reference in their entirety to the extent not inconsistent with this specification, including U.S. provisional application No. 63/141,915, filed at month 26 of 2021; U.S. provisional application No. 63/142,779, filed on 28 at 1 in 2021; U.S. provisional application No. 63/209,875, filed on day 11, 6, 2021; U.S. provisional application No. 63/255,921 filed on day 10 and 14 of 2021; and U.S. provisional application No. 63/280,971 filed on day 18 of 11 of 2021.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. The invention, therefore, is not to be restricted except in the spirit of the appended claims.

Sequence listing

<110> Weir Biotech Co., ltd (Vir Biotechnology, inc.)

<120> compositions and methods for treating hepatitis B virus infection

<130> 930485.433WO

<150> US 63/141,915

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<150> US 63/209,875

<151> 2021-06-11

<150> US 63/255,921

<151> 2021-10-14

<150> US 63/280,971

<151> 2021-11-18

<160> 139

<170> patent In version 3.5

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<220>

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<400> 1

Xaa Xaa Xaa Thr Cys Xaa Xaa Xaa Ala Xaa Gly

1 5 10

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<213> hepatitis B Virus

<220>

<221> VARIANT

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<221> VARIANT

<222> (2)..(2)

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<220>

<221> VARIANT

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Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln

1 5 10 15

Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu

20 25 30

Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Thr Thr Val Cys

35 40 45

Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser

50 55 60

Cys Pro Pro Thr Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe

65 70 75 80

Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val

85 90 95

Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly

100 105 110

Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Met Thr Thr Ala

115 120 125

Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp

130 135 140

Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys

145 150 155 160

Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu

165 170 175

Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu

180 185 190

Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile

195 200 205

Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val

210 215 220

Tyr Ile

225

<210> 4

<211> 226

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(226)

<223> S domain of HBsAg (Genbank accession number FJ 899792)

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Met Glu Asn Val Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln

1 5 10 15

Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu

20 25 30

Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Thr Thr Val Cys

35 40 45

Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser

50 55 60

Cys Pro Pro Thr Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe

65 70 75 80

Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val

85 90 95

Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly

100 105 110

Ser Ser Thr Thr Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala

115 120 125

Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp

130 135 140

Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys

145 150 155 160

Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu

165 170 175

Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu

180 185 190

Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Thr

195 200 205

Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val

210 215 220

Tyr Ile

225

<210> 5

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> J02203 (D, ayw3)

<400> 5

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 6

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> FJ899792 (D, adw2)

<400> 6

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 7

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> AM282986 (A)

<400> 7

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Thr Thr Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser

20 25 30

Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu

50 55 60

Trp Ala Ser Val Arg Phe Ser Trp

65 70

<210> 8

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> D23678 (B1)

<400> 8

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu

50 55 60

Trp Ala Ser Val Arg Phe Ser Trp

65 70

<210> 9

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> AB117758 (C1)

<400> 9

Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly Thr Ser Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala Gln Gly Thr Ser

20 25 30

Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg Phe Leu Trp Glu

50 55 60

Trp Ala Ser Val Arg Phe Ser Trp

65 70

<210> 10

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> AB205192 (E)

<400> 10

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Arg Thr Cys Thr Thr Leu Ala Gln Gly Thr Ser

20 25 30

Met Phe Pro Ser Cys Cys Cys Ser Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 11

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> X69798 (F4)

<400> 11

Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly Ser Thr Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Leu Ala Gln Gly Thr Ser

20 25 30

Met Phe Pro Ser Cys Cys Cys Ser Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Leu Gly Lys Tyr Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 12

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> AF160501 (G)

<400> 12

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu

50 55 60

Trp Ala Ser Val Arg Phe Ser Trp

65 70

<210> 13

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> AY090454 (H)

<400> 13

Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly Ser Thr Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Leu Ala Gln Gly Thr Ser

20 25 30

Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Tyr Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 14

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> AF241409 (I)

<400> 14

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 15

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> AB486012 (J)

<400> 15

Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly Ser Thr Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Arg Thr Cys Thr Ile Thr Ala Gln Gly Thr Ser

20 25 30

Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Val Arg Phe Ser Trp

65 70

<210> 16

<211> 73

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(73)

<223> HBsAg Y100C/P120T

<400> 16

Cys Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr

1 5 10 15

Thr Gly Thr Gly Thr Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr

20 25 30

Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys

35 40 45

Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp

50 55 60

Glu Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 17

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg P120T

<400> 17

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Thr Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 18

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg P120T/S143L

<400> 18

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Thr Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Leu Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 19

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg C121S

<400> 19

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Ser Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 20

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg R122D

<400> 20

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Asp Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 21

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg R122I

<400> 21

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Ile Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 22

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg T123N

<400> 22

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Asn Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 23

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg Q129H

<400> 23

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala His Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 24

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg Q129L

<400> 24

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Leu Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 25

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg M133H

<400> 25

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

His Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 26

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg M133L

<400> 26

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Leu Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 27

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg M133T

<400> 27

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Thr Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 28

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg K141E

<400> 28

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Glu Pro Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 29

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg P142S

<400> 29

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Ser Ser Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 30

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg S143K

<400> 30

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Lys Asp Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 31

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg D144A

<400> 31

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Ala Gly Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 32

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg G145R

<400> 32

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Arg Asn Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 33

<211> 72

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(72)

<223> HBsAg N146A

<400> 33

Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr

1 5 10 15

Gly Thr Gly Pro Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser

20 25 30

Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Ala Cys Thr

35 40 45

Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu

50 55 60

Trp Ala Ser Ala Arg Phe Ser Trp

65 70

<210> 34

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRH1

<400> 34

Gly Arg Ile Phe Arg Ser Phe Tyr

1 5

<210> 35

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRH2

<400> 35

Asn Gln Asp Gly Ser Glu Lys

1 5

<210> 36

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRH3

<400> 36

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val

1 5 10

<210> 37

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL1

<400> 37

Lys Leu Gly Asn Lys Asn

1 5

<210> 38

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL2

<400> 38

Glu Val Lys

1

<210> 39

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL2 Length

<400> 39

Val Ile Tyr Glu Val Lys Tyr Arg Pro

1 5

<210> 40

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL3

<400> 40

Gln Thr Trp Asp Ser Thr Thr Val Val

1 5

<210> 41

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequences HBC34, HBC34-V7, HBC34-V34, HBC34-V35 VH

<400> 41

Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Ser Val Ser Ser

115

<210> 42

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 VL

<400> 42

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Cys Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Trp Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 43

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRH1

<400> 43

ggacgcatct ttagaagttt ttac 24

<210> 44

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRH2

<400> 44

ataaaccaag atggaagtga gaaa 24

<210> 45

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRH3

<400> 45

gcggcttgga gcggcaatag tgggggtatg gacgtc 36

<210> 46

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL1

<400> 46

aaattgggga ataaaaat 18

<210> 47

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL2

<400> 47

gaggttaaa 9

<210> 48

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL2 Length

<400> 48

gtcatctatg aggttaaata ccgcccc 27

<210> 49

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 CDRL3

<400> 49

cagacgtggg acagcaccac tgtggtg 27

<210> 50

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 VH

<400> 50

gaactgcagc tggtggagtc tgggggaggc tgggtccagc cgggggggtc ccagagactg 60

tcctgtgcag cctctggacg catctttaga agtttttaca tgagctgggt ccgccaggcc 120

ccagggaagg ggctggagtg ggtggccact ataaaccaag atggaagtga gaaattatat 180

gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactattt 240

ctgcaaatga acaacctgag agtcgaggac acggccgttt attactgcgc ggcttggagc 300

ggcaatagtg ggggtatgga cgtctggggc caggggacca cggtctccgt ctcctca 357

<210> 51

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 VL

<400> 51

tcctatgagc tgactcagcc accctcagtg tccgtgtccc caggacagac agtcagcatc 60

ccctgctctg gagataaatt ggggaataaa aatgtttgct ggtttcagca taagccaggc 120

cagtcccctg tgttggtcat ctatgaggtt aaataccgcc cctcggggat tcctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg cctatttctg tcagacgtgg gacagcacca ctgtggtgtt cggcggaggg 300

accaggctga ccgtccta 318

<210> 52

<211> 33

<212> PRT

<213> hepatitis B Virus

<220>

<221> VARIANT

<222> (1)..(33)

<223> Xaa = any amino acid

<400> 52

Xaa Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Met Thr

1 5 10 15

Xaa Pro Ser Asp Gly Asn Ala Thr Ala Ile Pro Ile Pro Ser Ser Trp

20 25 30

Xaa

<210> 53

<211> 15

<212> PRT

<213> hepatitis B Virus

<400> 53

Thr Ser Thr Gly Pro Cys Arg Thr Cys Met Thr Thr Ala Gln Gly

1 5 10 15

<210> 54

<211> 26

<212> PRT

<213> hepatitis B Virus

<400> 54

Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser

1 5 10 15

Thr Gly Pro Cys Arg Thr Cys Met Thr Thr

20 25

<210> 55

<211> 33

<212> PRT

<213> hepatitis B Virus

<220>

<221> VARIANT

<222> (1)..(33)

<223> Xaa = any amino acid

<400> 55

Xaa Ser Met Tyr Pro Ser Ala Ser Ala Thr Lys Pro Ser Asp Gly Asn

1 5 10 15

Xaa Thr Gly Pro Cys Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser

20 25 30

Xaa

<210> 56

<211> 11

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(11)

<223> amino acids 120-130 of the S domain of HBsAg (HBV-D J02203)

<400> 56

Pro Cys Arg Thr Cys Met Thr Thr Ala Gln Gly

1 5 10

<210> 57

<211> 11

<212> PRT

<213> hepatitis B Virus

<220>

<221> VARIANT

<222> (3)..(3)

<223> xaa=r or K

<220>

<221> VARIANT

<222> (6)..(6)

<223> xaa=m or T

<220>

<221> VARIANT

<222> (7)..(7)

<223> Xaa=T or I

<220>

<221> VARIANT

<222> (8)..(8)

<223> xaa= T, P or L

<400> 57

Pro Cys Xaa Thr Cys Xaa Xaa Xaa Ala Gln Gly

1 5 10

<210> 58

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V7 CDRL3 and HBC34-V23 CDRL3

<400> 58

Gln Thr Phe Asp Ser Thr Thr Val Val

1 5

<210> 59

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V7 VL

<400> 59

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Cys Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 60

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34V7 CDRL1 and HBC-V23 CDRL1

<400> 60

aagctgggga acaaaaat 18

<210> 61

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V7 CDRL2 and HBC34V23 CDRL2

<400> 61

gaggtgaaa 9

<210> 62

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V7 CDRL2 Length and CDRL2 HBC34-V23 Length

<400> 62

gtcatctacg aggtgaaata tcggcct 27

<210> 63

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences CDRL3 HBC34-V7 and CDRL3 HBC34-V23

<400> 63

cagacattcg attccaccac agtggtc 27

<210> 64

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V7, HBC34-V34 and HBC34-V35 VL

<400> 64

tcttacgagc tgacacagcc acctagcgtg tccgtctctc caggacagac cgtgtccatc 60

ccttgctctg gcgacaagct ggggaacaaa aatgtctgtt ggttccagca caagccaggg 120

cagagtcccg tgctggtcat ctacgaggtg aaatatcggc cttcaggaat tccagaacgg 180

ttcagcggat caaacagcgg caatactgca accctgacaa ttagcgggac ccaggccatg 240

gacgaagccg cttatttctg ccagacattc gattccacca cagtggtctt tggcggggga 300

actaggctga ccgtgctg 318

<210> 65

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V23 VL

<400> 65

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Ala

20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 66

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34wt CDRH2

<400> 66

Ile Asn Gln Asp Gly Ser Glu Lys

1 5

<210> 67

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V31, HBC34-V32 and HBC34-V33 VH

<400> 67

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 68

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V31, HBC34-V32 and HBC34-V33 VH

<400> 68

gaggtgcagc tggtggaatc cggcggggga ctggtgcagc ctggcggctc actgagactg 60

agctgtgcag cttctggaag aatcttcaga tctttttaca tgagttgggt gagacaggct 120

cctgggaagg gactggagtg ggtcgcaaac atcaatcagg acggatcaga aaagctgtat 180

gtggatagcg tcaaaggcag gttcactatt tcccgcgaca acgccaaaaa ttctctgttt 240

ctgcagatga acaatctgcg ggtggaggat accgctgtct actattgtgc agcctggtct 300

ggcaacagtg gaggcatgga cgtgtgggga cagggaacca cagtgacagt cagctcc 357

<210> 69

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v23 VL

<400> 69

tcttacgagc tgacacagcc ccctagcgtg tccgtctctc caggccagac agcatccatc 60

acttgctctg gcgacaagct ggggaacaaa aatgcctgtt ggtatcagca gaagccaggg 120

cagagtcccg tgctggtcat ctacgaggtg aaatatcggc cttcaggaat tccagaaaga 180

ttcagtggat caaacagcgg caatactgct accctgacaa ttagcgggac ccaggccatg 240

gacgaagctg attactattg ccagacattc gattccacca cagtggtctt tggcggggga 300

actaagctga ccgtgctg 318

<210> 70

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt VH codon optimized

<400> 70

gaactgcagc tggtcgaatc aggaggaggg tgggtccagc ccggagggag ccagagactg 60

tcttgtgccg catcagggag gatcttcagg agcttctaca tgtcctgggt gcgccaggca 120

ccaggcaagg gactggagtg ggtcgccacc atcaaccagg acggatctga aaagctgtat 180

gtggatagtg tcaaaggccg gttcacaatt agcagagaca acgctaaaaa ttctctgttt 240

ctgcagatga acaatctgcg agtggaggat accgccgtct actattgcgc cgcttggtct 300

ggcaacagcg gcgggatgga tgtctggggg cagggcacaa cagtgagcgt ctcttcc 357

<210> 71

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt VL codon optimized

<400> 71

tcatacgaac tgactcagcc tccctccgtc tccgtctcac ctggacagac cgtctcaatc 60

ccctgctccg gcgataaact gggcaacaag aacgtgtgct ggttccagca caaacccgga 120

cagagtcctg tgctggtcat ctacgaggtc aagtatcggc caagcggcat tcccgaaaga 180

ttcagcggct ccaactctgg gaataccgca acactgacta tctctggaac ccaggcaatg 240

gacgaggcag cttacttttg ccagacttgg gattcaacta ctgtcgtgtt cggcggcgga 300

actagactga ctgtcctg 318

<210> 72

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt CDRH1 codon optimized

<400> 72

gggaggatct tcaggagctt ctac 24

<210> 73

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt CDRH2 codon optimized

<400> 73

atcaaccagg acggatctga aaag 24

<210> 74

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt CDRH3 codon optimized

<400> 74

gccgcttggt ctggcaacag cggcgggatg gatgtc 36

<210> 75

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt CDRL1 codon optimized

<400> 75

aaactgggca acaagaac 18

<210> 76

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt CDRL2 codon optimized

<400> 76

gaggtcaag 9

<210> 77

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt CDRL2 Long codon optimized

<400> 77

gtcatctacg aggtcaagta tcggcca 27

<210> 78

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34 wt CDRL3 codon optimized

<400> 78

cagacttggg attcaactac tgtcgtg 27

<210> 79

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic linker sequence

<400> 79

Gly Gly Ser Gly Gly

1 5

<210> 80

<211> 7

<212> PRT

<213> hepatitis B Virus

<400> 80

Thr Gly Pro Cys Arg Thr Cys

1 5

<210> 81

<211> 7

<212> PRT

<213> hepatitis B Virus

<400> 81

Gly Asn Cys Thr Cys Ile Pro

1 5

<210> 82

<211> 25

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(25)

<223> discontinuous epitope mimetic

<220>

<221> VARIANT

<222> (2)..(2)

<223> cysteine coupling to acetamidomethyl

<220>

<221> VARIANT

<222> (21)..(21)

<223> cysteine coupling to acetamidomethyl

<220>

<221> VARIANT

<222> (24)..(24)

<223> cysteine coupling to acetamidomethyl

<400> 82

Cys Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Cys Ser Thr Thr

1 5 10 15

Ser Thr Gly Pro Cys Arg Thr Cys Cys

20 25

<210> 83

<211> 28

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(28)

<223> discontinuous epitope mimetic

<220>

<221> VARIANT

<222> (4)..(4)

<223> Cys and acetamidomethyl coupling

<220>

<221> VARIANT

<222> (6)..(6)

<223> Cys and acetamidomethyl coupling

<220>

<221> VARIANT

<222> (24)..(24)

<223> Cys and acetamidomethyl coupling

<220>

<221> VARIANT

<222> (27)..(27)

<223> Cys and acetamidomethyl coupling

<400> 83

Cys Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Cys

1 5 10 15

Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Cys

20 25

<210> 84

<211> 17

<212> PRT

<213> hepatitis B Virus

<220>

<221> MISC_FEATURE

<222> (1)..(17)

<223> cyclic epitope mimetic

<220>

<221> VARIANT

<222> (13)..(16)

<223> Cys and acetamidomethyl coupling

<400> 84

Cys Gly Gly Gly Cys Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys

1 5 10 15

Cys

<210> 85

<211> 11

<212> PRT

<213> hepatitis B Virus

<400> 85

Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys

1 5 10

<210> 86

<211> 15

<212> PRT

<213> hepatitis B Virus

<400> 86

Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Cys

1 5 10 15

<210> 87

<211> 14

<212> PRT

<213> hepatitis B Virus

<400> 87

Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe

1 5 10

<210> 88

<211> 5

<212> PRT

<213> hepatitis B Virus

<220>

<221> VARIANT

<222> (1)..(5)

<223> Xaa = any amino acid

<400> 88

Pro Cys Arg Xaa Cys

1 5

<210> 89

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V35 VL

<400> 89

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ala Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 90

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V34 VL

<400> 90

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ser Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 91

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> HBC34-V35-MLNS-GAALIE and HBC34-V34-MLNS-GAALIE (g 1M17, 1)

Is the synthetic sequence HC of (2)

<400> 91

Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 92

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequences HC of HBC34-V35-MLNS and HBC34-V34-MLNS (g 1M17, 1)

<400> 92

Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 93

<211> 212

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence LC of HBC34-V35

<400> 93

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ala Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ala Ala

100 105 110

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn

115 120 125

Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val

130 135 140

Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

145 150 155 160

Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

165 170 175

Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser

180 185 190

Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro

195 200 205

Thr Glu Cys Ser

210

<210> 94

<211> 212

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence LC of HBC34-V34

<400> 94

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ser Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ala Ala

100 105 110

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn

115 120 125

Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val

130 135 140

Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

145 150 155 160

Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

165 170 175

Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser

180 185 190

Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro

195 200 205

Thr Glu Cys Ser

210

<210> 95

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC24 VH

<400> 95

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Thr Lys Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Val Pro Gly Phe Gly Ile Asp Thr Tyr Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys

65 70 75 80

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

85 90 95

Leu Tyr Tyr Cys Ala Lys Asp Val Gly Val Ile Gly Ser Tyr Tyr Tyr

100 105 110

Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser

115 120 125

<210> 96

<211> 109

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC24 VL

<400> 96

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Leu Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Tyr Ser Pro

85 90 95

Arg Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 97

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence CDRH1 of HBC24

<400> 97

Gly Ser Thr Phe Thr Lys Tyr Ala

1 5

<210> 98

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence CDRH2 of HBC24

<400> 98

Ile Ser Gly Ser Val Pro Gly Phe

1 5

<210> 99

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence CDRH3 of HBC24

<400> 99

Leu Tyr Tyr Cys Ala Lys Asp Val Gly Val Ile Gly Ser Tyr Tyr Tyr

1 5 10 15

Tyr Ala Met Asp Val

20

<210> 100

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence CDRL1 of HBC24

<400> 100

Gln Gly Leu Ser Ser Ser Tyr

1 5

<210> 101

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence CDRL2 of HBC24

<400> 101

Ser Ala Ser

1

<210> 102

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence CDRL3 of HBC24

<400> 102

Gln Gln Tyr Ala Tyr Ser Pro Arg Trp Thr

1 5 10

<210> 103

<211> 357

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences VH (codon optimized) of HBC34-V7, HBC34-V35 and HBC34-V34

<400> 103

gagctgcagc tggtggagtc cggcggcggc tgggtgcagc ctggcggctc ccagaggctg 60

agctgtgccg cttctggcag gatcttccgg tccttttaca tgtcttgggt gcggcaggct 120

ccaggcaagg gcctggagtg ggtggctacc atcaaccagg acggctccga gaagctgtat 180

gtggatagcg tgaagggcag attcacaatc tctcgcgaca acgccaagaa ctccctgttt 240

ctgcagatga acaatctgag ggtggaggat accgccgtgt actattgcgc cgcttggtct 300

ggcaatagcg gcggcatgga cgtgtgggga cagggcacca ccgtgtccgt gtccagc 357

<210> 104

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V34 VL (codon optimized)

<400> 104

agctacgagc tgacacagcc cccttccgtg tccgtgtccc ctggacagac cgtgtccatc 60

ccatgcagcg gcgacaagct gggcaacaag aacgtgtcct ggtttcagca taagcctggc 120

cagtcccccg tgctggtcat ctacgaggtg aagtataggc ccagcggcat ccctgagcgg 180

ttctctggct ccaacagcgg caatacagcc accctgacaa tctctggcac acaggctatg 240

gacgaggccg cttatttctg ccagaccttt gattccacca cagtggtgtt cggcggcggc 300

accagactga cagtgctg 318

<210> 105

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V35 VL (codon optimized)

<400> 105

agctacgagc tgacacagcc cccttccgtg tccgtgtccc ctggacagac cgtgtccatc 60

ccatgcagcg gcgacaagct gggcaacaag aacgtggcct ggtttcagca taagcctggc 120

cagtcccccg tgctggtcat ctacgaggtg aagtataggc ccagcggcat ccctgagcgg 180

ttctctggct ccaacagcgg caatacagcc accctgacaa tctctggcac acaggctatg 240

gacgaggccg cttatttctg ccagaccttt gattccacca cagtggtgtt cggcggcggc 300

accagactga cagtgctg 318

<210> 106

<211> 360

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC24 VH (wild type)

<400> 106

gaggtgcagt tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatc cacttttacc aaatatgcca tgagctgggt ccgtcaggct 120

ccagggaagg ggctggagtg ggtcgcaagt attagtggaa gtgttcctgg ttttggtatt 180

gacacatact acgcagactc cgttaagggc cggttcacca tctccagaga cacttccaag 240

aacaccctgt atctgcaaat gaacagcctg agagccgagg acacggcctt atattactgt 300

gcgaaagatg tcggggttat cgggtcatac tattactacg ctatggacgt ctggggtcaa 360

<210> 107

<211> 327

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC24 VL (wild-type)

<400> 107

aaattgtgtt gacgcagtct ccaggcaccc tgtctttgtc tccaggggaa agagccaccc 60

tctcctgcag ggccagtcag ggtcttagca gcagttactt agcctggtac cagcagaaac 120

ctggccaggc tcccaggctc ctcatctata gtgcgtccac cagggccact ggcatcccag 180

acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240

ctgaagattt tgcagtgtat tactgtcaac agtatgctta ctcacctcgg tggacgttcg 300

gccaagggac caaggtggag atcaaac 327

<210> 108

<211> 384

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC24 VH (codon optimized)

<400> 108

gaggtgcagc tgctggaaag cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60

tcttgcgccg cctctggcag caccttcaca aagtatgcaa tgtcttgggt gcgccaggca 120

ccaggcaagg gcctggagtg ggtggcctcc atctctggca gcgtgcctgg cttcggcatc 180

gacacctact atgccgattc cgtgaagggc cggtttacaa tcagcagaga cacctccaag 240

aacacactgt atctgcagat gaattctctg cgggccgagg acaccgccct gtactattgt 300

gccaaggatg tgggcgtgat cggcagctac tattactatg caatggacgt gtggggacag 360

ggaacagcag tgacagtgag ctcc 384

<210> 109

<211> 327

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC24 VL (codon optimized)

<400> 109

gagatcgtgc tgacccagtc tcctggcaca ctgtccctgt cccctggaga gagagccacc 60

ctgtcctgca gagcctctca gggcctgagc tcctcttacc tggcctggta tcagcagaag 120

cctggacagg cccctcggct gctgatctac tctgcctcca ccagagcaac aggcattcct 180

gaccgcttct ccggatctgg aagcggcaca gacttcaccc tgacaatcag ccggctggag 240

cctgaggact tcgccgtgta ctattgtcag cagtacgcct attccccaag gtggaccttt 300

ggccagggca caaaggtgga gatcaag 327

<210> 110

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V7 VL (codon optimized)

<400> 110

agctacgagc tgacacagcc cccttccgtg tccgtgtccc ctggacagac cgtgtccatc 60

ccatgcagcg gcgacaagct gggcaacaag aacgtgtgct ggtttcagca taagcctggc 120

cagtcccccg tgctggtcat ctacgaggtg aagtataggc ccagcggcat ccctgagcgg 180

ttctctggct ccaacagcgg caatacagcc accctgacaa tctctggcac acaggctatg 240

gacgaggccg cttatttctg ccagaccttt gattccacca cagtggtgtt cggcggcggc 300

accagactga cagtgctg 318

<210> 111

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v23-L_C40S

<400> 111

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 112

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v23-L_C40A

<400> 112

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Ala

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 113

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v31-L_C40S

<400> 113

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ser Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Trp Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 114

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v31-L_C40A

<400> 114

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ala Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Trp Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 115

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v32-L_C40S

<400> 115

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ser Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 116

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v32-L_C40A

<400> 116

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ala Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Trp Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 117

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v33-L_C40S

<400> 117

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 118

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34v33-L_C40A

<400> 118

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Ala

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 119

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-L_C40S

<400> 119

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ser Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Trp Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 120

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-L_C40A

<400> 120

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ala Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Trp Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu

100 105

<210> 121

<211> 458

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC24-MLNS

<400> 121

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Thr Lys Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Val Pro Gly Phe Gly Ile Asp Thr Tyr Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys

65 70 75 80

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

85 90 95

Leu Tyr Tyr Cys Ala Lys Asp Val Gly Val Ile Gly Ser Tyr Tyr Tyr

100 105 110

Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser

115 120 125

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

355 360 365

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 122

<211> 458

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC24-MLNS-GAALIE

<400> 122

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Thr Lys Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Val Pro Gly Phe Gly Ile Asp Thr Tyr Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys

65 70 75 80

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

85 90 95

Leu Tyr Tyr Cys Ala Lys Asp Val Gly Val Ile Gly Ser Tyr Tyr Tyr

100 105 110

Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser

115 120 125

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

355 360 365

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 123

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V7-mu (IgG 2 a) HC

<400> 123

Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Ser Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser

180 185 190

Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys

210 215 220

Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser

245 250 255

Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp

260 265 270

Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr

275 280 285

Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val

290 295 300

Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu

305 310 315 320

Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg

325 330 335

Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val

340 345 350

Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr

355 360 365

Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr

370 375 380

Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys

405 410 415

Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu

420 425 430

Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly

435 440 445

Lys

<210> 124

<211> 212

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V7-mu (IgG 2 a) LC

<400> 124

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Cys Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ser Ser

100 105 110

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Glu Thr Asn

115 120 125

Lys Ala Thr Leu Val Cys Thr Ile Thr Asp Phe Tyr Pro Gly Val Val

130 135 140

Thr Val Asp Trp Lys Val Asp Gly Thr Pro Val Thr Gln Gly Met Glu

145 150 155 160

Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn Lys Tyr Met Ala Ser Ser

165 170 175

Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu Arg His Ser Ser Tyr Ser

180 185 190

Cys Gln Val Thr His Glu Gly His Thr Val Glu Lys Ser Leu Ser Arg

195 200 205

Ala Asp Cys Ser

210

<210> 125

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V35-mu (IgG 2 a) HC

<400> 125

Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Ser Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser

180 185 190

Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys

210 215 220

Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser

245 250 255

Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp

260 265 270

Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr

275 280 285

Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val

290 295 300

Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu

305 310 315 320

Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg

325 330 335

Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val

340 345 350

Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr

355 360 365

Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr

370 375 380

Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys

405 410 415

Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu

420 425 430

Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly

435 440 445

Lys

<210> 126

<211> 212

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V35-mu (IgG 2 a) LC

<400> 126

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Val Ser Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Asn Val

20 25 30

Ala Trp Phe Gln His Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Ala Tyr Phe Cys Gln Thr Phe Asp Ser Thr Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ser Ser

100 105 110

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Glu Thr Asn

115 120 125

Lys Ala Thr Leu Val Cys Thr Ile Thr Asp Phe Tyr Pro Gly Val Val

130 135 140

Thr Val Asp Trp Lys Val Asp Gly Thr Pro Val Thr Gln Gly Met Glu

145 150 155 160

Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn Lys Tyr Met Ala Ser Ser

165 170 175

Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu Arg His Ser Ser Tyr Ser

180 185 190

Cys Gln Val Thr His Glu Gly His Thr Val Glu Lys Ser Leu Ser Arg

195 200 205

Ala Asp Cys Ser

210

<210> 127

<211> 458

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC24-mu (IgG 2 a) HC

<400> 127

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Thr Lys Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Val Pro Gly Phe Gly Ile Asp Thr Tyr Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys

65 70 75 80

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

85 90 95

Leu Tyr Tyr Cys Ala Lys Asp Val Gly Val Ile Gly Ser Tyr Tyr Tyr

100 105 110

Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser

115 120 125

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

130 135 140

Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

145 150 155 160

Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

165 170 175

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

180 185 190

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

195 200 205

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

210 215 220

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

225 230 235 240

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

245 250 255

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

260 265 270

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

275 280 285

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

290 295 300

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

305 310 315 320

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

325 330 335

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

340 345 350

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

355 360 365

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

370 375 380

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

385 390 395 400

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

405 410 415

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

420 425 430

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

435 440 445

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

450 455

<210> 128

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence HBC24-mu (IgG 2 a) LC

<400> 128

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Leu Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Tyr Ser Pro

85 90 95

Arg Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Asp Ala

100 105 110

Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser

115 120 125

Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp

130 135 140

Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val

145 150 155 160

Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met

165 170 175

Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser

180 185 190

Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys

195 200 205

Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 129

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V7, HBC34-V34, HBC34-V35 HC (wild type)

<400> 129

Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 130

<211> 990

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V7, HBC34-V34, HBC34-V35 CH 1-hinge-CH 2-CH3

(codon optimized)

<400> 130

gcctccacaa agggcccaag cgtgtttcca ctggctccct cttccaagtc tacctccggc 60

ggcacagccg ctctgggatg tctggtgaag gattacttcc cagagcccgt gaccgtgtct 120

tggaactccg gcgccctgac cagcggagtg catacatttc cagctgtgct gcagagctct 180

ggcctgtact ctctgtccag cgtggtgacc gtgccctctt ccagcctggg cacccagaca 240

tatatctgca acgtgaatca caagccaagc aatacaaagg tggacaagaa ggtggagccc 300

aagtcttgtg ataagaccca tacatgccct ccatgtccag ctccagagct gctgggcggc 360

ccaagcgtgt tcctgtttcc acccaagcct aaggataccc tgatgatctc cagaaccccc 420

gaggtgacat gcgtggtggt ggacgtgagc cacgaggatc ctgaggtgaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgct aagaccaagc ccagggagga gcagtacaac 540

tctacctatc gggtggtgtc cgtgctgaca gtgctgcacc aggattggct gaacggcaag 600

gagtataagt gcaaggtgtc taataaggcc ctgcccgctc ctatcgagaa gaccatctcc 660

aaggccaagg gccagcctag agagccacag gtgtacacac tgcctccatc tcgcgatgag 720

ctgaccaaga accaggtgtc cctgacatgt ctggtgaagg gcttctatcc ttccgacatc 780

gctgtggagt gggagagcaa tggccagcca gagaacaatt acaagaccac accccctgtg 840

ctggacagcg atggctcttt ctttctgtat agcaagctga ccgtggacaa gtctcgctgg 900

cagcagggca acgtgtttag ctgttctgtg atgcatgagg ccctgcacaa tcattataca 960

cagaagtccc tgagcctgtc tcctggcaag 990

<210> 131

<211> 1417

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V7, HBC34-V34, HBC34-V35 VH-CH 1-hinge-CH 2-CH3

(codon optimized)

<400> 131

gagctgcagc tggtggagtc cggcggcggc tgggtgcagc ctggcggctc ccagaggctg 60

agctgtgccg cttctggcag gatcttccgg tccttttaca tgtcttgggt gcggcaggct 120

ccaggcaagg gcctggagtg ggtggctacc atcaaccagg acggctccga gaagctgtat 180

gtggatagcg tgaagggcag attcacaatc tctcgcgaca acgccaagaa ctccctgttt 240

ctgcagatga acaatctgag ggtggaggat accgccgtgt actattgcgc cgcttggtct 300

ggcaatagcg gcggcatgga cgtgtgggga cagggcacca ccgtgtccgt gtccagcgcc 360

tccacaaagg gcccaagcgt gtttccactg gctccctctt ccaagtctac ctccggcggc 420

acagccgctc tgggatgtct ggtgaaggat tacttcccag agcccgtgac cgtgtcttgg 480

aactccggcg ccctgaccag cggagtgcat acatttccag ctgtgctgca gagctctggc 540

ctgtactctc tgtccagcgt ggtgaccgtg ccctcttcca gcctgggcac ccagacatat 600

atctgcaacg tgaatcacaa gccaagcaat acaaaggtgg acaagaaggt ggagcccaag 660

tcttgtgata agacccatac atgccctcca tgtccagctc cagagctgct gggcggccca 720

agcgtgttcc tgtttccacc caagcctaag gataccctga tgatctccag aacccccgag 780

gtgacatgcg tggtggtgga cgtgagccac gaggatcctg aggtgaagtt caactggtac 840

gtggacggcg tggaggtgca taatgctaag accaagccca gggaggagca gtacaactct 900

acctatcggg tggtgtccgt gctgacagtg ctgcaccagg attggctgaa cggcaaggag 960

tataagtgca aggtgtctaa taaggccctg cccgctccta tcgagaagac catctccaag 1020

gccaagggcc agcctagaga gccacaggtg tacacactgc ctccatctcg cgatgagctg 1080

accaagaacc aggtgtccct gacatgtctg gtgaagggct tctatccttc cgacatcgct 1140

gtggagtggg agagcaatgg ccagccagag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtatagc aagctgaccg tggacaagtc tcgctggcag 1260

cagggcaacg tgtttagctg ttctgtgatg catgaggccc tgcacaatca ttatacacag 1320

aagtccctga gcctgtctcc tggcaagtga tgaggtaccg tgcgacggcc ggcaagcccc 1380

cgctccccgg gctctcgcgg tcgtacgagg aaagctt 1417

<210> 132

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V7 CL (codon optimized)

<400> 132

ggacagccaa aggctgctcc atctgtgacc ctgtttccac cctcttccga ggagctgcag 60

gccaacaagg ccaccctggt gtgcctgatc tctgacttct accctggagc tgtgacagtg 120

gcttggaagg ctgatagctc tcccgtgaag gctggcgtgg agacaacaac ccctagcaag 180

cagtctaaca ataagtacgc cgcttccagc tatctgtctc tgacaccaga gcagtggaag 240

tcccaccgct cttattcctg ccaggtgacc catgagggca gcaccgtgga gaagacagtg 300

gcccccaccg agtgttct 318

<210> 133

<211> 636

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V7 LC (VL-CL) (codon optimized)

<400> 133

agctacgagc tgacacagcc cccttccgtg tccgtgtccc ctggacagac cgtgtccatc 60

ccatgcagcg gcgacaagct gggcaacaag aacgtgtgct ggtttcagca taagcctggc 120

cagtcccccg tgctggtcat ctacgaggtg aagtataggc ccagcggcat ccctgagcgg 180

ttctctggct ccaacagcgg caatacagcc accctgacaa tctctggcac acaggctatg 240

gacgaggccg cttatttctg ccagaccttt gattccacca cagtggtgtt cggcggcggc 300

accagactga cagtgctggg acagccaaag gctgctccat ctgtgaccct gtttccaccc 360

tcttccgagg agctgcaggc caacaaggcc accctggtgt gcctgatctc tgacttctac 420

cctggagctg tgacagtggc ttggaaggct gatagctctc ccgtgaaggc tggcgtggag 480

acaacaaccc ctagcaagca gtctaacaat aagtacgccg cttccagcta tctgtctctg 540

acaccagagc agtggaagtc ccaccgctct tattcctgcc aggtgaccca tgagggcagc 600

accgtggaga agacagtggc ccccaccgag tgttct 636

<210> 134

<211> 318

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequences HBC34-V34, HBC34-V35 CL (codon optimized)

<400> 134

ggacagccaa aggctgctcc atctgtgacc ctgtttccac cctcttccga ggagctgcag 60

gccaacaagg ccaccctggt gtgcctgatc tctgacttct accctggagc tgtgacagtg 120

gcttggaagg ctgatagctc tcccgtgaag gctggcgtgg agacaacaac ccctagcaag 180

cagtctaaca ataagtacgc cgcttccagc tatctgtctc tgacaccaga gcagtggaag 240

tcccaccgct cttattcctg ccaggtgacc catgagggca gcaccgtgga gaagacagtg 300

gcccccaccg agtgttct 318

<210> 135

<211> 636

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V34 LC (VL-CL) (codon optimized)

<400> 135

agctacgagc tgacacagcc cccttccgtg tccgtgtccc ctggacagac cgtgtccatc 60

ccatgcagcg gcgacaagct gggcaacaag aacgtgtcct ggtttcagca taagcctggc 120

cagtcccccg tgctggtcat ctacgaggtg aagtataggc ccagcggcat ccctgagcgg 180

ttctctggct ccaacagcgg caatacagcc accctgacaa tctctggcac acaggctatg 240

gacgaggccg cttatttctg ccagaccttt gattccacca cagtggtgtt cggcggcggc 300

accagactga cagtgctggg acagccaaag gctgctccat ctgtgaccct gtttccaccc 360

tcttccgagg agctgcaggc caacaaggcc accctggtgt gcctgatctc tgacttctac 420

cctggagctg tgacagtggc ttggaaggct gatagctctc ccgtgaaggc tggcgtggag 480

acaacaaccc ctagcaagca gtctaacaat aagtacgccg cttccagcta tctgtctctg 540

acaccagagc agtggaagtc ccaccgctct tattcctgcc aggtgaccca tgagggcagc 600

accgtggaga agacagtggc ccccaccgag tgttct 636

<210> 136

<211> 636

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence HBC34-V35 LC (VL-CL) (codon optimized)

<400> 136

agctacgagc tgacacagcc cccttccgtg tccgtgtccc ctggacagac cgtgtccatc 60

ccatgcagcg gcgacaagct gggcaacaag aacgtggcct ggtttcagca taagcctggc 120

cagtcccccg tgctggtcat ctacgaggtg aagtataggc ccagcggcat ccctgagcgg 180

ttctctggct ccaacagcgg caatacagcc accctgacaa tctctggcac acaggctatg 240

gacgaggccg cttatttctg ccagaccttt gattccacca cagtggtgtt cggcggcggc 300

accagactga cagtgctggg acagccaaag gctgctccat ctgtgaccct gtttccaccc 360

tcttccgagg agctgcaggc caacaaggcc accctggtgt gcctgatctc tgacttctac 420

cctggagctg tgacagtggc ttggaaggct gatagctctc ccgtgaaggc tggcgtggag 480

acaacaaccc ctagcaagca gtctaacaat aagtacgccg cttccagcta tctgtctctg 540

acaccagagc agtggaagtc ccaccgctct tattcctgcc aggtgaccca tgagggcagc 600

accgtggaga agacagtggc ccccaccgag tgttct 636

<210> 137

<211> 217

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<222> (1)..(217)

<223> IgG1, WT hIgG1 Fc

<400> 137

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210> 138

<211> 449

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequences HBC34, HBC34v7, HBC34v23, HBC34v34, HBC34v35,

HBC34_C40S、HBC34_C40A、HBC34v23_C40S、HBC34v23_C40A，

HC with GAALIE mutation in hIgG1 Fc

<400> 138

Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly

1 5 10 15

Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Arg Ser Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Asn Gln Asp Gly Ser Glu Lys Leu Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Trp Ser Gly Asn Ser Gly Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro Glu Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 139

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> chimeric hinge sequences

<400> 139

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro

Claims (55)

1. A method of treating Hepatitis B Virus (HBV) infection in a subject, the method comprising administering a single dose of a pharmaceutical composition comprising an antibody to the subject, wherein the antibody comprises the heavy chain amino acid sequence of SEQ ID No. 91 and the light chain amino acid sequence of SEQ ID No. 93, and

(a) Said single dose of said pharmaceutical composition comprises at least 6mg of said antibody; and is also provided with

(b) After administration of the single dose, the subject's serum HBsAg is reduced by at least 1.0log from baseline ¹⁰ IU/mL、1.5log ¹⁰ IU/mL or more; and is also provided with

(c) The reduction in serum HBsAg of the subject after administration of the single dose lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or more.

2. A method of treating Hepatitis B Virus (HBV) infection in a subject, the method comprising administering a single dose of a pharmaceutical composition comprising an antibody to the subject, wherein the antibody comprises the heavy chain amino acid sequence of SEQ ID No. 91 and the light chain amino acid sequence of SEQ ID No. 93, and

(a) Said single dose of said pharmaceutical composition comprises at least 75mg of said antibody; and is also provided with

(b) After administration of the single dose, the subject's serum HBsAg is reduced by at least 1.0log from baseline ¹⁰ IU/mL, at least 1.5log ¹⁰ IU/mL or more; and is also provided with

(c) The reduction in serum HBsAg in the subject after administration of the single dose lasts at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days or more.

3. A method of treating Hepatitis B Virus (HBV) infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody, wherein:

(a) The antibody comprises a heavy chain amino acid sequence of SEQ ID NO. 91 and a light chain amino acid sequence of SEQ ID NO. 93,

(b) At least 1000ng/mL of antibody remains unbound to serum HBsAg for at least 14 days after administration of a single dose; and is also provided with

(c) The subject has a baseline serum HBsAg level of less than 3000IU/mL.

4. The method of any one of claims 1-3, wherein the serum HBsAg of the subject is reduced by at least 1.0log from baseline over a single dose of 8 days of administration ¹⁰ IU/mL。

5. The method of any one of claims 1-4, wherein the single dose of the pharmaceutical composition comprises at least 75mg of the antibody, and within 8 days of administration of the single dose, the subject's serum HBsAg is reduced by at least 1.5log from baseline ¹⁰ IU/mL。

6. The method of any one of claims 1-5, wherein the serum HBsAg of the subject is reduced by at least 0.5log compared to baseline 56 days after administration of the single dose ¹⁰ IU/mL。

7. The method of any one of claims 1-6, wherein the subject has a C of the antibody between 300ng/mL and 6,000ng/mL _max 。

8. According to claim7, wherein the subject has the C of the antibody _max Is at least 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, 1000ng/mL, 1100ng/mL, 1200ng/mL, 1300ng/mL, 1400ng/mL, 1500ng/mL, 1600ng/mL, 1700ng/mL, 1800ng/mL, 1900ng/mL, 2000ng/mL, 2100ng/mL, 2200ng/mL, 2300ng/mL, 2400ng/mL, 2500ng/mL, 2600ng/mL, 2700ng/mL, 2800ng/mL, 2900ng/mL, 3000ng/mL, 3100ng/mL, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800 or 5900ng/mL.

9. The method of any one of claims 1 to 8, wherein the single dose of the pharmaceutical composition comprises at most 10mg, at most 15mg, at most 18mg, at most 25mg, at most 30mg, at most 35mg, at most 40mg, at most 45mg, at most 50mg, at most 55mg, at most 60mg, at most 75mg, at most 90mg, at most 300mg, at most 900mg, or at most 3000mg of the antibody,

Or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 3000mg, or ranging from 10mg to 3000mg, or ranging from 25mg to 3000mg, or ranging from 30mg to 3000mg, or ranging from 50mg to 3000mg, or ranging from 60mg to 3000mg, or ranging from 75mg to 3000mg, or ranging from 90mg to 3000mg, or ranging from 100mg to 3000mg, or ranging from 150mg to 3000mg, or ranging from 200mg to 3000mg, or ranging from 300mg to 3000mg, or ranging from 500mg to 3000mg, or ranging from 750mg to 3000mg, or ranging from 900mg to 3000mg, or ranging from 1500mg to 3000mg, or ranging from 2000mg to 3000mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 900mg, or ranging from 10mg to 900mg, or ranging from 25mg to 900mg, or ranging from 30mg to 900mg, or ranging from 50mg to 900mg, or ranging from 60mg to 900mg, or ranging from 75mg to 900mg, or ranging from 90mg to 900mg, or ranging from 100mg to 900mg, or ranging from 150mg to 900mg, or ranging from 200mg to 900mg, or ranging from 300mg to 900mg, or ranging from 500mg to 900mg, or ranging from 750mg to 900mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 500mg, or ranging from 10mg to 500mg, or ranging from 25mg to 500mg, or ranging from 30mg to 500mg, or ranging from 50mg to 500mg, or ranging from 60mg to 500mg, or ranging from 75mg to 500mg, or ranging from 90mg to 500mg, or ranging from 100mg to 500mg, or ranging from 150mg to 500mg, or ranging from 200mg to 500mg, or ranging from 300mg to 500mg, or ranging from 400mg to 500mg,

Or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 300mg, or ranging from 10mg to 300mg, or ranging from 25mg to 300mg, or ranging from 30mg to 300mg, or ranging from 50mg to 300mg, or ranging from 60mg to 300mg, or ranging from 75mg to 300mg, or ranging from 90mg to 300mg, or ranging from 100mg to 300mg, or ranging from 150mg to 300mg, or ranging from 200mg to 300mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 200mg, or ranging from 10mg to 200mg, or ranging from 25mg to 200mg, or ranging from 30mg to 200mg, or ranging from 50mg to 200mg, or ranging from 60mg to 200mg, or ranging from 75mg to 200mg, or ranging from 90mg to 200mg, or ranging from 100mg to 200mg, or ranging from 150mg to 200mg,

or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount ranging from 6mg to 100mg, or ranging from 10mg to 100mg, or ranging from 25mg to 100mg, or ranging from 30mg to 100mg, or ranging from 50mg to 100mg, or ranging from 60mg to 100mg, or ranging from 75mg to 100mg, or ranging from 90mg to 100mg,

Or wherein said single dose of said pharmaceutical composition comprises said antibody in an amount ranging from 6mg to 25mg, or ranging from 10mg to 25mg, or ranging from 15mg to 25mg, or ranging from 20mg to 25mg,

or wherein said single dose of said pharmaceutical composition comprises said antibody in an amount ranging from 6mg to 50mg, or ranging from 6mg to 25mg, or ranging from 6mg to 50mg, or ranging from 10mg to 25mg, or ranging from 6mg to 15mg, or ranging from 10mg to 15mg, or wherein the single dose of the pharmaceutical composition comprises 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 985mg, 990mg, 995mg or 1000mg or more of said antibody,

Or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount of less than 3000mg, less than 2500mg, less than 2000mg, less than 1500mg, less than 1000mg, less than 900mg, less than 500mg, less than 300mg, less than 200mg, less than 100mg, less than 90mg, less than 75mg, less than 50mg, less than 25mg, or less than 10 mg.

10. The method according to any one of claims 1 to 9, wherein the single dose of the pharmaceutical composition comprises the antibody in a concentration in the range of 100mg/mL to 200mg/mL, such as 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL or 200mg/mL, preferably 150 mg/mL.

11. The method of any one of claims 1-10, wherein the single dose of the pharmaceutical composition comprises about 75mg of the antibody.

12. The method of any one of claims 1-10, wherein the single dose of the pharmaceutical composition comprises about 90mg of the antibody.

13. The method of any one of claims 1 to 10, wherein the single dose of the pharmaceutical composition comprises up to 300mg of the antibody.

14. The method of any one of claims 1 to 10, wherein the single dose of the pharmaceutical composition comprises up to 900mg of the antibody.

15. The method of any one of claims 1 to 10, wherein the single dose of the pharmaceutical composition comprises up to 3,000mg of the antibody.

16. The method of any one of claims 1 to 15, wherein the method comprises administering the single dose by subcutaneous injection, optionally wherein the single dose comprises, or consists of, 6mg of the antibody, 18mg of the antibody, or 75mg of the antibody.

17. The method of any one of claims 1 to 16, wherein the method comprises administering the single dose by intravenous injection.

18. The method of any one of claims 1 to 17, wherein the pharmaceutical composition further comprises water, optionally USP water.

19. The method according to any one of claims 1 to 18, wherein the pharmaceutical composition further comprises histidine, optionally in the pharmaceutical composition at a histidine concentration in the range of 10mM to 40mM, such as 20mM.

20. The method according to any one of claims 1 to 19, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally 5%, 6%, 7%, 8% or 9%, preferably about 7% (w/v).

21. The method of any one of claims 1 to 20, wherein the pharmaceutical composition further comprises a surfactant or triblock copolymer, optionally polysorbate or poloxamer-188, preferably polysorbate 80 (PS 80), wherein optionally the polysorbate or poloxamer-188 is present in a range of 0.01% to 0.05% (w/v), preferably 0.02% (w/v).

22. The method of any one of claims 1 to 21, wherein the pharmaceutical composition has a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or 5.8, 5.9, 6.0, 6.1, or 6.2.

23. The method of claim 22, wherein the pharmaceutical composition comprises:

(i) 150mg/mL of the antibody;

(ii) USP water;

(iii) 20mM histidine;

(iv) 7% sucrose; and

(v)0.02％PS80，

wherein the pharmaceutical composition has a pH of 6.

24. The method of any one of claims 1-23, wherein the subject is an adult.

25. The method of claim 24, wherein the subject has an age ranging from 18 years to 65 years.

26. The method of any one of claims 1 to 25, wherein the subject has a body weight of 40kg to 125kg and/or the subject has a Body Mass Index (BMI) of 18 to 35kg/m ² 。

27. The method of any one of claims 1 to 26, wherein the subject has chronic HBV infection; the HBV infection is for example defined by 2 serum HBsAg, HBV DNA and/or HBeAg positives, wherein the 2 intervals are at least 6 months apart.

28. The method of any one of claims 1-27, wherein the subject is not suffering from cirrhosis.

29. The method of claim 28, wherein the absence of cirrhosis is determined by:

fibrincan assessment (e.g., within 6 months prior to administration of the single dose of the pharmaceutical composition); or (b)

Liver biopsy (e.g., within 12 months prior to administration of the single dose of the pharmaceutical composition),

wherein preferably the absence of cirrhosis is determined by absence of Metavir F3 fibrosis or absence of F4 cirrhosis.

30. The method of any one of claims 1-29, wherein the subject has received a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI) optionally within 120 days, further optionally within 60 days, prior to administration of the single dose.

31. The method of claim 30, wherein the NRTI comprises one or more of: tenofovir; tenofovir disoproxil (e.g., tenofovir disoproxil fumarate); tenofovir alafenamide; entecavir; lamivudine; adefovir; adefovir dipivoxil.

32. The method of any one of claims 1-31, wherein the subject's serum HBV DNA concentration is less than 100IU/mL no more than 28 days prior to administration of the single dose.

33. The method of any one of claims 1 to 32, wherein the subject's serum HBsAg concentration is below 3,000iu/mL prior to administration of the single dose, and optionally the subject's serum HBsAg concentration is below 1,000iu/mL prior to administration of the single dose.

34. The method of any one of claims 1 to 32, wherein the subject's serum HBsAg concentration is greater than or equal to 3,000iu/mL for no more than 28 days prior to administration of the single dose, and optionally the subject's serum HBsAg concentration is greater than or equal to 1,000iu/mL for no more than 28 days prior to administration of the single dose.

35. The method of any one of claims 1-34, wherein the subject is negative for HBe antigen (HBeAg) no more than 28 days prior to administration of the single dose.

36. The method of any one of claims 1-35, wherein the subject is negative for anti-HB antibodies no more than 28 days prior to administration of the single dose.

37. The method of any one of claims 1 to 36, wherein prior to administration of the single dose,

(i) The subject is not suffering from fibrosis and/or is not suffering from cirrhosis; and/or

(ii) Alanine Aminotransferase (ALT) <2x upper normal limit (ULN) for the subject.

38. The method of any one of claims 1 to 37, wherein the serum HBsAg of the subject is reduced by > 2-fold 56 days after administration of the single dose compared to the serum HBsAg of the subject (e.g., the concentration of HBsAg in serum, e.g., determined using the yaban ARCHITECT assay) 0 to 28 days prior to administration of the single dose.

39. The method of any one of claims 1 to 38, wherein after administration of the single dose (e.g., 56 days after administration of the single dose),

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

(ii) The subject has an adaptive immune response against HBV.

40. The method of any one of claims 1-39, wherein the subject is male.

41. The method of any one of claims 1-39, wherein the subject is female.

42. A pharmaceutical composition comprising an antibody, wherein the antibody comprises the heavy chain amino acid sequence of SEQ ID NO:91 and the light chain amino acid sequence of SEQ ID NO:93, wherein the pharmaceutical composition comprises the antibody in a concentration in the range of 100mg/mL to 200mg/mL, such as 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL or 200mg/mL, preferably 150mg/mL, and wherein after administration of the composition to a subject in need thereof, at least 1000ng/mL of the antibody remains unbound to serum HBsAg for at least 14 days after administration of a single dose. And the subject's baseline serum HBsAg level is less than 3000IU/mL.

43. The pharmaceutical composition of claim 42, wherein the pharmaceutical composition comprises at most 6mg, at most 18mg, at most 75mg, at most 90mg, at most 300mg, at most 900mg, or at most 3000mg of the antibody.

44. The pharmaceutical composition of claim 42 or 43, wherein the pharmaceutical composition comprises about 75mg of the antibody.

45. The pharmaceutical composition of claim 42 or 43, wherein the pharmaceutical composition comprises about 90mg of the antibody.

46. The pharmaceutical composition of claim 42 or 43, wherein the pharmaceutical composition comprises about 300mg of the antibody.

47. The pharmaceutical composition of claim 42 or 43, wherein the pharmaceutical composition comprises about 900mg of the antibody.

48. The pharmaceutical composition of claim 42 or 43, wherein the pharmaceutical composition comprises about 3,000mg of the antibody.

49. The pharmaceutical composition of any one of claims 42 to 48, wherein the pharmaceutical composition further comprises water, optionally USP water.

50. The pharmaceutical composition according to any one of claims 42 to 49, wherein the pharmaceutical composition further comprises histidine, optionally in the pharmaceutical composition at a histidine concentration of 10mM to 40mM, such as 20mM.

51. The pharmaceutical composition according to any one of claims 42 to 50, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally 5%, 6%, 7%, 8% or 9%, preferably about 7% (w/v).

52. The pharmaceutical composition according to any one of claims 42 to 51, wherein the pharmaceutical composition further comprises a surfactant, optionally a polysorbate, preferably polysorbate 80 (PS 80), wherein optionally the polysorbate is present in the range of 0.01% to 0.05% (w/v), preferably 0.02% (w/v).

53. The pharmaceutical composition of any one of claims 42 to 52, wherein the pH of the pharmaceutical composition is in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or 5.8, 5.9, 6.0, 6.1, or 6.2.

54. The pharmaceutical composition of any one of claims 42 to 53, wherein the pharmaceutical composition comprises:

(i) 150mg/mL of the antibody;

(ii) USP water;

(iii) 20mM histidine;

(iv) 7% sucrose; and

(v)0.02％PS80，

wherein the pharmaceutical composition has a pH of 6.

55. The method of any one of claims 1 to 41, wherein the subject is negative for HBeAg or positive for HBeAg.