Combined HBV Therapy

Abstract

The present disclosure provides methods for treating HBV infection using combination therapies, as well as related kits and compositions for use. The components of the combination therapy include an inhibitor of HBV gene expression or an agent that reduces HBV antigen load, and an anti-HBV antibody.

Description

[0001] Statement regarding sequence listing

[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 930485.401WO_SEQUENCE_LISTING.txt. This text file is 111 KB, was created on December 17, 2019, and is being submitted electronically through EFS-Web. Background Art

[0003] Globally, over 400 million people are living with chronic HBV infection (CHB), placing them at increased risk for serious liver diseases such as chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC), resulting in an estimated 600,000 deaths per person. Longitudinal studies of patients with CHB have shown a 5-year cumulative incidence of cirrhosis ranging from 8% to 20%, while the 5-year cumulative incidence of hepatic decompensation is approximately 20%. The global incidence of HCC has increased and is now the third leading cause of cancer-related death worldwide (El-Serag HB and Rudolph KL, Gastroenterology 132:2557–76 (2007)).

[0004] HBV is a DNA virus with a lipid envelope and an icosahedral nucleocapsid that encloses the viral DNA genome and DNA polymerase. The HBV capsid is formed in the cytosol of infected cells during the packaging of the RNA pre-genomic replication complex and is composed of the core protein (also known as HBcAg) and its cleaved variant, HBeAg. When the viral DNA dissociates from the capsid upon entry into new cells, it can be converted into covalently closed circular DNA (cccDNA), which may be retained in hepatocytes after HBV treatment and may reactivate infection. The lipid envelope contains the hepatitis B surface antigen (HBsAg), which refers to three different proteins, S-HBsAg (small antigen), M-HBsAg (middle antigen), and L-HBsAg (large antigen), which are encoded by the same open reading frame but utilize different start codons. HBsAg is the antigen present in currently available hepatitis B vaccines. HBV also encodes the protein HBx, which inhibits the tumor suppressor p53, promotes cell cycle progression, and increases the production of reactive oxygen species.

[0005] In addition to producing virions, HBV also produces subviral particles (SVPs), which contain the lipid envelope of the HBV virion but are replication-incompetent and typically lack a nucleocapsid. SVPs can produce a 3-4 log excess of replication-competent virions. The high levels of HBsAg present on SVPs deplete HBsAg-specific T cell responses, which may be a key factor in the immune system's inability to clear HBV infection during chronic hepatitis B (Chisari, FV et al., Pathologie Biologie 58:258-66 (2010)).

[0006] The natural evolution of CHB infection consists of four successive stages: (1) an early "immune tolerant" stage, which is associated with high levels of viral replication and minimal liver inflammation; (2) an immunoreactive stage, which is associated with significant liver inflammation and elevated serum transaminases; some patients progress to (3) a "non-replicating" or "inactive" stage, which is associated with: seroconversion to anti-HBe; undetectable or low-level viremia (less than 2000 IU / ml by PCR-based assays); and resolution of liver inflammation; and for some, (4) viral reactivation. Reactivation of HBV infection may be associated with the emergence of specific viral mutations that prevent HBeAg production but do not hinder viral replication, which is known as HBeAg-negative chronic hepatitis B. HBeAg-negative chronic hepatitis B (also known as anti-HBe-positive or pre-core mutant hepatitis) is characterized by fluctuating serum HBV DNA and serum transaminase (ALT and AST) levels, as well as progressive liver disease.

[0007] The primary goals of currently available HBV treatments are to permanently suppress HBV replication and improve liver disease. Clinically important short-term goals include achieving HBeAg seroconversion, normalizing serum ALT and AST, resolving liver inflammation, and preventing liver decompensation. The ultimate long-term goal of HBV treatment is to achieve a durable immune response to prevent the development of cirrhosis and hepatocellular carcinoma, thereby prolonging survival. Because cccHBV DNA persists in the nuclei of infected hepatocytes, currently available HBV treatments do not completely eliminate the virus. However, treatment-induced serum HBsAg clearance is a marker of termination of chronic HBV infection and is associated with the best long-term outcomes.

[0008] Although all three major HBV proteins (HBsAg, HBeAg, and HBcAg) have immunosuppressive properties, HBsAg is the predominant circulating HBV protein in HBV-infected individuals. Furthermore, although HBeAg removal (through seroconversion) or reduction in serum viremia has not been associated with the development of sustained control of HBV infection after treatment, the removal of serum HBsAg from the blood (and seroconversion) in HBV infection is a well-established prognostic indicator of antiviral response on treatment, which will lead to control of HBV infection after treatment (although this occurs in only a small fraction of patients receiving immunotherapy). Therefore, HBsAg removal may be an important strategy to overcome viral suppression of immune function in HBV-infected individuals.

[0009] Current standard approaches to treating HBV include immunotherapy based on interferon or thymosin α1 and suppression of viral production by inhibiting HBV polymerase. HBV polymerase inhibitors are effective in reducing viral production but have little effect on rapid HBsAg reduction or, in a limited number of patients, can slowly reduce HBsAg with long-term treatment (as in the case of tenofovir disoproxil fumarate). Interferon-based immunotherapy can reduce viral production and early clearance of HBsAg from the blood, but only in a small proportion of treated subjects. The generally accepted role of HBsAg in the blood is to sequester anti-HBsAg antibodies and allow infectious viral particles to evade immune detection, which may be one of the reasons why HBV infection remains a chronic disease. Furthermore, HBsAg, HBeAg, and HBcAg all have immunosuppressive properties, and the persistent presence of these viral proteins in the blood of patients receiving any currently available HBV treatment may have a significant impact in preventing patients from achieving immune control of their HBV infection.

[0010] Currently available treatments do not restore immune control of HBV in a substantial proportion of patients. Therefore, there remains a need for effective treatments for HBV infection that can suppress viral replication and restore immune control in the majority of patients. Summary of the Invention

[0011] In some embodiments, the present disclosure provides an HBV gene expression inhibitor for use in treating chronic HBV infection in a subject, wherein an anti-HBV antibody is subsequently administered to the subject.

[0012] The present disclosure also provides an agent for reducing HBV antigen load for use in treating chronic HBV infection in a subject, wherein an anti-HBV antibody is subsequently administered to the subject.

[0013] The present disclosure also provides a composition for treating chronic HBV infection in a subject, wherein (a) the composition comprises anti-HBV antibodies and the subject has previously been administered an inhibitor of gene expression; or (b) the composition comprises anti-HBV antibodies and the subject has previously been administered an agent that reduces HBV antigen load.

[0014] In some embodiments, the present disclosure provides the use of HBV gene expression inhibitors and anti-HBV antibodies in the preparation of a medicament for treating chronic HBV infection. The present disclosure also provides the use of agents for reducing HBV antigen load and anti-HBV antibodies in the preparation of a medicament for treating chronic HBV infection.

[0015] In some embodiments, the present disclosure provides a method for treating chronic HBV infection in a subject in need thereof, comprising: administering to the subject an agent that reduces HBV antigen load; and administering to the subject an anti-HBV antibody. The present disclosure also provides a method for treating chronic HBV infection in a subject in need thereof, comprising: administering to the subject an HBV gene expression inhibitor; and administering to the subject an anti-HBV antibody.

[0016] In some methods, compositions, or uses described herein, the anti-HBV antibody is HBC34 or a non-natural variant of HBC34.

[0017] In some methods, compositions, or uses described herein, the agent that reduces HBV antigen load or the HBV gene expression inhibitor is an RNAi agent (eg, siRNA, such as HBV02).

[0018] In some embodiments, the present disclosure also provides kits comprising an RNAi agent and an anti-HBV antibody as disclosed herein, and optionally instructions for performing the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 depicts the dosing regimen for the combination therapy study in a mouse HBV model (AAV-HBV model). Entecavir was administered orally once daily. HBV-specific siRNA was injected subcutaneously once at the start of the study, and a chimeric anti-HBV mouse antibody was administered intraperitoneally twice weekly during the third and fourth weeks of the study. A subset of mice was sacrificed at the fourth week, and another subset was sacrificed at the sixth week of the study.

[0020] FIG2A depicts the results of measuring HBV viral load in mouse serum samples using HBV DNA copy number as an indicator, and mice were treated with HBV02 siRNA (squares, solid line); mouse chimeric HBC34 antibody (HBC34v7; 15 mg / kg) (circles, solid line); or saline (triangles, solid line).

[0021] Figure 2B depicts the results of measuring HBV viral load in mouse serum samples using HBV DNA copy number as an indicator, with mice treated with control siRNA and control antibody (squares, dashed line); entecavir alone ("ETV", diamonds, dashed line); HBV02 siRNA and mouse chimeric HBC34v7 antibody (15 mg / kg) (circles, dashed line); or HBV02 siRNA, HBC34v7 antibody (15 mg / kg) and entecavir (triangles, solid line).

[0022] FIG3A depicts HBsAg levels measured from mouse sera after treatment with HBV02 siRNA (squares, solid line); mouse chimeric HBC34v7 antibody (15 mg / kg) (circles, solid line); or saline (triangles, solid line).

[0023] Figure 3B depicts HBsAg levels measured from mouse serum after treatment with control siRNA and control antibody (squares, dashed line); entecavir alone ("ETV", diamonds, dashed line); HBV02 siRNA and mouse chimeric HBC34v7 antibody (15 mg / kg) (circles, dashed line); or HBV02 siRNA, mouse chimeric HBC34v7 antibody (15 mg / kg) and entecavir (triangles, dashed line).

[0024] Figure 4 depicts the levels of free antibodies measured from mouse sera between days 14 and 42 after siRNA administration. The following treatment groups are depicted: mouse chimeric HBC34v7 antibody alone (circles); HBV02 siRNA, mouse chimeric HBC34v7 antibody, and entecavir (squares); and HBV02 siRNA and mouse chimeric HBC34v7 antibody (triangles).

[0025] FIG5A depicts the results of the determination of HBV viral load in mouse serum samples after treatment with siRNA, antibody, and / or control, using HBV DNA copy number as an indicator. Mice were injected with AAV / HBV virus on day -28. AAV / HBV-infected C57Bl / 6 mice were administered one of 11 different treatments on day 0: (1) HBV-specific siRNA (HBV02, having the antisense strand of SEQ ID NO: 8; see description in Example 1); (2)-(3) anti-HBV antibody (whole mouse HBC24) at one of two doses; (4)-(5) one dose of HBV02 siRNA and one of two doses of whole mouse HBC24; (6-9) one of two doses of HBV02 siRNA and one of three antibody doses of whole mouse anti-HBV antibody HBC34 (HBC34v35); (10) control siRNA and control antibody; or (11) PBS alone, administered intraperitoneally. Results for treatments 1-5, 10, and 11 are shown.

[0026] FIG5B depicts the results of the determination of HBV viral load in mouse serum samples after treatment with siRNA, antibody, and / or control, using HBV DNA copy number as an indicator. Mice were injected with AAV / HBV virus on day -28. AAV / HBV-infected C57Bl / 6 mice were administered one of 11 different treatments on day 0: (1) HBV-specific siRNA (HBV02, having the antisense strand of SEQ ID NO: 8; see description in Example 1); (2)-(3) anti-HBV antibody (whole mouse HBC24) at one of two doses; (4)-(5) one dose of HBV02 siRNA and one of two doses of whole mouse HBC24; (6-9) one of two doses of HBV02 siRNA and one of three antibody doses of whole mouse anti-HBV antibody HBC34 (HBC34v35); (10) control siRNA and control antibody; or (11) PBS alone, administered intraperitoneally. Results for treatments 1 and 6-11 are shown.

[0027] FIG. 6A depicts HBsAg levels measured from mouse sera following treatment with siRNA, antibodies, and / or controls, as described above for FIG. 5A .

[0028] FIG. 6B depicts HBsAg levels measured from mouse sera following treatment with siRNA, antibodies, and / or controls, as described above for FIG. 5B .

[0029] FIG. 7A depicts HBeAg levels measured from mouse serum following treatment with siRNA, antibodies, and / or controls, as described above for FIG. 5A .

[0030] FIG. 7B depicts HBeAg levels measured from mouse serum following treatment with siRNA, antibodies, and / or controls, as described above for FIG. 5B .

[0031] Figure 8 shows the experimental design for the study described in Example 3, including the dosing regimens used to evaluate HBsAG serum clearance and viral entry inhibition in a mouse model following treatment with anti-HBV antibodies and HBV-specific siRNA. AAV / HBV-infected SCID mice transplanted with primary human hepatocytes (n = 4 mice per treatment group) were administered one of seven different treatments: (1) PBS alone; (2-4) one of three doses of anti-HBV antibody (whole-murine HBC34v35) administered intraperitoneally twice weekly for two to three weeks; or (5-7) one subcutaneous administration of HBV-specific siRNA (HBV02, with the antisense strand of SEQ ID NO: 8; see description in Example 1) at the beginning of the study and two intraperitoneal administrations of whole-murine HBC34v35 at one of three antibody doses per week for two to three weeks. Mice were sacrificed at week 6.

[0032] FIG. 9 shows serum HBV DNA concentrations in mice after treatment with PBS (control); HBV34v35 antibody; or HBV34v35 antibody and HBV02 siRNA in SCID mice transplanted with primary human hepatocytes.

[0033] FIG. 10 shows serum HBsAg concentrations in SCID mice transplanted with primary human hepatocytes after treatment with PBS (control); HBV34v35 antibody; or HBV34v35 antibody and HBV02 siRNA.

[0034] Figure 11 Shown are serum HBeAg concentrations in SCID mice transplanted with primary human hepatocytes after treatment with PBS (control); HBV34v35 antibody; or HBV34v35 antibody and HBV02 siRNA.

[0035] FIG. 12 shows serum HBcrAg concentrations in SCID mice transplanted with primary human hepatocytes after treatment with PBS (control); HBV34v35 antibody; or HBV34v35 antibody and HBV02 siRNA.

[0036] FIG. 13 depicts the treatment regimen designed in a Phase 2 study to evaluate the effectiveness of siRNA-antibody combination therapy in treating HBV. DETAILED DESCRIPTION

[0037] The present disclosure provides methods and compositions for treating hepatitis B virus (HBV) infection using HBV protein expression inhibitors and anti-HBV antibodies, as well as related kits. Combination therapy can be used to treat chronic hepatitis B (CHB).

[0038] In some embodiments, the method comprises treating HBV infection in a subject in need thereof by: (i) administering to the subject an inhibitor of HBV gene expression; and (ii) administering to the subject an anti-HBV antibody. In a specific embodiment, after administration of the HBV gene expression inhibitor, expression of at least one HBV gene is reduced, and when expression of the at least one HBV gene is reduced, administering to the subject an anti-HBV antibody.

[0039] In certain embodiments, the HBV gene expression inhibitor is an RNAi agent that inhibits the expression of HBV transcripts. In a specific embodiment, the RNAi agent is siRNA (also referred to herein as "double-stranded RNA" or "dsRNA"), which targets and inhibits the expression of mRNA encoded by the X gene of HBV.

[0040] In certain embodiments, the anti-HBV antibody recognizes HBV genotypes A, B, C, D, E, F, G, H, I, and J and / or is a human antibody. In a specific embodiment, the anti-HBV antibody is selected from: an HBC34 wild-type antibody, an unnatural variant of an HBC34 antibody, and / or an HBC24 antibody.

[0041] In some embodiments described herein, HBV gene expression inhibitors and anti-HBV antibodies act synergistically to reduce viral load and circulating HBsAg. This combination therapy may provide a functional cure for chronic HBV and may allow for administration of lower doses of antibodies, thereby reducing the likelihood of antibody-induced toxicity.

[0042] I. Glossary

[0043] The following section provides a detailed description of HBV combination therapy, including: an HBV protein expression inhibitor; an anti-HBV antibody; a method for treating a subject using the combination of an HBV protein expression inhibitor and an anti-HBV antibody; and a kit related to the combination therapy. Before describing this disclosure in more detail, it may be helpful to provide definitions of certain terms used herein. Additional definitions are set forth throughout this disclosure.

[0044] In this specification, unless otherwise stated, the term "about" refers to ±20% of the indicated range, value or structure.

[0045] The term "comprising" refers to the presence of the features, integers, steps, or components recited in a claim, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0046] It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean any one, two, or any combination of the alternatives and can be used synonymously with "and / or." As used herein, the terms "including" and "having" are used synonymously, and these terms and variations thereof are intended to be interpreted as non-limiting.

[0047] for example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definitions provided herein.

[0048] As used herein, the term "disease" is intended to be generally synonymous with the terms "disorder" and "condition" (as in medical condition) and may be used interchangeably because they all reflect an abnormal condition of the human or animal body or one of its parts that impairs normal function, usually manifests itself by distinctive signs and symptoms, and results in a decrease in the duration or quality of life of the human or animal.

[0049] As used herein, the terms "peptide", "polypeptide" and "protein" and variants of these terms refer to molecules, particularly peptides, oligopeptides, polypeptides or proteins, including fusion proteins, respectively comprising at least two amino acids that are interconnected by a normal peptide bond or by a modified peptide bond, such as in the case of isosteric peptides. For example, a peptide, polypeptide or protein can be made up of the 20 amino acid residues defined by the genetic code, connected to each other by a normal peptide bond ("classical" polypeptide). A peptide, polypeptide or protein can be made up of L-amino acids and / or D-amino acids. In particular, the terms "peptide", "polypeptide" and "protein" also include "peptide mimics", which are defined as peptide analogs containing non-peptide structural elements that can simulate or antagonize the biological effects of natural parent peptides. Peptide mimics lack classical peptide features, such as enzymatic cleavage peptide bonds. In particular, in addition to these amino acids, a peptide, polypeptide or protein can comprise the amino acids in addition to the 20 amino acids defined by the genetic code, or it can be made up of the amino acids in addition to the 20 amino acids defined by the genetic code. In particular, peptides, polypeptides or proteins in the context of the present disclosure may also be composed of amino acids modified by natural processes, such as post-translational maturation processes or chemical processes, which are well known to those skilled in the art. Such modifications are described in detail in the literature. These modifications may occur anywhere in the polypeptide: the peptide backbone, the amino acid chain, or even the carboxyl or amino termini. In particular, the peptide or polypeptide may be branched or cyclic with or without branches after ubiquitination. This type of modification may be the result of natural or synthetic post-translational processes well known to those skilled in the art. In the context of the present disclosure, the terms "peptide," "polypeptide," or "protein" also include modified peptides, polypeptides, and proteins. For example, peptide, polypeptide or protein modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositols, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, seneloylation, sulfation, amino acid addition such as arginylation, or ubiquitination.Such modifications are well described in the literature (Proteins Structure and Molecular Properties, 2nd Ed., E. Creighton, New York (1993); Post-translational Covalent Modifications of Proteins, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182:626-46 (1990); and Rattan et al., Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci 663:48-62 (1992)). Thus, the terms "peptide," "polypeptide," and "protein" include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like.

[0050] As used herein, a "(poly)peptide" comprises a single chain of amino acid monomers linked by peptide bonds as described above. As used herein, a "protein" comprises one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (poly)peptides, i.e., a chain of one or more amino acid monomers linked by peptide bonds as explained above. In a specific embodiment, a protein according to the present disclosure comprises 1, 2, 3 or 4 polypeptides.

[0051] As used herein, the term "recombinant" (e.g., recombinant antibodies, recombinant proteins, recombinant nucleic acids, etc.) refers to any molecule (antibody, protein, nucleic acid, siRNA, etc.) that is prepared, expressed, formed, or isolated by recombinant means and is not naturally occurring. As used herein, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably and are intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded. In specific embodiments, the nucleic acid molecule is double-stranded RNA.

[0052] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all of these designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be identical in DNA content, due to intentional or unintentional mutations. Variant progeny screened for the same function or biological activity as the originally transformed cell are included. Where a different designation is intended, this will be clear from the context.

[0053] As used herein, the term "sequence variant" refers to any sequence that has one or more changes compared to a reference sequence, wherein the reference sequence is any sequence listed in the sequence listing, i.e., SEQ ID NO: 1 to SEQ ID NO: 104. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. For sequence variants in the context of nucleotide sequences, the reference sequence is also a nucleotide sequence, and for sequence variants in the context of amino acid sequences, the reference sequence is also an amino acid sequence. As used herein, a "sequence variant" has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a reference sequence. Unless otherwise indicated, sequence identity is typically calculated over the entire length of the reference sequence (i.e., the sequence described in the application). Percent identity as referred to herein can be determined, for example, using BLAST using the default parameters specified by NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap opening penalty = 11 and gap extension penalty = 1]. " Sequence variant " in the context of nucleic acid (nucleotide) sequence has the sequence of change, wherein one or more nucleotides in the reference sequence are deleted or replaced, or one or more nucleotides are inserted in the sequence of the reference nucleotide sequence. Nucleotide is referred to by standard single-letter name (A, C, G or T) in this article. Due to the degeneracy of genetic code, the " sequence variant " of nucleotide sequence can cause the change of corresponding reference amino acid sequence, i.e. amino acid " sequence variant " or do not change. In some embodiments, nucleotide sequence variant is the variant that does not cause generation amino acid sequence variant (that is, silent mutation). However, the nucleotide sequence variant that causes " non-silent " sudden change is also within the scope, particularly such nucleotide sequence variant, it produces the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical with the reference amino acid sequence. " Sequence variant " in the context of amino acid sequence has the sequence of change, wherein compared with the reference amino acid sequence, one or more amino acids are deleted, replaced or inserted. As a result of the alterations, such sequence variants have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having no more than 10 alterations (i.e., any combination of deletions, insertions, or substitutions) for every 100 amino acids in the reference sequence is "at least 90% identical" to the reference sequence.

[0054] In certain embodiments, although non-conservative amino acid substitutions are possible, the substitutions are conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the reference sequence. For example, conservative amino acid substitutions involve replacing an aliphatic or hydrophobic amino acid (e.g., alanine, valine, leucine, and isoleucine) with another; replacing an amino acid containing a hydroxyl group (e.g., serine and threonine) with another; replacing an acidic residue (e.g., glutamic acid or aspartic acid) with another; replacing an amide-containing residue (e.g., asparagine and glutamine) with another; replacing an aromatic residue (e.g., phenylalanine and tyrosine) with another; replacing a basic residue (e.g., lysine, arginine, and histidine) with another; and replacing a small amino acid (e.g., alanine, serine, threonine, methionine, and glycine) with another.

[0055] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusing the N-terminus or C-terminus of an amino acid sequence to a reporter molecule or enzyme.

[0056] Unless otherwise specified, the changes in the sequence variants do not eliminate the function of the corresponding reference sequence, for example, in the present case, the function of the sequence of the anti-HBV antibody or HBV gene expression inhibitor (e.g., siRNA) is sufficient to neutralize HBV infection or reduce HBV protein expression, respectively. Guidance on determining which nucleotides and amino acid residues can be substituted, inserted, or deleted without eliminating such function can be found by using computer programs well known in the art.

[0057] As used herein, "derived from" specifies the nucleic acid sequence or amino acid sequence of a nucleic acid, peptide, polypeptide or protein and refers to the source of the nucleic acid, peptide, polypeptide or protein. In some embodiments, the nucleic acid sequence or amino acid sequence derived from a specific sequence has an amino acid sequence substantially identical to the sequence or part thereof, wherein "substantially identical" includes sequence variants as defined above. In certain embodiments, the nucleic acid sequence or amino acid sequence derived from a specific peptide or protein is derived from the corresponding domain in the specific peptide or protein. Therefore, "corresponding to" particularly refers to identical function. For example, an "extracellular domain" corresponds to another "extracellular domain" of another (another protein), or a "transmembrane domain" corresponds to another "transmembrane domain" of (another protein). Therefore, "corresponding to" the part of a peptide, protein and nucleic acid is identifiable to one of ordinary skill in the art. Similarly, the sequence of "derived from" other sequences is typically identifiable to one of ordinary skill in the art because there is its source in the sequence.

[0058] In some embodiments, the nucleotide sequence or amino acid sequence that is derived from another nucleic acid, peptide, polypeptide or protein can be identical with initial nucleic acid, peptide, polypeptide or protein (from which it derives). However, the nucleotide sequence or amino acid sequence that is derived from another nucleic acid, peptide, polypeptide or protein can also have one or more sudden changes relative to (from which it derives), and in particular, the nucleotide sequence or amino acid sequence that is derived from another nucleic acid, peptide, polypeptide or protein can be the variant of the above-mentioned functional sequence of initial nucleic acid, peptide, polypeptide or protein (from which it derives). For example, in peptide / protein, one or more amino acid residues can be replaced by other amino acid residues or one or more amino acid residues can be inserted or deleted.

[0059] As used herein, the term "mutation" refers to a change in a nucleic acid sequence and / or an amino acid sequence compared to a reference sequence (e.g., a corresponding genomic sequence). For example, compared to a genomic sequence, a mutation can be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation (e.g., induced by an enzyme, a chemical substance, or radiation), or a mutation obtained by site-directed mutagenesis (a molecular biological method for preparing specific and deliberate changes in a nucleic acid sequence and / or an amino acid sequence). Therefore, the term "mutation" or "mutating" should be understood to also include, for example, physically making a mutation in a nucleic acid sequence or an amino acid sequence. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. To achieve mutations in an amino acid sequence, mutations can be introduced into the nucleotide sequence encoding the amino acid sequence to express (recombinantly) a mutated polypeptide. Mutations can be achieved, for example, by changing (e.g., by site-directed mutagenesis) the codons of a nucleic acid molecule encoding one amino acid to produce codons encoding a different amino acid, or by synthesizing sequence variants (e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and by designing the synthesis of a nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide variant) without mutating one or more nucleotides of the nucleic acid molecule.

[0060] As used herein, the term "coding sequence" is intended to refer to a polynucleotide molecule that encodes the amino acid sequence of a protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon.

[0061] As used herein, the term "expression" refers to any step involved in the production of a polypeptide including transcription, post-transcriptional modification, translation, post-translational modification, secretion, and the like.

[0062] In some aspects, the present disclosure relates to the purposes of HBV gene expression inhibitors. As used herein, "HBV gene expression inhibitor" is any agent that causes HBV gene expression to be at least partially reduced, such as by being able to separate or detect HBV mRNA amount from a first cell or cell group (wherein the HBV gene is transcribed and has been treated with an HBV gene expression inhibitor so that the expression of the HBV gene has been always) and a second cell or cell group (substantially the same as the first cell or cell group but not subjected to such treatment (control cells)) compared to achieve. In some embodiments, the HBV gene expression inhibitor is an RNAi agent (e.g., siRNA). HBV gene expression can be measured by methods well known in the art. Unless otherwise indicated, "HBV gene expression" as used herein refers to determining using rtPCR or by measuring protein expression using enzyme-linked immunosorbent assay (ELISA) or immunohistochemistry.

[0063] In some aspects, the disclosure relates to the purposes of the medicament for reducing HBV antigen load. As used herein, " medicament for reducing HBV antigen load " refers to any medicament that causes the HBV antigen amount that can be separated or detected from the first cell or cell group (treated with the medicament) to be reduced compared with the second cell or cell group (substantially the same as the first cell or cell group but without such treatment (control cells)). In some embodiments, the medicament for reducing HBV antigen load is an RNAi agent (e.g., siRNA). Antigen load can be measured by methods well known in the art. Unless otherwise stated, " HBV antigen complex " as used herein refers to determining by using ELISA to measure the amount of antigen (e.g., HBsAg).

[0064] The present disclosure provides a combination therapy for treating HBV, comprising an anti-HBV antibody. In certain embodiments, the anti-HBV antibody or antigen-binding fragment thereof binds to the antigenic loop region of HBsAg and neutralizes hepatitis B virus infection.

[0065] As used herein, the term "antibody" encompasses various forms of antibodies, including but not limited to full-length antibodies, antibody fragments, antigen-binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variants or mutant antibodies), as long as the characteristic properties of the antibody are retained. In some embodiments, the antibody is a human antibody and / or a monoclonal antibody. In specific embodiments, the antibody is a human monoclonal antibody. In certain specific embodiments, the antibody is a recombinant human monoclonal antibody. As used herein, the terms "antigen-binding fragment," "fragment," and "antibody fragment" can be used interchangeably to refer to any fragment of an antibody for a combination therapy that retains the antigen-binding activity of the antibody. Examples of antibody fragments include but are not limited to single-chain antibodies, Fab, Fab', F(ab')2, Fv, or scFv. Furthermore, as used herein, the term "antibody" includes both antibodies and antigen-binding fragments thereof.

[0066] As used herein, "neutralizing antibodies" are antibodies that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of a pathogen to initiate and / or persist an infection in a host. The terms "neutralizing antibody" and "antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used alone or in combination as prophylactic or therapeutic agents after appropriate formulation, in combination with active vaccination, as diagnostic tools, or as production tools as described herein.

[0067] Human antibodies are well known in the art (van Dijk, MA and van de Winkel, JC, Curr. Opin. Chem. Biol. 5:368-74 (2001)). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire or selection of human antibodies upon immunization in the absence of endogenous immunoglobulin production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90:2551-55 (1993); Jakobovits, A. et al., Nature 362:255-258 (1993); Bruggemann, M. et al., Year Immunol. 7:3340 (1993)). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR and Winter, G., Mol. Biol. 227: 381-88 (1992); Marks, JD et al., Mol Biol. 222: 581–97 (1991)). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner, P. et al., Immunol. 147: 86-95 (1991)). In some embodiments, human monoclonal antibodies are prepared by immortalizing EBV-B cells modified as described in Traggiai, E. et al. (Nat Med. 10 (8): 871-5 (2004)). The term "human antibody," as used herein, also includes such antibodies that have been modified, for example, in the variable regions, to produce the properties described herein.

[0068] The antibody of the combination therapy can be of any isotype (e.g., IgA, IgG, IgM, i.e., kappa, gamma, or mu heavy chain), but in certain specific embodiments, the antibody is IgG. Within the IgG isotype, the antibody can be of IgG1, IgG2, IgG3, or IgG4 subclass. In a specific embodiment, the antibody is IgG1. The antibody of the combination therapy can have a kappa or lambda light chain. Based on the antigen-independent uptake of IgG into hepatocytes through the FcRN-IgG receptor, IgG-type HBsAg-specific antibodies can also advantageously block the release of HBV and HBsAg from infected cells. Therefore, IgG-type HBsAg-specific antibodies can bind intracellularly, thereby blocking the release of HBV virions and HBsAg.

[0069] As used herein, the term "variable region" (light chain variable region (V L ), heavy chain variable region (V H )) represents the portion of an antibody light chain (LC) or heavy chain (HC) (generally around the 105-120 amino-terminal amino acids of a mature antibody heavy or light chain) that contains the complementarity determining region ("CDR") and framework region ("FR") and is directly involved in binding the antibody to an antigen. The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and refer to non-contiguous sequences of amino acids within an antibody variable region that are known in the art to confer antigen specificity and / or binding affinity. Typically, there are three CDRs in each variable region of an immunoglobulin binding protein; for example, for an antibody, V H and V L The region typically contains six CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3). Immunoglobulin sequences can be aligned with numbering schemes (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which can allow the use of the Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (Bioinformatics 15:298-300 (2016)) to mark equivalent positions and compare different molecules. It should be understood that in certain embodiments, the antibodies or antigen-binding fragments of the present disclosure may contain all or part of the heavy chain (HC), light chain (LC), or both. For example, a full-length intact IgG antibody monomer typically contains V H , CH1, CH2, CH3, VL and CL.

[0070] In certain embodiments, the anti-HBV antibody, or antigen-binding fragment thereof, of the combination therapy according to the present disclosure is a purified antibody, single-chain antibody, Fab, Fab', F(ab')2, Fv or scFv. Therefore, the antibody of the combination therapy can be a human antibody, a monoclonal antibody, a human monoclonal antibody, a recombinant antibody and / or a purified antibody. The present disclosure also provides fragments of antibodies, in particular fragments that retain the antigen-binding activity of the antibody. Examples of such fragments include, but are not limited to, single-chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Although in some places, the present disclosure may explicitly refer to one or more antigen-binding fragments, one or more antibody fragments, one or more variants and / or one or more derivatives of an antibody, as used herein, the term "antibody" or "antibody for combination therapy" includes all classes of antibodies, i.e., one or more antigen-binding fragments, one or more antibody fragments, one or more variants and one or more derivatives of an antibody.

[0071] Fragments of antibodies can be obtained from antibodies by digestion including with enzymes (such as pepsin or papain), and / or by chemical reduction methods for cleaving disulfide bonds. Alternatively, fragments of antibodies can be obtained by cloning and expressing partial sequences of heavy or light chains. The present disclosure also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of antibodies of the present disclosure. For example, the present disclosure includes scFv, which comprises the CDRs of antibodies from the present disclosure. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single-chain antibodies, for example, single-chain Fv, in which the heavy and light chain variable domains are joined by a peptide linker.

[0072] The antibody fragments of the present disclosure can impart monovalent or multivalent interactions and be included in various structures as described above. For example, scFv molecules can be synthesized to produce trivalent "tribodies" or tetravalent "tetrabodies". ScFv molecules can include domains that result in the production of bivalent miniantibodies in the Fc region. In addition, the sequences of antibodies / antibody fragments can be components of multispecific molecules, wherein the sequence targets expression as described herein, and other regions of the multispecific molecules bind to other targets. Exemplary multispecific molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, Nature Biotechnology 9: 1126-36 (2005)).

[0073] Antibodies according to the present disclosure can be provided in purified form. Typically, the antibodies 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 constituted by other polypeptides.

[0074] In embodiments, the antibodies and antigen-binding fragments of the present disclosure may be multispecific (e.g., bispecific, trispecific, tetraspecific, etc.) and may be provided in any multispecific format as disclosed herein. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are multispecific antibodies, such as bispecific or trispecific antibodies. Bispecific antibody formats are disclosed in, for example, Spiess et al. (Mol. Immunol. 67(2):95 (2015)), and Brinkmann and Kontermann (mAbs 9(2):182-212(2017)), the bispecific formats and methods of making the same are incorporated herein by reference and include, for example, bispecific T cell engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, TandAbs, triplet, TriBi minibody, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCab, endosomes, CrossMab, dual-action Fab (DAF) (two-in-one or four-in-one), DutaMab, DT-IgG, charge pairing, Fab arm exchange, SEED body, Triomab, LUZ-Y assembly, Fcab, κλ body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody and DVI-IgG (four-in-one). Bispecific or multispecific antibodies may comprise a HBV and / or HDV-specific binding domain of the present disclosure in combination with another such binding domain of the present disclosure, or a combination of different binding domains that specifically bind to HBV and / or HDV (e.g., at the same or different epitopes), or a combination of binding domains that specifically bind to different antigens.

[0075] As used herein, the term "vaccine" is generally understood to mean a preventive or therapeutic substance that provides at least one antigen or immunogen. Antigens or immunogens can be derived from any substance suitable for vaccination. For example, antigens or immunogens can be derived from pathogens, such as from bacteria or virions, or from tumors or cancerous tissues. Antigens or immunogens stimulate the body's adaptive immune system to provide an adaptive immune response. In particular, "antigen" or "immunogen" generally refers to a substance that can be recognized by the immune system (e.g., an adaptive immune system), and that can trigger an antigen-specific immune response (e.g., by forming antibodies and / or antigen-specific T cells as part of the adaptive immune system). Typically, an antigen can be or can comprise a peptide or protein that is presented to a T cell by MHC.

[0076] Doses are usually expressed relative to body weight. Thus, a dose expressed in [g, mg or other unit] / kg (or g, mg, etc.) usually means [g, mg or other unit] "per kg (or g, mg, etc.) of body weight" even if the term "body weight" is not explicitly mentioned.

[0077] As used herein, "hepatitis B virus," which is used interchangeably with the term "HBV," refers to a well-known non-cytopathic, hepatotropic DNA virus of the family Hepadnaviridae. The HBV genome is a partially double-stranded circular DNA with four overlapping reading frames (which may be referred to herein as "genes," "open reading frames," or "transcripts"): C, X, P, and S. The core protein (HBcAg) is encoded by gene C. Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the pre-core (pre-C) protein. The DNA polymer is encoded by gene P. Gene S is the gene encoding the surface antigen (HBsAg). The HBsAg gene is a long open reading frame containing three "start" (ATG) codons in frame, producing three different sized polypeptides, referred to as large, medium, and small S antigens (pre-S1+pre-S2+S, pre-S2+S, or S). In addition to decorating the envelope of HBV, surface antigens are also part of subviral particles, which are overproduced compared to virions and play a role in immune tolerance and chelating anti-HBsAg antibodies, thereby allowing infectious particles to escape immune detection. The function of the nonstructural protein encoded by gene X is not fully understood, but it plays a role in transcriptional transactivation and replication and is associated with the development of liver cancer. Eight genotypes of HBV have been determined (designated as A to H) and two other genotypes, I and J, have been proposed, each of which has a different geographical distribution. The term "HBV" includes any genotype (A to J) of HBV. The complete coding sequence of the reference sequence of the HBV genome can be found in, for example, GenBank gene accession numbers GI:21326584 and GI:3582357. The amino acid sequences of the C, X, P, and S proteins can be found, for example, in NCBI accession numbers YP_009173857.1 (C protein); YP_009173867.1 and BAA32912.1 (X protein); YP_009173866.1 and BAA32913.1 (P protein); and YP_009173869.1, YP_009173870.1, YP_009173871.1, and BAA32914.1 (S protein). Additional examples of HBV mRNA sequences can be readily obtained using publicly available databases, such as GenBank, UniProt, and OMIM. The International Hepatitis B Virus Strain Database can be accessed at http: / / www.hpa-bioinformatics.org.uk / HepSEQ / main.php. As used herein, the term "HBV" also refers to naturally occurring DNA sequence variants of the HBV genome, ie, genotypes A to J and variants thereof.

[0078] II. HBV Protein Expression Inhibitors and Delivery Systems

[0079] The present disclosure provides HBV protein expression inhibitors for the combined therapy for treating HBV. In certain embodiments, the HBV gene expression inhibitor is an RNAi agent. As used herein, the term "RNA interference agent" or "RNAi agent" refers to an agent containing RNA as defined herein, and it mediates the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent described herein affects the inhibition of HBV gene expression.

[0080] In one aspect, RNA interference agent includes single-stranded RNA, which interacts with the target RNA sequence to direct the cutting of the target RNA. Without wishing to be bound by a particular theory, the long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is to be broken into siRNA (Sharp et al., Genes Dev.15:485 (2001)) by type III endonuclease (called Dicer). Dicer is a ribonuclease III sample enzyme that processes dsRNA into short interfering RNA (siRNA) (Bernstein et al., Nature 409:363 (2001)) of 19 to 23 base pairs with characteristic two base 3' overhangs. Then, the siRNA is introduced into RNA-induced silencing complex (RISC), where one or more helicases untie the siRNA double helix, and the complementary antisense strand is able to guide the identification of target (Nykanen et al., Cell 107:309 (2001)). When bound to a suitable target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., Genes Dev. 15:188 (2001)). Thus, in one aspect, the technology described herein relates to a single-stranded RNA that promotes the formation of a RISC complex to affect the silencing of a target gene.

[0081] As long as they refer to the HBV gene, the terms "silencing", "inhibiting its expression", "downregulating its expression", "suppressing its expression" and the like refer herein to at least a partial reduction in HBV gene expression, such as by a reduction in the amount of HBV mRNA that can be isolated or detected from a first cell or cell population (in which the HBV gene is transcribed and has been treated with an inhibitor of HBV gene expression such that expression of the HBV gene has been inhibited) compared to a second cell or cell population that is substantially identical to the first cell or cell population but has not been treated in this manner (control cells). The degree of inhibition can be measured, for example, by subtracting the difference between the mRNA expression levels in the treated cells from the mRNA expression levels in the control cells. Alternatively, the degree of inhibition can be given by a reduction in a parameter functionally linked to HBV gene expression, for example, the amount of protein edited by the HBV gene, or data on cells exhibiting certain phenotypes, for example, HBV infection phenotypes (such as HBV infection), HBV protein expression (such as hepatitis B surface antigen, HBsAg) or changes in cellular gene expression that reflect HBV gene expression (e.g., Smc5 / 6 expression and localization). The degree of inhibition can also be measured using cells engineered to express a reporter gene reflecting HBV RNA expression. In theory, HBV gene silencing can be detected in any cell expressing an HBV gene, e.g., an HBV-infected cell or a cell engineered to express an HBV gene, and by any appropriate assay.

[0082] The level of HBV RNA expressed by a cell or cell population or the level of circulating HBV RNA can be measured using any method known in the art for evaluating mRNA expression, such as the rtPCR method provided in Example 2 of International Application Publication No. WO2016 / 077321A1 and U.S. Patent Application No. US2017 / 0349900A1, which are incorporated herein by reference. In some embodiments, the level of HBV gene expression in a sample (e.g., total HBV RNA, HBV transcripts, e.g., 3.5 kb transcripts) is measured by detecting transcribed polynucleotides or portions thereof (e.g., RNA of HBV genes). RNA can be extracted from cells using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit, or the like. Or PAXgene (PreAnalytix, Switzerland). Common detection formats using ribonuclease hybridization include nuclear transcription activity assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), northern blotting, in situ hybridization, and microarray analysis. Circulating HBV mRNA can be measured using the methods described in International Application Publication No. WO 2012 / 177906A1 and U.S. Patent Application No. US2014 / 0275211A1, which are incorporated herein by reference.

[0083] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an HBV gene, including mRNAs of RNA processing products of primary transcripts. The target portion of the sequence will be at least long enough to serve as a receptor for RNAi-directed cleavage at or near that portion. For example, the target sequence will typically be 9-36 nucleotides in length, for example, 15-30 nucleotides in length, including all subranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19 -26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides or 21-22 nucleotides.

[0084] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by referring to the sequence using standard nucleotide nomenclature.

[0085] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a double helical structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as will be understood by those skilled in the art. Such conditions may, for example, be stringent conditions, wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours, followed by washing. Other conditions may be applied, such as physiologically relevant conditions that may occur in an organism. Those skilled in the art will be able to determine the setting of the optimal conditions for the complementarity test of the two sequences based on the ultimate application of the hybridizing nucleotides.

[0086] The complementary sequence in an RNAi agent (e.g., in an siRNA as described herein) includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" relative to each other. However, when a first sequence is referred to herein as "substantially complementary" relative to a second sequence, the two sequences may be fully complementary, or they may form one or more, but typically no more than 5, 4, 3, or 2 mismatched base pairs after hybridization with a double helix of up to 30 base pairs, while retaining hybridization capability under conditions most relevant to their ultimate application (e.g., inhibition of gene expression by a RISC pathway). However, when two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs should not be considered to be mismatches for purposes of determining complementarity. For example, an siRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide, can still be referred to as "fully complementary" for the purposes described herein.

[0087] As used herein, "complementary" sequences may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, as long as the above requirements for their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein pairings.

[0088] The terms "complementary," "fully complementary," and "substantially complementary" may be used herein with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an RNAi agent and a target sequence, as will be understood from the context of their use.

[0089] As used herein, a polynucleotide that is "substantially complementary" to at least a portion of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an HBV protein). For example, if the sequence is substantially complementary to an uninterrupted portion of the HBV mRNA, then the polynucleotide is complementary to at least a portion of the HBV mRNA.

[0090] a.siRNA

[0091] In some embodiments, the RNAi agent comprises siRNA. As used herein, the term "siRNA" refers to an RNAi comprising an RNA molecule or molecular complex having a hybrid double helix region comprising two antiparallel and substantially complementary nucleic acid strands, which will be referred to as having "sense" and "antisense" orientations relative to the target RNA. The double helix region can be of any length, as long as it allows for specific degradation of the desired target RNA by the RISC pathway, but typically will range from 9 to 36 base pairs in length, e.g., 15-30 splice pairs in length. Considering that the duplex is between 9 and 36 splice pairs, the duplex can be any length within this range, for example, 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 or 36 and any subranges therebetween including, but not limited to, 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 1 8-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs and 21-22 base pairs. siRNAs generated in cells by treatment with Dicer and similar enzymes are typically in the range of 19 to 22 base pairs in length.The term "double-stranded RNA" or "dsRNA" is also used synonymously herein to refer to siRNAs as described above.

[0092] One chain of the double helix region of siRNA comprises a sequence that is substantially complementary to the region of the target RNA. The two chains forming the double helix structure can be from a single RNA molecule with at least one self-complementary region, or can be formed from two or more different RNA molecules. When the double helix structure region is formed from a double strand of a single molecule, the molecule can have a double helix region separated by a single nucleotide chain (referred to herein as a "hairpin loop"), between the 3' end of a chain and the 5' end of the corresponding other chain forming the double helix structure. The hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. When the two substantially complementary chains of siRNA are composed of different RNA molecules, these molecules do not need to be, but can be covalently linked. When these two chains are covalently linked in a manner other than the hairpin loop, then the connecting structure is referred to as a "joint."

[0093] The term "antisense strand" or "guide strand" refers to a strand in an RNAi agent, e.g., siRNA, that includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence). When the region of complementarity is not completely complementary to the target sequence, mismatches may be in internal or terminal regions of the molecule.

[0094] Typically, the maximum tolerable mismatch is in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5' and / or 3' termini.

[0095] As used herein, the term "sense strand" or "follower strand" refers to the strand of an RNAi that includes a region that is substantially complementary to a region of the antisense strand (as that term is defined herein).

[0096] In another aspect, the agent is a single-stranded antisense RNA molecule. The antisense RNA molecule can have 15 to 30 nucleotides complementary to the target. For example, the antisense RNA molecule can have a sequence of at least 15, 16, 17, 18, 19, 20, 21 or more adjacent nucleotides from one of the antisense sequences disclosed herein.

[0097] What those skilled in the art will recognize is that the term "RNA molecule" or "ribonucleic acid molecule" not only encompasses the RNA molecule expressed or found in nature, but also encompasses analogs and derivatives of RNA, which comprise one or more ribonucleotides / ribonucleoside analogs or derivatives as described herein or as well known in the art. Strictly speaking, "ribonucleoside" comprises nucleoside bases and ribose, and "ribonucleotide" refers to a ribonucleoside with one, two or three phosphate moieties. However, as used herein, the terms "ribonucleoside" and "ribonucleotide" can be considered to be equal. RNA can be modified in a core base structure or in a ribose phosphate backbone structure, such as described in more detail below. However, the siRNA molecule comprising a ribonucleoside analog or derivative retains the ability to form a double helix. As non-limiting examples, RNA molecules may also include at least one modified ribonucleoside, including but not limited to 2'-O-methyl modified nucleosides, nucleosides comprising 5' thiophosphate groups, terminal nucleosides connected to cholesterol derivatives or dodecyl bisdecylamide groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino-modified nucleosides, 2'-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates or the non-natural bases comprising nucleosides, or any combination thereof. Alternatively, RNA molecules may include at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the siRNA molecule. For each of such multiple modified ribonucleosides in RNA molecules, modification need not be identical. In some embodiments, the modified RNA contemplated for use in the methods and compositions described herein is a peptide nucleic acid (PNA), which has the ability to form a desired double helical structure and which allows or mediates the specific degradation of the target RNA via the RISC pathway.

[0098] In some embodiments, the modified ribonucleosides include deoxyribonucleosides. For example, an RNAi agent can include one or more deoxyribonucleosides, including, for example, one or more deoxyribonucleoside overhangs, or one or more deoxyribonucleosides within the double-stranded portion of the siRNA. However, as used herein, the term "RNAi agent" does not include a complete DNA molecule.

[0099] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double helical structure of an RNAi agent (e.g., siRNA). For example, a nucleotide overhang is present when the 3' end of one strand in the siRNA extends beyond the 5' end of the other strand, or vice versa. The siRNA may comprise an overhang of at least one nucleotide, alternatively, the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. One or more overhangs may be located on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0100] In some embodiments, the antisense strand of the siRNA has an overhang of 1-10 nucleotides at the 3' end and / or the 5' end. In some embodiments, the sense strand of the siRNA has an overhang of 1 to 10 nucleotides at the 3' end and / or the 5' end. In some other embodiments, one or more nucleotides in the overhang are replaced by nucleoside phosphorothioates.

[0101] In some embodiments, at least one end of the siRNA has an overhang of 1 to 4 single-stranded nucleotides, typically 1 or 2 nucleotides. siRNAs having an overhang of at least one nucleotide can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts.

[0102] As used herein with reference to siRNA, the term "blunt-ended" or "flat-ended" means that there are no unpaired nucleotides or nucleotide analogs at a given end of the siRNA, i.e., no nucleotide overhangs. One or both ends of the siRNA can be blunt-ended. When both ends of the siRNA are blunt-ended, the siRNA is referred to as "blunt-ended." A "blunt-ended" siRNA is an siRNA that is blunt-ended at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. Most commonly, such molecules will be double-stranded throughout their entire length.

[0103] In certain embodiments, the combination therapy described herein includes one or more RNAi agents that inhibit HBV gene expression. In some embodiments, an RNAi agent comprising a short interfering ribonucleic acid (siRNA) is used to inhibit the expression of an HBV gene in a mammal (e.g., a human infected with HBV), wherein the siRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of an mRNA formed during HBV gene expression, and wherein the complementary region is 30 nucleotides long or less, typically 19 to 24 nucleotides long, and when the siRNA is contacted with a cell expressing the HBV gene, the expression of the HBV gene is inhibited by at least 10%, as detected by, for example, PCR or branched DNA (bDNA)-based methods, or by protein-based methods such as Western blotting. Expression of HBV genes in cell culture or expression of cellular genes that serve as surrogates for HBV gene expression (e.g., Smc5 / 6) (e.g., in COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells, or in a biological sample from a subject) can be measured by measuring HBV mRNA levels (e.g., by bDNA or TaqMan assays) or by measuring protein levels (e.g., by immunofluorescence analysis, using, for example, Western blotting or flow cytometry).

[0104] siRNA comprises two RNA chains, which are complementary and hybridize to form a duplex structure under the conditions of using the siRNA. One chain (antisense strand) of siRNA comprises a complementary region, which is substantially complementary to the target sequence, and is typically completely complementary. The target sequence can be derived from the sequence of the mRNA formed during the expression of the HBV gene. The other chain (sense strand) comprises a region complementary to the antisense strand so that when merging these two chains under suitable conditions, the two chains hybridize and form a duplex structure. Typically, the duplex structure is between 15 and 30 (including end values), more typically between 18 and 25 (including end values), more typically between 19 and 24 (including end values), and typically between 19 and 21 base pairs in length (including end values). Similarly, the complementary region of the target sequence is between 15 and 30 (including the end value), more generally between 18 and 25 (including the end value), more generally between 19 and 24 (including the end value), and generally between 19 and 21 nucleotides in length (including the end value). In some embodiments, siRNA is a length between 15 and 20 nucleotides (including the end value), and in other embodiments, siRNA is a length between 25 and 30 nucleotides (including the end value). As will be appreciated by those of ordinary skill in the art, the most common target region of the RNA of targeted cutting is a part of a larger RNA molecule (typically an mRNA molecule). Relatedly, "a part" of an mRNA target is an adjacent sequence of an mRNA target with sufficient length to become a substrate for RNAi directed cutting (that is, cutting by RISC approach). siRNA with a double helix as short as 9 base pairs can mediate RNAi directed RNA cutting under some circumstances. The most common target will be a length of at least 15 nucleotides. In certain embodiments, the target is a length of 15-30 nucleotides.

[0105] It will be appreciated by those skilled in the art that this duplex region is the main functional part of siRNA, for example, a duplex region of 9 to 36 (for example, 15-30) base pairs. Therefore, in some embodiments, in order to reach the degree of being processed into a functional duplex (for example, 15 to 30 base pairs) of the required RNA cutting of the targeting, the RNA molecule or the complex of the RNA molecule with a duplex region greater than 30 base pairs is siRNA. Therefore, then, it will be appreciated by those of ordinary skill in the art that in some embodiments, miRNA is siRNA. In some other embodiments, siRNA is not naturally occurring miRNA. In some embodiments, RNAi agents that can be used for targeting HBV gene expression are not produced in target cells by cutting larger double-stranded RNA.

[0106] The siRNAs described herein can be synthesized by standard methods well known in the art, for example, by using an automated DNA synthesizer, such as commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0107] In some embodiments, the RNAi agent comprises an siRNA that targets and inhibits HBV mRNA expression. In some embodiments, the RNAi agent comprises an siRNA that targets and inhibits expression of mRNA encoded by the HBV genome according to the NCBI reference sequence NC_003977.2 (GenBank accession number GI:21326584) (SEQ ID NO:1). Transcription of the HBV genome produces polycistronic, overlapping RNAs. Therefore, in some embodiments, a combination therapy of siRNAs targeting a single HBV gene can result in significant inhibition of expression of most or all HBV transcripts. In some embodiments, the mRNA target of the siRNA can be an mRNA encoded by the following genes: P gene, nucleotides 2309-3182 and 1-1625 of NC_003977.1; S gene (encoding L, M, and S proteins), nucleotides 2850-3182 and 1-837 of NC_003977; X protein, nucleotides 1376-1840 of NC_003977; and / or C gene, nucleotides 1816-2454 of NC_003977.

[0108] In some embodiments, the siRNA targets and inhibits expression of mRNA encoded by the HBV X gene. In some embodiments, the RNAi agent or siRNA targets mRNA encoded by a portion of the HBV genome comprising the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO: 2), which corresponds to nucleotides 1579-1597 of NC_003977.2 (GenBank Accession No. GI: 21326584) (SEQ ID NO: 1).

[0109] In yet another embodiment, the siRNA has a sense strand comprising 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3) and an antisense strand comprising 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0110] In certain embodiments, the HBV gene expression inhibitor comprises an siRNA containing a sense chain and an antisense chain, wherein the sense chain comprises SEQ ID NO: 3, or a sequence that differs from SEQ ID NO: 3 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides; and wherein the antisense chain comprises SEQ ID NO: 4, or a sequence that differs from SEQ ID NO: 4 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides.

[0111] In one aspect, the siRNA will include at least two nucleotide sequences, a sense and an antisense sequence, whereby the sense sequence comprises SEQ ID NO: 3 and the corresponding antisense sequence comprises SEQ ID NO: 4. In this aspect, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the mRNA produced during HBV gene expression. Thus, in this aspect, the siRNA will include two oligonucleotides, one of which is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere herein and as is well known in the art, the complementary sequence of the siRNA can also include a self-complementary region as a single nucleic acid molecule, as opposed to being located on a separate oligonucleotide.

[0112] In yet another embodiment, the siRNA has a sense strand comprising 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 106), and an antisense strand comprising 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107).

[0113] In certain embodiments, the HBV gene expression inhibitor comprises an siRNA containing a sense chain and an antisense chain, wherein the sense chain comprises SEQ ID NO: 106, or a sequence that differs from SEQ ID NO: 106 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides; and wherein the antisense chain comprises SEQ ID NO: 107, or a sequence that differs from SEQ ID NO: 107 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides.

[0114] In one aspect, the siRNA will comprise at least two nucleotide sequences, a sense and an antisense sequence, whereby the sense sequence comprises SEQ ID NO: 106 and the corresponding antisense sequence comprises SEQ ID NO: 107. In this aspect, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of an mRNA produced during HBV gene expression. Thus, in this aspect, the siRNA will comprise two oligonucleotides, one of which is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere herein and as is well known in the art, the complementary sequence of the siRNA can also comprise a self-complementary region as a single nucleic acid molecule, as opposed to being located on a separate oligonucleotide.

[0115] It will be appreciated by those skilled in the art that siRNAs having a duplex structure of between about 20 and 23, but particularly 21, base pairs have been described as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 20:6877-88 (2001)). However, others have found that shorter or longer RNA duplex structures may also be effective. In the embodiments described above, the siRNAs described herein may include at least one strand having a length of at least 21 nucleotides. In some embodiments, shorter duplexes having a sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:106, or SEQ ID NO:107, with only a few nucleotides missing on one or both ends, are similarly effective as compared to the above siRNAs. Thus, siRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one or both of SEQ ID NO: 3 or SEQ ID NO: 4 are expected to have an ability to inhibit HBV gene expression that differs by no more than 5, 10, 15, 20, 25, or 30% compared to an siRNA comprising the full-length sequence, according to the techniques described herein. Also within the present disclosure are siRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one or both of SEQ ID NO: 106 and SEQ ID NO: 107, and according to the techniques described herein, are expected to have an ability to inhibit HBV gene expression that differs by no more than 5, 10, 15, 20, 25, or 30% compared to an siRNA comprising the full-length sequence.

[0116] In addition, the siRNA provided herein identifies sites in HBV gene transcripts that are susceptible to cleavage by RISC. Thus, the technology described herein further characterizes RNAi agents that target one of these sequences. As used herein, if RNAi promotes cleavage of a transcript anywhere within a specific site, the RNAi agent is said to target a specific site within the RNA transcript. In some embodiments, the RNAi agent includes at least 15 adjacent nucleotides from one or both of the sequences of SEQ ID NO: 3 and SEQ ID NO: 4, which are coupled to additional nucleotide sequences obtained from a region adjacent to the selected sequence in the HBV gene. In some embodiments, the RNAi agent includes at least 15 adjacent nucleotides from one or both of the sequences of SEQ ID NO: 106 and SEQ ID NO: 107, which are coupled to additional nucleotide sequences obtained from a region adjacent to the selected sequence in the HBV gene.

[0117] Although target sequences are typically 15-30 nucleotides in length, there is wide variation in the suitability of specific sequences within this range for directed cleavage of any given target RNA. Various software packages and guidelines are listed herein to provide guidance for identifying the best target sequence for any given gene target, but an empirical approach can also be employed in which a "window" or "mask" of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, for example, in silico) placed on the target RNA sequence to identify sequences within a size range that can serve as target sequences. By gradually moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform best, can identify those RNA sequences that, when targeted with an RNAi agent, result in the best inhibition of target gene expression. It is expected that further optimization of inhibition efficiency can be achieved by gradually "window walking" one nucleotide upstream or downstream along a given sequence to identify sequences with equal or better inhibition properties.

[0118] In addition, it is contemplated that any sequence identified (e.g., SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 106, or SEQ ID NO: 107) can be further optimized by systematically adding or removing nucleotides to generate longer or shorter sequences and testing these and those sequences generated by moving the window of longer or shorter sequences up or down the target RNA from this point. Again, combining this method of generating new candidate targets with testing the effectiveness of RNAi agents based on those target sequences in inhibition assays known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further, such optimized sequences can be adjusted by, for example, introducing modified nucleotides as described herein or as known in the art, adding or changing overhangs, or other modifications as known in the art and / or discussed herein to further optimize the molecule as an expression inhibitor (e.g., to increase serum stability or circulation half-life, increase thermal stability, enhance transmembrane delivery, target specific locations or cell types, increase interaction with silencing pathway enzymes, increase release from endosomes, etc.).

[0119] RNAi agents as described herein may include one or more mismatches with the target sequence. In some embodiments, RNAi agents as described herein include no more than 3 mismatches. In some embodiments, if the antisense strand of the RNAi agent includes a mismatch with the target sequence, the mismatch region will not be located at the center of the complementarity region. In a specific embodiment, if the antisense strand of the RNAi agent includes a mismatch with the target sequence, the mismatch is confined to the last 5 nucleotides from the 5' or 3' end of the complementarity region. For example, for the RNA chain of a 23-nucleotide RNAi agent complementary to the region of the HBV gene, the RNA chain must not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch with the target sequence effectively inhibits HBV gene expression. It is important to consider the efficacy of RNAi agents with mismatches in inhibiting the expression of the HBV gene, particularly if it is known that the specific complementary region in HBV has polymorphic sequence variations.

[0120] b. Chemically modified RNAi agents

[0121] In some embodiments, the RNA of the RNAi agent (e.g., siRNA) is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids characterized in the technology described herein can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, the methods of which are incorporated herein by reference.

[0122] For example, modification includes (a) terminal modification, for example, 5' terminal modification (phosphorylation, conjugation, inverted connection, etc.), 3' terminal modification (conjugation, DNA nucleotide, inverted connection, etc.), (b) base modification, for example, substituted with stabilized base, destabilized base or base that base pairs with the expanded partner library, removed base (abasic nucleotide) or conjugated base, (c) sugar modification (for example, at the 2' position or the 4' position) or the replacement of sugar, and (d) backbone modification, including modification or replacement of phosphodiester linkage. The specific examples of RNA compounds used in the embodiments described herein include but are not limited to RNA containing a modified backbone or without natural internucleotide connection. RNA with a modified backbone especially includes those without a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes mentioned in the art, a modified RNA without a phosphorus atom in its internucleoside backbone can also be considered as an oligonucleoside. In a specific embodiment, the modified RNA will have a phosphorus atom in its internucleoside backbone.

[0123] Not all positions in a given compound need be uniformly modified, and in fact, more than one of the aforementioned modifications may be introduced into a single compound or even into a single nucleoside within an RNAi agent. The technology described herein also includes RNAi agent compounds that are chimeric compounds. In the context of the present disclosure, "chimeric" RNAi agent compounds or "chimeras" are RNAi compounds, such as siRNAs, that contain two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., a nucleotide in the case of siRNA compounds. These RNAi agents typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increase cellular uptake, and / or increase binding affinity to the target nucleic acid. Additional regions of the RNAi agent can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double helix. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly enhancing the efficiency of RNAi inhibitor gene expression. Therefore, when using chimeric siRNAs, comparable results can usually be achieved with shorter RNAi agents compared to phosphorothioate deoxy siRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

[0124] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphates (including 3'-alkylene phosphates and chiral phosphates), phosphites, phosphoamidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thiophosphoramidates, thioalkylphosphates, thioalkylphosphotriesters and boranophosphates, 2'-5' linked analogs of these esters, and those with reversed polarity, wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5 to '5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0125] Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799 ; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,59 0; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464; each of which is incorporated herein by reference.

[0126] Modified RNA backbones that do not include a phosphorus atom have internucleoside linkages formed by short-chain alkyl or cycloalkyl groups, internucleoside linkages between mixed heteroatoms and alkyl or cycloalkyl groups, or internucleoside linkages between one or more short-chain heteroatoms or heterocycles. These include backbones with morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; alkylene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazinyl backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 components.

[0127] Representative U.S. patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489, 677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439; each of which is incorporated herein by reference for its teachings relating to such methods of preparation.

[0128] In other embodiments, it is considered appropriate to use suitable RNAi mimics in RNAi agents, wherein both the sugar and the internucleoside connection (i.e., the backbone) of the nucleotide unit are replaced by novel groups. The base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, which is an RNA mimic that has been shown to have excellent hybridization properties, is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and is directly or indirectly bonded to the nitrogen atom of the backbone amide portion. Representative U.S. patents that teach the preparation of RNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262; each of which is incorporated herein by reference for teachings related to such preparation methods. Other teachings of PNA compounds can be found, for example, in Nielsen et al. (Science, 254: 1497-1500 (1991)).

[0129] Some embodiments characterized in the technology described herein include RNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, and in particular the backbones are -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as methylene (methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-], as well as amide backbones in U.S. Pat. No. 5,602,240. In some embodiments, the RNA characterized herein has a morpholine backbone structure in U.S. Pat. No. 5,034,506.

[0130] The modified RNA may also comprise one or more substituted sugar moieties. The RNAi agents (e.g., siRNA) featured herein may include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl; wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable substitutions include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are from 1 to about 10. In other embodiments, the siRNA comprises one of the following at the 2' position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silicon, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of RNAi agent, or group for improving the pharmacodynamic properties of RNAi agent and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta 78:486-504 (1995)), i.e., alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE and 2'-dimethylaminoethoxyethyl (also known in the art as 2*-O-dimethylaminoethoxyethyl or 2*-DMAEOE), i.e., 2*-O-CH2-O-CH2-N(CH2)2.

[0131] Other exemplary modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the RNAi agent, particularly the 3' position of the sugar on the 3' terminal nucleotide or in a 2'-5' linked siRNA and the 5' position of the 5' terminal nucleotide. The RNAi agent can also have a sugar mimetic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0132] Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920; each of which is incorporated herein by reference for its teachings relating to such methods of preparation.

[0133] RNAi agents can also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and thymine. uracil, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine (Herdewijn, P. ed. Wiley-VCH, (2008)); those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering (pp. 858-859, Kroschwitz, JL ed. John Wiley & Sons (1990)), those disclosed by Englisch et al. (Angewandte Chemie, International Edition, 30, 613 (1991)), and those disclosed by Sanghvi, Y S. (Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B. eds., CRC Press (1993)). Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds characterized in the technology described herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, pp. 276-278 (1993)) and are an exemplary base substitution, even particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0134] Representative U.S. patents that teach the preparation of some of the modified nucleobases described above, as well as other modified nucleobases, include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,59 each of which is incorporated herein by reference for its teachings relating to such methods of preparation.

[0135] The RNA of an RNAi agent can also be modified to include one or more locked nucleic acids (LANs). Locked nucleic acids are nucleotides with a modified ribose moiety that contains an additional bridge connecting the 2' carbon and the 4' carbon. The result effectively "locks" the nucleic acid in a 3'-endo configuration. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., Nucleic Acids Research 33(1):439-47 (2005); Mook, OR et al., Mol Cane Ther 6(3):833-43 (2007); Grunweller, A. et al., Nucleic Acids Research 31(12):3185-93 (2003)).

[0136] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845; each of which is incorporated herein by reference for its teachings relating to such preparation methods.

[0137] In certain embodiments, the combination therapy includes an siRNA modified to include one or more adenosine-glycol nucleic acids ("GNAs") Adenosine-GNAs are described, for example, in Zhang et al. (JACS 127(12):4174-75 (2005)).

[0138] In some embodiments, the present disclosure provides methods and related compositions wherein the RNAi is a siRNA comprising an oligonucleotide sequence having one or more modified nucleotides. Table 1 provides abbreviations for nucleotide monomers in modified nucleic acid sequences as used herein.

[0139] Table 1: Abbreviations of nucleotide monomers used in the representation of modified nucleic acid sequences It will be understood that, unless otherwise indicated, these monomers, when present in an oligonucleotide, are linked to each other by 5'-3'-phosphodiester bonds.

[0140]

[0141]

[0142] In some embodiments, the HBV gene expression inhibitor comprises siRNA, wherein the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand comprising 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6).

[0143] In yet another embodiment, the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 7) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 8).

[0144] In certain embodiments, the HBV gene expression inhibitor comprises an siRNA comprising a sense chain and an antisense chain, wherein the sense chain comprises SEQ ID NO: 5 or SEQ ID NO: 7, or a sequence that differs from SEQ ID NO: 5 or SEQ ID NO: 7 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively.

[0145] In certain embodiments, the HBV gene expression inhibitor comprises an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises SEQ ID NO: 6 or SEQ ID NO: 8, or a sequence that differs from SEQ ID NO: 6 or SEQ ID NO: 8 by no more than 4, no more than 3, no more than 2, or no more than 2 nucleotides, respectively.

[0146] In some embodiments, the HBV gene expression inhibitor comprises siRNA, wherein the siRNA has a sense strand comprising 5'-gsgsuggaCfuUfCfUfcucaAfUfuuuaL96-3' (SEQ ID NO: 108), and an antisense strand comprising 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 109).

[0147] In certain embodiments, the HBV gene expression inhibitor comprises an siRNA comprising a sense chain and an antisense chain, wherein the sense chain comprises SEQ ID NO: 108, or a sequence that differs from SEQ ID NO: 108 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide.

[0148] c. RNAi Agents Conjugated to Ligands

[0149] In some embodiments, the RNAi agent includes a modification involving chemically linking the RNA to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the RNAi agent. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA 86:6553-56 (1989)), cholic acid (Manoharan et al., Biorg. Med. Chem. Let. 4:1053-60 (1994)), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci. 660:306-9 (1992); Manoharan et al., Biorg. Med. Chem. Let. 3:2765-70 (1993)), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 20:533-38 (1992)), an aliphatic chain, e.g., dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J 10:1111-18 (1991); Kabanov et al., FEBS Lett. 259:327-30 (1990); Svinarchuk et al., Biochimie 75:49-54 (1993)), phospholipids, for example, di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett. 36:3651-54 (1995); Shea et al., Nucl. Acids Res. 18:3777-83 (1990)), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides 14:969-73 (1995)), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 36:3651-54 (1995); Shea et al., Nucl. Acids Res. 18:3777-83 (1990)). Lett. 36:3651-54 (1995)), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1264:229-37 (1995)), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 277:923-37 (1996)).

[0150] In some embodiments, the ligand alters the distribution, targeting, or lifespan of the RNAi agent into which it is introduced. In some embodiments, the ligand provides enhanced affinity for a selected target, such as a molecule, cell, cell type, compartment (e.g., a compartment of a cell or organ, a tissue, organ, or region of the body), as compared to a substance in the absence of such a ligand. In such embodiments, the ligand will not participate in duplex pairing in duplex nucleic acids.

[0151] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipids. Lipids can also include recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0152] The ligand can also comprise a targeting group, for example, a cell or tissue targeting agent that binds to a specific cell type (such as a hepatocyte), for example, a lectin, a glycoprotein, a lipid or a protein, for example, an antibody. The targeting group can be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipid, cholesterol, a steroid, a bile acid, a folic acid, vitamin B12, vitamin A, biotin or an RGD peptide or RGD peptide mimetic. Other examples of lipids include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), synthetic endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0 (hexadecyl) glycerol, geranyloxyhexyl group, hexadecyl glycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl group, palmitic acid, myristic acid,

[0015] Examples of the present invention include, but are not limited to, oligonucleotides (e.g., oligoacylcholine ...

[0153] The ligand can be a protein (e.g., a glycoprotein), or a peptide (e.g., a molecule with a specific affinity for a co-ligand), or an antibody (e.g., an antibody that binds to a specific cell type (e.g., a hepatocyte). The ligand can also include hormones and hormone receptors. It can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose and multivalent fucose. The ligand can be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[0154] The ligand can be a substance (e.g., a drug) that can increase the uptake of the RNAi agent into the cell, for example, by disrupting the cell's cytoskeleton (e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments). The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0155] In another aspect, the ligand is a moiety that is taken up by a target molecule (e.g., a hepatocyte), for example, a vitamin. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, for example, folic acid, B12, riboflavin, biotin, pyridoxal, or other microorganisms or nutrients that are taken up by target cells (e.g., hepatocytes). HSA and low-density lipoprotein (LDL) are also included.

[0156] In some embodiments, the ligand attached to the RNAi agent described herein acts as a pharmacokinetic (PK) regulator. As used herein, "PK regulator" refers to a pharmacokinetic regulator. PK regulators include lipophilic substances, bile acid, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK regulators include, but are not limited to, cholesterol, fatty acids, bile acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides comprising multiple thiophosphate connections are also known to bind to serum proteins, so short oligonucleotides (e.g., oligonucleotides comprising multiple thiophosphate connections in the backbone) are also suitable for use in the technology described herein as ligands (e.g., as PK regulators). In addition, aptamers in conjunction with serum components (e.g., serum proteins) are also suitable for use as PK regulators in the embodiments described herein.

[0157] (i) lipid conjugates. In some embodiments, the ligand or conjugate is a lipid or a lipid-based molecule. Lipid or lipid-based ligands can (a) increase resistance to conjugate degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) can be used to adjust binding to serum proteins (e.g., HSA). Such lipids or lipid-based molecules can bind serum proteins, such as human serum albumin (HSA). The ligand in conjunction with HSA allows the conjugate to be distributed to target tissues such as non-renal target tissues of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules in conjunction with HSA can also be used as ligands. For example, naproxen or aspirin can be used.

[0158] Lipid-based ligands can be used to inhibit (e.g., control) the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA will be less likely to be targeted to the kidney and, therefore, less likely to be cleared from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

[0159] In some embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate will distribute to non-renal tissues. In certain specific embodiments, the HSA-ligand binding is irreversible.

[0160] In some other embodiments, the lipid-based ligand binds HSA weakly or not at all, such that the conjugate will distribute to the kidney.Other moieties that target renal cells can also be used instead of or in addition to the lipid-based ligand.

[0161] (ii) Cell penetrating peptides and cell penetrants. In another aspect, the ligand is a cell penetrant, such as a helical cell penetrant. In some embodiments, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennapedia peptides. If the agent is a peptide, it can be modified, including peptidyl mimetics, inverse isomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. In some embodiments, the helical agent is an alpha helical agent. In certain specific embodiments, the helical agent has a lipophilic phase and a lipophobic phase.

[0162] "Cell penetrating peptides" are capable of penetrating cells, e.g., microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. The penetrating microbial cell peptide can be, for example, an alpha-helical linear peptide (e.g., LL-37 or CeropinPI), a disulfide bond-containing peptide (e.g., alpha-defensin, beta-defensin, or cathelicidin), or a peptide containing only one or two main amino acids (e.g., PR-39 or indolicidin).

[0163] The ligand can be a peptide or peptide mimetic. Peptide mimics (also referred to herein as oligopeptide mimics) are molecules that can fold into a limited three-dimensional structure similar to a natural peptide. The attachment of peptides and peptide mimics to RNAi agents can affect the pharmacokinetic distribution of RNAi, such as by enhancing cellular recognition and absorption. The peptide or peptide mimetic portion can be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0164] The peptide or peptide mimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., primarily composed of Tyr, Trp, or Phe). The peptide portion can be a dendritic peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide portion can be a "delivery" peptide that can carry large polar molecules containing peptides, oligonucleotides, and proteins across the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWK (SEQ ID NO: 12)) have been found to function as delivery peptides. The peptide or peptide mimetic can be encoded by a random DNA sequence, such as a peptide identified from a phage display library or one-bead-one-compound (OBOC) (Lam et al., Nature 354:82-84 (1991)).

[0165] The cell penetrating peptide can also include a nuclear localization signal (NLS). For example, the cell penetrating peptide can be a biphanic peptide, such as MPG, which is a fusion protein domain derived from HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31: 2717-24 (2003)).

[0166] (iii) Carbohydrate conjugates. In some embodiments, the RNAi agent oligonucleotides described herein further include a carbohydrate conjugate. Carbohydrate conjugates can be advantageous for in vivo nucleic acid delivery and compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is itself composed of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic), wherein oxygen, nitrogen, or sulfur atoms are bonded to each carbon atom; or a compound having as a part thereof a carbohydrate that is composed of one or more monosaccharide units each having at least 6 carbon atoms (which may be linear, branched, or cyclic), wherein oxygen, nitrogen, or sulfur atoms are bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing from about 4-9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of C5 and above (in some embodiments, C5-C8); and disaccharides and trisaccharides include sugars having two or three monosaccharide units (in some embodiments, C5-C8).

[0167] In some embodiments, the carbohydrate conjugate is selected from the group consisting of:

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

[0175]

[0176] (Formula XXII), wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0177] In some embodiments, the carbohydrate conjugate further comprises another ligand, such as, but not limited to, a PK modulator, an endosomolytic ligand, or a cell penetrating peptide.

[0178] (iv) Linkers. In some embodiments, the conjugates described herein can be attached to RNAi agent oligonucleotides with various linkers, which can be cleavable or non-cleavable.

[0179] The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound. A linker typically comprises a direct bond or an atom (such as oxygen or sulfur), a unit (such as NR8, C(O), C(O)NH, SO, SO2, SO2NH) or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroaryl wherein one or more methylene radicals may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle; and wherein R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In certain embodiments, the linker is between 1-24 atoms, between 4-24 atoms, between 6-18 atoms, between 8-18 atoms, or between 8-16 atoms.

[0180] A cleavable linking group is a linking group that is sufficiently stable outside the cell, but is cleaved upon entry into the target cell to release the two parts of the linker held together. In certain embodiments, the cleavable linking group is cleaved at least 10 times or at least 100 times faster in the target cell or under a first reference condition (which can, for example, be selected to simulate or represent intracellular conditions) than in the blood of the subject or under a second reference condition (which can, for example, be selected to simulate or represent conditions found in blood or serum).

[0181] Cleavable linking groups are sensitive to cleavage agents, for example, pH, redox potential, or the presence of degradation molecules. Typically, cleavage agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such cleavage agents include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidizing or reductases or reducing agents, such as thiols present in cells, which can degrade redox-cleavable linking groups by reduction; endosomes or agents that can produce an acidic environment, for example, those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate-specific), and phosphatases. Cleavable linking groups, such as disulfide bonds, may be sensitive to pH. The pH of human serum is 7.4, while the average pH inside cells is slightly lower, at approximately 7.1-7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, at approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a specific pH, thereby releasing the cationic lipid from the ligand inside the cell, or into a desired cellular compartment.

[0182] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group introduced into the linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker comprising an ester group. Hepatocytes are rich in esterases, so the linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung cells, renal cortical cells, and testicular cells.

[0183] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0184] In general, the suitability of a candidate's cleavable linking group can be assessed by detecting the ability of a degradation agent (or condition) to cut the candidate linking group. It may also be necessary to detect the ability of a candidate's cleavable linking group to resist cutting in blood or when in contact with other non-target tissues. Therefore, the relative sensitivity to cutting can be determined between the first and second conditions, wherein the first is selected to indicate the cutting in target cells, and the second is selected to indicate the cutting in other tissues or biofluids (e.g., blood or serum). Assessment can be carried out in a cell-free system, cell, cell culture, organ or tissue culture or whole animal. Preliminary assessment is carried out under cell-free or culture conditions and may be useful by confirming the further assessment of the whole animal. In some embodiments, compared with blood or serum (or under the in vitro conditions selected to simulate extracellular conditions), the cutting of useful candidate compounds in cells (or under the in vitro conditions selected to simulate intracellular conditions) is at least 2 times, at least 4 times, at least 10 times or at least 100 times faster.

[0185] A class of cleavable linking groups is a redox cleavable linking group that is cleaved when reduced or oxidized. An example of a linking group that can be reduced is a disulfide bond linking group (-SS-). In order to determine whether a candidate's cleavable linking group is a suitable "reducible cleavage linking group", or for example whether it is suitable for use with a specific RNAi portion and a specific targeting agent, reference can be made to the methods described herein. For example, candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents well known in the art, which simulate the cleavage rate observed in cells (e.g., target cells). Candidates can also be evaluated under conditions that simulate blood or serum conditions. In some embodiments, candidate compounds cut up to 10% in blood. In certain embodiments, useful candidate compounds are degraded at least 2 times, at least 4 times, at least 10 times, or at least 100 times faster in cells (or under conditions selected to simulate intracellular conditions) than blood (or under conditions selected to simulate extracellular conditions). The cleavage rate of candidate compounds can be determined using standard enzyme kinetic analysis under conditions that simulate intracellular mediators, and compared with conditions that simulate extracellular mediators.

[0186] Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups intracellularly are enzymes, such as intracellular phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In certain embodiments, the phosphate-based linking group is selected from the group consisting of: -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -O-P(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In a specific embodiment, the phosphate linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0187] Acid cleavable linking group is a linking group that is cut under acidic conditions. In some embodiments, acid cleavable linking group is cut in an acidic environment of about pH 6.5 or lower (for example, about 6.0, 5.5, 5.0 or lower), or is cut by a reagent (such as an enzyme) that can be used as a general acid. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cutting environment for the linking group that can be acid-cleaved. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and amino acid esters. Acid cleavable groups have the general formula -C=N-, C (O) O, or -OC (O). In some embodiments, the carbon attached to the oxygen (alkoxy) of the ester is an aryl, substituted alkyl, or tertiary alkyl, such as dimethylpentyl or tert-butyl. These candidates can be evaluated using methods similar to those described above.

[0188] Ester-based cleavable linking groups are cleaved by enzymes in the cell, such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0189] The cleavable linking group based on peptide is cut by enzyme (such as peptidase and protease in cell). The cleavable linking group based on peptide is formed between amino acids to produce oligopeptides (for example, dipeptides, tripeptides, etc.) and polypeptide peptide bonds. The cleavable group based on peptide does not include amide groups (-C (O) NH-). The amide group can be formed between any alkylene, alkenylene and alkynylene groups. The peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The cleavable group based on peptide is usually limited to the peptide bond (i.e., amide bond) formed between amino acids to produce peptides and proteins, and does not include the entire amide functional group. The cleavable linking group based on peptide has the general formula -NHCHRAC (O) NHCHRBC (O) -, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0190] Representative carbohydrate conjugates with linkers include, but are not limited to,

[0191]

[0192]

[0193] wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0194] In certain embodiments of the compositions and methods, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker. For example, in some embodiments, siRNA is conjugated to a GalNAc ligand as shown in the following structure:

[0195]

[0196] Wherein X is O or S.

[0197] In some embodiments, the sense strand of the siRNA is conjugated to a ligand attached at the 3' end of the sense strand via a linker as shown in the following structure:

[0198]

[0199] Wherein X is O or S.

[0200] In some embodiments, the combination therapy comprises an siRNA conjugated to a bivalent or trivalent branched linker selected from the group consisting of structures represented by any one of Formulae (XXXI)-(XXXIV):

[0201]

[0202] in:

[0203] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0-20 at each occurrence, and wherein the repeating units may be the same or different;

[0204] P 2A 、P 2B 、P 3A 、P 3B 、P 4A 、P 4B 、P 5A 、P 5B 、P 5C 、T 2A 、T 2B 、T 3A 、T 3B 、T 4A 、T 4B 、T 4A 、T 5B and T 5C Each occurrence is independently absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CHO;

[0205] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B and Q 5C Each occurrence is independently absent, alkylene, or substituted alkylene, wherein one or more methylene groups may be interrupted or terminated by one or more of: O, S, S(O), SO2, N(R N ), C(R')=C(R"), C≡C or C(O);

[0206] R 2A 、R 2B 、R 3A 、R 3B 、R 4A 、R 4B 、R 5A 、R 5B and R 5CEach occurrence is independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-、CO、CH=NO、 or a heterocyclic group;

[0207] L 2A 、L 2B 、L 3A 、L 3B 、L 4A 、L 4B 、L 5A 、L 5B and L 5C represents a ligand; that is, each occurrence is independently a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a Is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly suitable for use with RNAi agents to inhibit the expression of target genes, such as those of formula (XXXIV):

[0208]

[0209] Among them L 5A 、L 5B and L 5C Represents a monosaccharide, such as a GalNAc derivative.

[0210] Examples of suitable divalent or trivalent branched linker groups conjugated to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas I, VI, X, IX, and XII.

[0211] Representative U.S. patents that teach the preparation of RNA conjugates include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,60 3; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5, 112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567, each of which is incorporated herein by reference for its teachings relating to such methods of preparation.

[0212] In certain instances, the RNA of an RNAi agent can be modified with a non-ligand group. A variety of non-ligand molecules have been conjugated to RNAi agents to enhance the activity, cellular distribution, or cellular uptake of the RNAi agent, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm. 365(1):54-61 (2007); Letsinger et al., Proc. Natl. Acad. Sci. USA 86:6553 (1989)), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 4:1053 (1994)), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci. 660:306 (1992); Manoharan et al., Bioorg. Med. Chem. Let. 3:2765 (1993)), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 20:533 (1992)), an aliphatic chain, for example, dodecandiol or an undecyl residue (Saison-Behmoaras et al., EMBO J. 10:111 (1991); Kabanov et al., FEBS Lett. 259:327 (1990); Svinarchuk et al., Biochimie 75:49 (1993)), a phospholipid, for example, di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 36:3651 (1995); Shea et al., Nucl. Acids Res. 18:3777 (1990)), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides 14:969 (1995)) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett. 36:3651 (1195)), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1264:229 (1995)) or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 277:923 (1996)).

[0213] A typical conjugation protocol involves the synthesis of an RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate conjugating agent or activator. The conjugation reaction can be performed while the RNA is still bound to a solid support or in solution after RNA cleavage. Purification of the RNA conjugate by HPLC generally provides a pure conjugate.

[0214] d. Delivery of RNAi Agents

[0215] "Introduced into a cell," when referring to an RNAi agent, means facilitating or effectively uptake or absorption into the cell, as understood by those skilled in the art.

[0216] The absorption or uptake of RNAi agents can be by independent diffusion (unaided diffusive) or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to in vitro cells; RNAi agents can also be "introduced into cells", wherein the cells are part of a living organism. In this case, introducing cells will include delivery to the organism. For example, for in vivo delivery, RNAi agents can be injected into tissue sites or systemically administered. In vivo delivery can also be carried out by beta-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Patent Publication No. 2005 / 0281781, which are incorporated herein by reference for teachings related to such delivery systems. In vitro introduction into cells includes methods well known in the art, such as electroporation and lipofection. Other methods are described below or are well known in the art.

[0217] Delivery of RNAi agents to subjects in need can be achieved in a variety of different ways. In vivo delivery can be performed by directly administering a composition comprising an RNAi agent (e.g., siRNA) to the subject. Alternatively, delivery can be performed indirectly by administering one or more vectors encoding and directing the expression of the RNAi agent. These alternatives will be discussed further below.

[0218] In general, any method of delivering nucleic acid molecules can be applied to RNAi agents (see, e.g., Akhtar S. and Julian RL., Trends Cell. Biol. 2(5):139-44 (1992) and WO94 / 02595, which are incorporated herein by reference for teachings relating to such delivery methods). Three factors are particularly important for the successful delivery of RNAi agents in vivo: (a) biological stability of the delivered molecule, (2) prevention of nonspecific effects, and (3) accumulation of the delivered molecule in the target tissue. Nonspecific effects of RNAi agents can be minimized by local administration, for example, by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or by local administration of the formulation. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that might otherwise be harmed by the agent or that might degrade the agent, and allows for administration of a lower total dose of the RNAi agent. Several studies have demonstrated successful knockout of gene products when RNAi agents are administered locally. For example, intraocular delivery of VEGF siRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ et al., Retina 24: 132-38 (2004)) and subretinal injection in mice (Reich, SJ et al., Mol. Vis. 9: 210-16 (2003)) has been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of siRNA in mice reduced tumor volume (Pille, J. et al., Mol. Ther. 11: 267-74 (2005)) and the survival of tumor-bearing mice (Kim, WJ et al., Mol. Ther. 14: 343-50 (2006); Li, S. et al., Mol. Ther. 15: 515-23 (2007)).RNA interference has also been shown to be successfully delivered locally to the CNS by direct injection (Dorn, G. et al., Nucleic Acids 32:e49 (2004); Tan, PH et al., Gene Ther. 12:59-66 (2005); Makimura, H. et al., BMC Neurosci. 3:18 (2002); Shishkina, GT et al., Neuroscience 129:521-28 (2004); Thakker, ER et al., Proc. Natl. Acad. Sci. USA 101:17270-75 (2004); Akaneya, Y. et al., J. Neurophysiol. 93:594-602 (2005)), and successfully delivered to the lungs by intranasal administration (Howard, KA et al., Mol. Ther. 14:476-84 (2006); Zhang, X. et al., J. Biol. Chem. 279:10677-84 (2004); Bitko, V. et al., Nat. Med. 11:50-55 (2005)). For systemic administration of RNAi agents to treat disease, RNA can be modified or alternatively delivered using a drug delivery system; both methods work to prevent rapid degradation of siRNA by endonucleases and exonucleases in vivo. The modification of RNA or drug carrier can also allow RNAi pharmaceutical composition targeting target tissue and avoid unwanted off-target effect. RNAi medicament can be modified by chemical conjugation to lipophilic groups (such as cholesterol), to enhance cellular uptake and prevent degradation. For example, the RNAi medicament systemic injection of ApoB for being conjugated with lipophilic cholesterol moiety into mice leads to the knockout (Soutschek, J. etc., Nature 432:173-78 (2004)) of apoB mRNA in liver and jejunum. In some other embodiments, RNAi medicament can be delivered using delivery system, such as nanoparticles, dendrimers, polymers, liposomes or cationic delivery systems. Positively charged cationic delivery systems usually promote the combination of RNAi medicament (negatively charged) and enhance the interaction at negatively charged cell membrane to allow cells to effectively take up RNAi medicament. Cationic lipids, dendrimers, or polymers can bind to RNAi or be induced to form vesicles or micelles that encapsulate RNAi agents (see, e.g., Kim, S, H. et al., Journal of Controlled Release 129(2):107-16 (2008)). When administered systemically, the formation of vesicles or micelles further prevents degradation of the RNAi agent.Methods of preparing and administering cationic-RNAi agent complexes are within the capabilities of those skilled in the art (see, e.g., Sorensen, DR, et al., J. Mol. Biol 327:761-66 (2003); Verma, UN, et al., Clin. Cancer Res. 9:1291-1300 (2003); Arnold, AS, et al., J. Hypertens. 25:197-205 (2007); these methods are incorporated herein by reference). Some non-limiting examples of drug delivery systems for systemic delivery of RNAi agents include DOTAP (Sorensen, DR, et al., (2003), supra; Verma, UN, et al., (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS, et al., Nature 441: 111-14 (2006)), cardiolipin (Chien, PY, et al., Cancer Gene Ther. 12: 321-28 (2005); Pal, A, et al., Int J Adv Manuf Technol Eng, 2, 43: 111-112 (2006)), and oligofectamine. J. Oncol. 26: 1087-91 (2005)), polyethyleneimine (Bonnet, M.E. et al., Pharm. Res. 25(12): 2972-82; Aigner, A., J. Biomed. Biotechnol. 2006(4): 71659 (2006)), Arg-Gly-Asp (RGD) peptide (Liu, S., Mol. Pharm. 3: 472-487 (2006)) and polyamidoamine (Tomalia, D.A. et al., Biochem. Soc. Trans. 35: 61-7 (2007); Yoo, H. et al., Pharm. Res. 16: 1799-1804 (1999)).

[0219] As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. SNALP represents a lipid vesicle that coats a shrunken aqueous interior, which contains a nucleic acid such as an RNAi agent or a plasmid that transcribes the RNAi agent. For example, SNALP is described in U.S. Patent Application Publications US2006 / 0240093 and US2007 / 0135372 and in International Application Publication WO 2009 / 082817. These applications are incorporated herein by reference for the teachings relevant to SNALP.

[0220] In some embodiments, RNAi forms a complex with cyclodextrin for systemic administration. Methods of administering RNAi and cyclodextrin and pharmaceutical compositions can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference for teachings related to such compositions and methods. In some embodiments, the gene encoding RNAi is encoded and expressed from an expression vector. Examples of vectors and their use in delivering RNAi are described in U.S. Patent Application No. US2017 / 0349900A1, examples of which are incorporated herein by reference.

[0221] e. Pharmaceutical compositions and formulations of RNAi agents

[0222] In some embodiments, provided herein is a pharmaceutical composition comprising an RNAi agent as described herein and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition comprising an RNAi agent can be used in combination therapy to treat HBV infection in a subject or to reduce HBV viral load. Such pharmaceutical compositions are formulated based on the mode of delivery. For example, the composition can be formulated for systemic administration by parenteral delivery, for example, by intravenous (IV) delivery, or for direct delivery into the brain parenchyma, for example, by infusion into the brain, such as by continuous pump infusion.

[0223] A "pharmaceutically acceptable carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected based on the intended mode of administration so as to provide the desired volume, consistency, etc. when combined with the nucleic acid and the other ingredients of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers or excipients include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0224] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not adversely react with nucleic acids may also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like.

[0225] Preparations for topical administration of nucleic acid can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil bases. The solution can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that are applicable to non-parenteral administration and that do not have a deleterious reaction with the nucleic acid can be used.

[0226] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0227] In some embodiments, the pharmaceutical composition containing RNAi medicament as described herein is administered with a dosage sufficient to suppress HBV gene expression. Under normal circumstances, the suitable dosage range of RNAi medicament is 0.001 to 200.0 milligrams per kilogram of recipient body weight per day, more typically 1 to 50 mg per kilogram of body weight per day. For example, siRNA can be administered per single dose 0.01mg / kg, 0.05mg / kg, 0.5mg / kg, 1mg / kg, 1.5mg / kg, 2mg / kg, 3mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg or 50mg / kg. The pharmaceutical composition can be administered once a day, or the RNAi medicament can be administered with two, three or more sub-doses at appropriate intervals throughout the day, or even delivered by continuous infusion or controlled release formulation. In this case, the RNAi medicament included in each sub-dose must be correspondingly smaller to reach a total daily dose. Dosage units can also be mixed for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of RNAi over several days. Sustained-release formulations are well known in the art and are particularly suitable for delivering agents at specific locations, for example, as can be used with agents of the technology described herein. In this embodiment, the dosage unit contains a corresponding plurality of daily doses.

[0228] The effect of a single dose on HBV gene expression levels can be sustained, so that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart.

[0229] It will be understood by those skilled in the art that certain factors may affect the dosage and time required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatment, the subject's overall health and / or age, and other existing diseases. In addition, treating a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective dosage and in vivo half-life of a single RNAi agent encompassed by the technology described herein can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0230] Mouse models are useful for studying HBV infection. These models can be used to test RNAi in vivo and to determine the dose that is effective in reducing HBV gene expression.

[0231] In some embodiments, administration of the pharmaceutical compositions and formulations described herein can be topical (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including via a nebulizer); intratracheal; intranasal; epidermal and transdermal; oral; or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subcutaneous administration (e.g., via an implantable device); or intracranial administration (e.g., via intraparenchymal, intrathecal, or intraventricular administration).

[0232] In certain embodiments, the RNAi agent used in the combination therapy for treating HBV disclosed herein is delivered subcutaneously.

[0233] In some embodiments, RNAi agents can be delivered in a manner that targets a specific tissue, such as the liver (eg, hepatocytes of the liver).

[0234] Pharmaceutical compositions and preparations for topical administration may include transdermal patches, ointments, emulsions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, etc. may be necessary or desirable. Coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which RNAi characterized by the technology described herein is mixed with local delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). RNAi agents can be encapsulated in liposomes or can form complexes therewith, particularly with cationic liposomes. Alternatively, the RNAi agent can be complexed with a lipid, particularly a cationic lipid. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monooleic acid, dilaurate, 1-monocaprin, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, or C 1-20Alkyl esters (eg, isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Examples of topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference for its teachings regarding such topical formulations.

[0235] Vesicles, such as liposomes, can be used to deliver formulations of the RNAi agents disclosed herein; such formulations may have desired properties, such as specificity and duration of action. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or layers.

[0236] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with cell walls. Non-cationic liposomes, although not able to fuse effectively with cell walls, can be taken up by macrophages in vivo. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0237] In some embodiments, liposomal delivery can have the following advantageous properties: high deformability and ability to pass through fine pores in the skin; biocompatibility and biodegradability; ability to incorporate a wide variety of water-soluble and lipid-soluble drugs; the ability to protect the encapsulated drug in the internal compartment from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245 (1998)); for topical administration, reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target site, and the ability to administer a wide variety of hydrophilic and hydrophobic drugs into the skin; and the ability to deliver pharmaceutical agents including high molecular weight nucleic acids, analgesics, antibodies, and hormones to the skin.

[0238] Liposomes are divided into two major categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form a stable complex. The positively charged DNA / liposome complex binds to the negatively charged cell surface and is internalized. Due to the acidic pH inside the endosome, the liposomes rupture and release their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun. 147, 980-985 (1987)).

[0239] pH-sensitive or negatively charged liposomes will capture nucleic acids rather than complex with them. Since both DNA and lipids carry similar charges, repulsion rather than complex formation occurs. However, some DNA is encapsulated in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids to cultured cell monolayers (e.g., Zhou et al., Journal of Controlled Release 19, 269-74 (1992)).

[0240] In some embodiments, the liposome composition is formed from phosphatidylcholine (PC), for example, soybean PC and egg PC. In some embodiments, the liposome composition includes phospholipids other than naturally derived phosphatidylcholine. For example, a neutral liposome composition can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions can be formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes can be formed from dioleoylphosphatidylethanolamine (DOPE). In yet other embodiments, the liposome composition is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0241] In some embodiments, liposomal drug formulations are delivered topically to the skin.

[0242] In some embodiments, the RNAi agent for conjoint therapy described herein is fully encapsulated in lipid formulations, for example, to form SPLP, pSPLP, SNALP or other nucleic acid-lipid particles. As used herein, term " SNALP " refers to stable nucleic acid-lipid particles, including SPLP. As used herein, term " SPLP " refers to nucleic acid-lipid particles comprising the plasmid DNA encapsulated in lipid vesicles. SNALP and SPLP generally include cationic lipids, non-cationic lipids and the lipid (for example, PEG-lipid conjugates) preventing particle aggregation. SNALP and SPLP can be used for systemic application because they show prolonged circulation life after intravenous (iv) injection and accumulate in distal sites (for example, the position physically separated from the administration site). SPLP includes " pSPLP ", which includes the condensing agent-nucleic acid complex of the encapsulation as described in International Application Publication No. WO 00 / 03683. The particles of the technology described herein typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, in some embodiments, when present in nucleic acid-lipid particles, the nucleic acid resists degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and related preparation methods are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and International Application Publication No. WO 96 / 40964.

[0243] In some embodiments, the RNAi agent is delivered via liposomes or other lipid formulations in which the lipid to drug ratio (mass / mass ratio) (e.g., lipid to siRNA ratio) is in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0244] III. Anti-HBV Antibodies

[0245] The present disclosure provides anti-HBV antibodies for use in combination therapy for treating HBV.

[0246] a. Antibodies that bind to HBV proteins

[0247] In some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy binds to the antigenic loop region of HBsAg. The envelope of the hepatitis B virus contains three "HBV envelope proteins" (also known as "HBsAg", "hepatitis B virus surface antigen"): S protein (for "small", also known as S-HBsAg), M protein (for "medium", also known as M-HBsAg) and L protein (for "large", also known as L-HBsAg). S-HBsAg, M-HBsAg and L-HBsAg share the same C-terminal end (also known as "S domain", 226 amino acids), which corresponds to the S protein (S-HBsAg) and is involved in viral assembly and infectivity. S-HBsAg, M-HBsAg and L-HBsAg are synthesized, assembled and secreted as particles through the Golgi apparatus in the endoplasmic reticulum (ER). The S domain contains four predicted transmembrane (TM) domains, of which both the N-terminus and C-terminus of the S domain are exposed to the lumen. Both transmembrane domains TM1 and TM2 are required for co-translational protein integration into the ER membrane, while 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 HBsAg S domain, where the antigenic loop region comprises amino acids 101-172 of the S domain (Salisse J. and Sureau C. Journal of Virology 83:9321-8 (2009)). Important determinants of infectivity are present in the antigenic loop region of the HBV envelope protein. In particular, residues between 119 and 125 of HBsAg contain a CXXC motif, which has been shown to be the most important sequence required for HBV infectivity (Jaoude, G.A. and Sureau, C., Journal of Virology 79:10460-6 (2005)).

[0248] As used herein, the S domain of HBsAg refers to the amino acid sequence shown in SEQ ID NO: 13 (shown below) or a natural or artificial sequence variant thereof.

[0249] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTA QGTSMYPSCCCTKPSDGN CTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0250] (SEQ ID NO: 13; amino acids 101-172 are underlined)

[0251] For example, the expression "amino acids 101-172 of the S domain" refers to the amino acid residues at positions 101-172 of the polypeptide according to SEQ ID NO: 13. However, it will be understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions, for example, in HBsAg of different genotypes or different HBsAg mutants described herein) may occur naturally in the amino acid sequence of the HBsAg S domain or be artificially introduced into the amino acid sequence of the HBsAg S domain without affecting its biological properties. Therefore, the term "S domain of HBsAg" encompasses all such polypeptides, for example, including the polypeptide according to SEQ ID NO: 13 and its natural or artificial mutants. In addition, when a sequence fragment of the S domain of HBsAg is described herein (e.g., amino acids 101-172 or amino acids 120-130 of the S domain of HBsAg), it includes not only the corresponding sequence fragment of SEQ ID NO: 13, but also the corresponding sequence fragment of its natural or artificial mutants. For example, the expression "amino acid residues 101-172 from the S domain of HBsAg" includes amino acid residues 101-172 from SEQ ID NO: 13 and corresponding fragments of mutants thereof (natural or artificial mutants).

[0252] As used herein, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at the same position in the sequence when the sequences are optimally aligned, i.e., the sequences are aligned to obtain the highest percentage of identity. The M protein (M-HBsAg) corresponds to the S protein extended by a 55-amino acid N-terminal domain called "pre-S2". The L protein (L-HBsAg) corresponds to the M protein extended by a 108-amino acid N-terminal domain called "pre-S1" (genotype D). The pre-S1 and pre-S2 domains of the L protein can be present on the inner surface of the virus particle (on the cytoplasmic side of the ER), playing a key role in virus assembly, or on the outer surface (on the lumen side of the ER), where they can be used to interact with target cells and are essential for viral infectivity. In addition, the HBV surface protein (HBsAg) is not only incorporated into the virion envelope, but also spontaneously buds from the ER-Golgi intermediate compartment membrane to form empty "subviral particles" (SVPs), which are released from the cell by secretion.

[0253] Since all three HBV envelope proteins, S-HBsAg, M-HBsAg, and L-HBsAg, contain an S domain, all three HBV envelope proteins, S-HBsAg, M-HBsAg, and L-HBsAg, also contain an "antigenic loop region." Therefore, antibodies or antigen-binding fragments thereof that bind to the HBsAg antigenic loop region bind to all three HBV envelope proteins: S-HBsAg, M-HBsAg, and L-HBsAg.

[0254] Furthermore, in some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy neutralizes hepatitis B virus infection. In other words, the antibody or antigen-binding fragment thereof can reduce the viral infectivity of the hepatitis B virus.

[0255] In order to study and quantify viral infectivity (or "neutralization") in the laboratory, various standard "neutralization assays" are known to those skilled in the art. For neutralization assays, animal viruses are typically propagated in cells and / or cell lines. In the context of the present disclosure, for neutralization assays, cultured cells can be incubated with a fixed amount of HBV in the presence (or absence) of the antibody to be tested. As a readout, the level of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant can be used and / or HBcAg staining can be assessed. In one embodiment of the HBV neutralization assay, cultured cells, such as HepaRG cells, in particular differentiated HepaRG cells, are incubated with a fixed amount of HBV in the presence or absence of the antibody to be tested, for example at 37°C for 16 hours. The incubation can be carried out in a culture medium (e.g., supplemented with 4% PEG 8000). After incubation, the cells can 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 an immunofluorescence assay.

[0256] In some embodiments, antibodies and antigen-binding fragments have strong neutralizing ability. The concentration of antibodies of the present disclosure required for 50% neutralization of hepatitis B virus (HBV) is, for example, about 10pg / ml or less. In certain embodiments, the concentration of antibodies of the present disclosure required for 50% neutralization of HBV is about 5pg / ml, about 1pg / ml or about 750ng / ml. In certain embodiments, the concentration of antibodies of the present disclosure required for 50% neutralization of HBV is 500ng / ml or less, for example, 450, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60 or about 50ng / ml or less. This means that only a low concentration of antibody is required for 50% neutralization of HBV. Standard assays known to those skilled in the art can be used to measure specificity and efficacy.

[0257] In some embodiments, anti-HBV antibodies can be used as part of a combination therapy to prevent and / or treat hepatitis B.

[0258] In some embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure promote the clearance of HBsAg and HBV. In particular, the antibodies or antigen-binding fragments thereof according to the present disclosure can promote the clearance of both HBV and subviral particles (SVP) of hepatitis B virus. The clearance of HBsAg or subviral particles can be assessed by measuring, for example, the HBsAg level in a blood sample from a hepatitis B patient. Similarly, the clearance rate of HBV can be assessed by measuring, for example, the HBV level in a blood sample from a hepatitis B patient.

[0259] In the serum of patients infected with HBV, in addition to infectious particles (HBV), there is usually an excess (usually 1,000 to 100,000 times) of empty subviral particles (SVPs) consisting solely of HBV envelope protein (HBsAg), which are relatively small spheres and filaments of variable length. Subviral particles have been shown to strongly enhance intracellular viral replication and HBV gene expression (Bruns, M. et al., J Virol 72(2):1462-8(1998)). This is also important in terms of the infectivity of HBV-containing serum, because infectivity depends not only on the amount of virus but also on the amount of SVPs (Bruns, M. et al., J Virol 72(2):1462-8(1998)). In addition, excess subviral particles can act as bait by absorbing neutralizing antibodies, thereby delaying the clearance of infection. Generally, achieving the disappearance of hepatitis B surface antigen (HBsAg) is therefore considered the ideal treatment endpoint and the closest outcome to curing chronic hepatitis B (CHB). Thus, in some embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure, which promote the clearance of HBsAg, particularly the clearance of hepatitis B virus and HBV subviral particles, can improve the treatment of hepatitis B, particularly in the case of chronic hepatitis B. Thus, the antibodies or antigen-binding fragments thereof according to the present disclosure can effectively neutralize HBV because less antibody is absorbed by the SVP used as bait. Furthermore, in certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure promote the clearance of hepatitis B virus subviral particles and reduce the infectivity of HBV in serum.

[0260] According to the genome sequence, HBV can be divided into multiple genotypes. To date, eight well-known genotypes (AH) of the HBV genome have been defined. In addition, two new genotypes, I and J, have been identified (Sunbul, M., World J Gastroenterol 20 (18): 5427-34 (2014)). It is known that genotype affects the progression of the disease, and differences in response to antiviral treatment between genotypes have been determined. For example, genotype A has a tendency to become chronic, while genotype C often encounters viral mutations. The chronicity and mutation frequency of genotype D are both common. Moreover, there are differences in the distribution of HBV genotypes worldwide (Sunbul, M., 2014, supra). In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to at least 6, at least 8, or all 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind 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 genotypes for HBsAg include the following: GenBank Accession No. J02203 (HBV-D, ayw3), GenBank Accession No. FJ899792.1 (HBV-D, adw2), GenBank Accession No. AM282986 (HBV-A), GenBank Accession No. D23678 (HBV-B1 Japan), GenBank Accession No. AB117758 (HBV-C1 Cambodia), GenBank Accession No. AB205192 (HBV-E Ghana), GenBank Accession No. X69798 (HBV-F4 Brazil), GenBank Accession No. AF160501 (HBV-G USA), GenBank Accession No. AY090454 (HBV-H Nicaragua), GenBank Accession No. AF241409 (HBV-I Vietnam), and GenBank Accession No. AB486012 (HBV-J Borneo). The amino acid sequences of the antigenic loop regions of the HBsAg S domains of different genotypes are shown in Table 2 (SEQ ID NOs: 14-42).

[0261] Table 2: Antigenic loop sequences from different HBV genotypes

[0262]

[0263]

[0264] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the HBsAg mutants having mutations in the following antigenic loop regions: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121 S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141 E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAgG145R and HBsAgN146A. These mutants are naturally occurring mutants based on the S domain of HBsAg genotype D (SEQ ID NO: 43, Genbank accession number FJ899792), (wherein the mutated amino acid residue(s) are indicated in the name).

[0265] MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSD GNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI (SEQ ID NO: 43) (the antigenic loop region, i.e., amino acids 101-172, is underlined)

[0266] In a specific embodiment, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to at least 12, at least 15, or all 18 of the infectious HBsAg mutants having mutations in the following antigenic loop regions: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121 S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141 E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A.

[0267] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to an epitope comprising at least one, at least two, at least three, or at least four amino acids of an antigenic loop region of HBsAg, wherein the at least two, at least three, or at least four amino acids are 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. Notably, the amino acid positions (e.g., 115-133, 120-133, 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.

[0268] In a specific embodiment, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to an epitope in the antigenic loop region of HBsAg, whereby the epitope is formed by one or more amino acids located at a position selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg.

[0269] The term "formed by..." as used herein in the context of epitopes means that the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind can be linear (continuous) or conformational (discontinuous). A linear or continuous epitope is an epitope that an antibody recognizes by its linear amino acid sequence or primary structure. In contrast, a conformational epitope has a specific three-dimensional shape and protein structure. Therefore, 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 selected from the HBsAg S domain, the amino acids contained in the epitope can be located at adjacent positions of the primary structure (i.e., continuous amino acids in the amino acid sequence). In contrast, in the case of a conformational epitope (3D structure), the amino acid sequence typically forms a 3D structure as an epitope, and therefore, the amino acids forming the epitope (or the amino acids "comprising" the epitope) may or may not be located at adjacent positions of the primary structure (i.e., may or may not be continuous amino acids in the amino acid sequence). In certain embodiments, the epitope bound by the antibodies or antigen-binding fragments thereof of the present disclosure is formed solely by (one or more) amino acids selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. In a specific embodiment, (additional) amino acids outside of positions 115-133, positions 120-133, or positions 120-130 are not required to form the epitope bound by the antibodies or antigen-binding fragments thereof of the present disclosure.

[0270] In certain embodiments, the epitope in the antigenic loop of HBsAg to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed by two or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. In certain embodiments, the epitope in the antigenic loop of HBsAg to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed by three or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. In some embodiments, the epitope in the antigenic loop of HBsAg to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed by four or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. Thus, the antibodies or antigen-binding fragments thereof according to the present disclosure may bind to at least one, at least two, at least three amino acids, or at least four amino acids in the antigenic loop region of HBsAg 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 a specific embodiment, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to an epitope comprising at least two, at least three, or at least four amino acids in the antigenic loop region of HBsAg, wherein the at least two, at least three, or 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 the at least two, at least three, or at least four amino acids are located in adjacent positions (i.e., in a continuous sequence in the amino acid sequence / primary structure).

[0271] In certain embodiments, the epitope to which the antibodies or antigen-binding fragments thereof according to the present disclosure bind is a conformational epitope. Thus, the antibodies or antigen-binding fragments thereof according to the present disclosure may bind to an epitope comprising at least two, at least three, or at least four amino acids in the antigenic loop region of HBsAg, wherein the at least two, at least three, or 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, at least three, or at least four amino acids are not located in adjacent positions (of the primary structure).

[0272] In certain specific embodiments, the antibodies of the present disclosure are bispecific antibodies having a first specificity for HBsAg and a second specificity for stimulating immune effector cells (e.g., by targeting a T cell surface protein, such as, for example, the extracellular portion of the CD3 protein). The second specificity can elicit, for example, a cytotoxic effect or a vaccination effect.

[0273] b. Fc part

[0274] In some embodiments, the binding moiety (e.g., an antibody or antigen-binding fragment thereof) comprises an Fc portion. In certain embodiments, the Fc portion can be derived from a human source, e.g., from human IgG1, IgG2, IgG3, and / or IgG4. In a specific embodiment, the antibody or antigen-binding fragment can comprise an Fc portion derived from human IgG1.

[0275] As used herein, the term "Fc portion" refers to a sequence comprising or derived from a part of an immunoglobulin heavy chain located in the hinge region upstream of the papain cleavage site (e.g., residue 216 in native IgG, the first residue of the heavy chain constant region is 114) and ends at the C-terminus of the immunoglobulin heavy chain. Therefore, the Fc portion can be a complete Fc portion or a portion thereof (e.g., domain). In certain embodiments, a complete Fc portion comprises a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid position 216-446). Additional lysine residues (K) are sometimes present in the extreme C-terminus of the Fc portion, but are typically cut from mature antibodies. The amino acid position in the Fc portion is numbered according to the EU numbering system of Kabat (see, e.g., Kabat et al., " Sequences of Proteins of Immunological Interest ", U.S. Pat. Health and Human Services, 1983 and 1987). The amino acid position of the Fc portion can also be numbered according to the IMGT numbering system (including unique numbering and exon numbering for the C domain) and the Kabat numbering system.

[0276] In some embodiments, the Fc portion comprises at least one of the following: 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 other embodiments, the Fc portion is a complete Fc portion. The amino acid sequence of an exemplary Fc portion of a human IgG1 isotype is provided in SEQ ID NO: 96. The Fc portion may also comprise one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc portion. For example, at least one of the hinge domain, the CH2 domain, or the CH3 domain, or a portion thereof, may be deleted. For example, the Fc portion can 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.

[0277] The Fc portion of the present disclosure can be modified so that its amino acid sequence differs 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 fixation. Portions of the naturally occurring Fc portion associated with these functions have been described in the art.

[0278] For example, to activate the complement cascade, when an immunoglobulin molecule is attached to an antigenic target, the C1q protein complex can bind to at least two molecules of IgG1 or one molecule of IgM (Ward, ES and Ghetie, V., Ther. Immunol. 277-94 (1995)). The heavy chain region comprising amino acid residues 318 to 337 is involved in complement fixation (Burton, DR, Mol. Immunol. 22: 161-206 (1985)). Duncan, AR and Winter, G. (Nature 332: 738-40 (1988)) used site-directed mutagenesis to report that Glu318, Lys320, and Lys322 form a binding site for C1q. The role of Glu318, Lys320, and Lys322 residues in C1q binding was confirmed by the ability of short synthetic peptides containing these residues to inhibit complement-mediated lysis.

[0279] For example, FcR binding can be mediated by 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 have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes, as well as the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated with the corresponding antibody by antibody-dependent cell-mediated cytotoxicity (ADCC; Vande Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49:511-24 (1991)). FcRs are defined by their specificity for immunoglobulin classes; the Fc receptors for IgG antibodies are referred to as FcγRs, for IgE as FcεRs, for IgA as FcαRs, and so on, and the neonatal Fc receptor is referred to as FcRn. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9:457-92 (1991); Capel, PJ et al., Immunomethods 4:25-34 (1994); deHaas, M. et al., J Lab. Clin. Med. 126:330-41 (1995); and Gessner, JE et al., Ann. Hematol. 76:231-48 (1998).

[0280] Cross-linking of the Fc domain of a natural IgG antibody (FcγR) to a receptor triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and the release of inflammatory mediators, as well as regulation of immune complex clearance and antibody production. It is contemplated herein to provide an Fc portion of a receptor (e.g., FcγR) cross-linking. In humans, three types of FcγRs have been characterized: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) FcγRII (CD32), which binds complexed IgG with low to intermediate affinity, is widely expressed, especially on leukocytes, and is considered a core player in antibody-mediated immunity. It can be divided into FcγRIIA, FcγRIIB, and FcγRIIC, which perform different functions in the immune system but have similar low affinity for binding to IgG-Fc, and the extracellular domains of these receptors are highly homologous; and (iii) FcγRIII (CD16), which binds IgG with low to intermediate affinity and exists in two forms: FcγRIIIA, which has been found on NK cells, macrophages, eosinophils, and some monocytes and T cells and is believed to mediate ADCC; and FcγRIIIB, which is highly expressed on neutrophils.

[0281] FcγRIIA is present on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to activate the killing process. FcγRIIB appears to play a role in the suppression process and is present on B cells, macrophages, mast cells, and eosinophils. It has been shown that 75% of FcγRIIB is present in the liver (Ganesan, LP et al., Journal of Immunology 189:4981–8 (2012)). FcγRIIB is abundantly expressed in the liver sinusoidal endothelium (called LSEC) and liver Kupffer cells, which are the main sites of clearance of small immune complexes (Ganesan, LP et al., 2012, supra).

[0282] In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein comprise an Fc portion, particularly an Fc region, for binding to FcγRIIb, such as an IgG-type antibody. In addition, the Fc portion can be engineered to enhance binding to FcγRIIB by introducing mutations S267E and L328F, as described by Chu, SY et al. (Molecular Immunology 45:3926–33 (2008)). Thus, clearance of immune complexes can be enhanced (Chu, S. et al., Am J Respir Crit, American Thoracic Society International Conference Abstracts (2014)). 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 by Chu, SY et al. (2008, supra).

[0283] On B cells, FcγRIIB appears to inhibit further immunoglobulin production and isotype switching, such as to the IgE class. On macrophages, FcγRIIB is thought to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the b form may help inhibit activation of these cells through binding of IgE to its independent receptor.

[0284] For FcγRI binding, modifications of at least one of E233-G236, P238, D265, N297, A327, and P329 in native IgG reduce binding to FcγRI. Substitution of IgG2 residues at positions 233-236 with the corresponding IgG1 and IgG4 positions reduces IgG1 and IgG4 binding to FcγRI by 10 3fold, and abolished the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29: 2613-2624 (1999)).

[0285] For FcyRII binding, IgG mutations, for example, to at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 were found to reduce binding to FcyRIIA.

[0286] For FcγRIII binding, it was found that, for example, mutations to at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 reduced binding to FcγRIIIA.

[0287] Mapping of the Fc receptor binding site on human IgG1, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL et al. (J. Biol. Chem. 276: 6591-6604 (2001)).

[0288] Regarding binding to FcγRII, two regions of native IgG Fc appear to be involved in the interaction between FcγRII and IgG, namely (i) the lower hinge site of IgG Fc, specifically amino acid residues L, L, G, and G (234-237, EU numbering), and (ii) adjacent regions of the CH2 domain of IgG Fc, specifically loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example, in the region of P331 (Wines, BD et al., J. Immunol. 164: 5313-8 (2000)). In addition, 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 at the CH2-CH3 interface (Wines, BD et al., 2000, supra).

[0289] Mutations that increase the binding affinity of the Fc portion of the present disclosure to (i.e., one or more) Fcγ receptors (e.g., compared to a reference Fc portion or antibody that does not comprise the mutation) are also contemplated. See, e.g., Delillo and Ravetch, Cell 161(5):1035-45 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016), which Fc mutations and techniques are incorporated herein by reference. In any of the embodiments disclosed herein, the binding protein may comprise an Fc portion comprising a mutation selected from the group consisting of: G236A; S239D; A330L; and I332E; or a combination thereof, e.g., S239D / I332E; S239D / A330L / I332E; G236A / S239D / I332E; G236A / A330L / I332E; and G236A / S239D / A330L / I332E.

[0290] In certain embodiments, the Fc portion may comprise or consist of at least a portion of an Fc portion that participates in binding to 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., 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 and half-life-extending mutation comprising any one or more of the following: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; and E380A (EU encoding). In certain embodiments, the half-life-extending mutation comprises M428L / N434S. 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.

[0291] In a specific embodiment, the binding protein comprises an Fc portion comprising the substitution mutations: M428L / N434S and G236A / A330L / I332E. In certain embodiments, the antibody or antigen-binding fragment comprises an Fc portion comprising the substitution mutations: M428L / N434S and G236A / S239D / A330L / I332E.

[0292] In a specific embodiment, the binding protein comprises an Fc portion comprising the substitution mutations: G236A / A330L / I332E. In certain embodiments, the antibody or antigen-binding fragment comprises an Fc portion comprising the substitution mutations: G236A / S239D / A330L / I332E.

[0293] Alternatively or additionally, the Fc portion of the binding protein of the present disclosure may comprise at least a portion of a protein A binding region known in the art; and / or the Fc portion of the antibody of the present disclosure may comprise at least a portion of an Fc molecule known in the art to be required for protein G binding. In some embodiments, the retained function includes clearance of HBsAg and HBVg. Thus, in certain embodiments, the Fc portion comprises at least a portion of a protein G binding region known in the art. As described above, the Fc portion may comprise at least (i) the lower hinge site of a native IgG Fc, specifically amino acid residues L, L, G, and G (234–237, EU numbering), and (ii) an adjacent region of the CH2 domain of a native IgG Fc, specifically the loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example, in the region of P331, such as a region of at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upper CH2 domain of a native IgG Fc surrounding P331 (e.g., between amino acids 320 and 340 (EU numbering) of a native IgG Fc).

[0294] In some embodiments, the binding proteins according to the present disclosure include an Fc region. As used herein, the term "Fc region" refers to an immunoglobulin portion formed by two or more Fc portions of an antibody heavy chain. For example, the Fc region can be a monomer or a "single-chain" Fc region (i.e., an scFc region). A single-chain Fc region is composed of the Fc portion connected within a single polypeptide chain (e.g., encoded in a single continuous nucleic acid sequence). Exemplary scFc regions are disclosed in WO 2008 / 143954 A2 and are incorporated herein by reference. The Fc region can be or include a dimerized Fc region. A "dimerized Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two independent immunoglobulin heavy chains. The dimerizing Fc region can be a homodimer of two identical Fc moieties (e.g., the Fc regions of naturally occurring immunoglobulins) or a heterodimer of two different Fc moieties (e.g., one Fc monomer of the dimerizing 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).

[0295] The currently disclosed Fc portions 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 from any combination thereof. In certain embodiments, the Fc portions of the Fc region 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 Fc portions of a dimer or single-chain Fc region may be from different immunoglobulin classes and / or subclasses. In certain embodiments, the dimer Fc region may comprise sequences from two or more different isotypes or subclasses; for example, a SEED body ("chain exchange engineered domain") (see Davis et al., Protein Eng. Des. Sel. 23(4): 195 (2010)).

[0296] In addition or alternatively, the chimeric Fc region can comprise one or more chimeric Fc portions. For example, a chimeric Fc region or portion can comprise one or more portions of an immunoglobulin derived from a first subclass (e.g., IgG1, IgG2, or IgG3 subclass), while the remainder of the Fc region or Fc portion belongs to a different subclass. For example, the Fc region or Fc portion of an Fc polypeptide can comprise a CH2 and / or CH3 domain derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG3 subclass) and a hinge region from an immunoglobulin of a second subclass (e.g., IgG3 subclass). For example, the Fc region or Fc portion can comprise a hinge and / or CH2 domain derived from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and a CH2 domain from an immunoglobulin of a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). For example, a chimeric Fc region can comprise an Fc portion (e.g., a complete 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 Fc portion can comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or Fc portion can 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 Fc portion can comprise a portion of a CH2 domain from a specific subclass of an antibody, e.g., EU positions 292-340 of the CH2 domain. For example, the Fc region or Fc portion can comprise amino acid positions 292-340 of the CH2 derived from the IgG4 portion and the remainder of the CH2 derived from the IgG1 portion (or, 292-340 of CH2 can be derived from the IgG1 portion and the remainder of the CH2 derived from the IgG4 portion).

[0297] In addition, the Fc region or Fc moiety can (in addition or alternatively) for example comprise a chimeric hinge region. For example, a chimeric hinge can be, for example, partially derived from an IgG1, IgG2 or IgG4 molecule (e.g., upper and lower middle hinge sequences) and partially derived from an IgG3 molecule (e.g., middle hinge sequence). In another example, the Fc region or Fc moiety can comprise a chimeric hinge partially derived from an IgG1 molecule and partially derived from an IgG4 molecule. In another example, a chimeric hinge can comprise upper and lower hinge domains from an IgG4 molecule and middle hinge domains from an IgG1 molecule. Such a chimeric hinge can be prepared, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the middle hinge domain of the IgG4 hinge region. In some other embodiments, the chimeric hinge can comprise amino acids at EU positions 233-236 from an IgG2 antibody and / or a Ser228Pro mutation, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g., a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP (SEQ ID NO: 105)). Other chimeric hinges that can be used in the Fc portion of an antibody according to the present disclosure are described in US 2005 / 0163783A1.

[0298] In some embodiments, 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 derived 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 may be included in the subject polypeptide. Alternatively, one or more mouse amino acids may be present in the Fc portion or Fc region.

[0299] c. HBC34 and HBC24 antibodies

[0300] In certain embodiments, the anti-HBV antibody is HBC34 or an engineered variant thereof, or HBC24 or an engineered variant thereof. HBC34 and HBC24 are human antibodies against HBsAg with high neutralizing activity. HBC34 binds to the antigenic loop of HBsAg with high affinity (in the pM range), recognizes all 10 HBV genotypes and 18 mutants, and binds to the spherical SVP with low stoichiometry. The activity of HBC34, as determined by immunoassay, is 5000 IU / mg. In comparison, the activity of HBIG is ~1 IU / mg.

[0301] As referred to herein, unless otherwise indicated, the terms "HBC34 antibody" and "HBC antibody" may include wild-type HBC34 antibody or engineered variants thereof (eg, HBC34 and HBC34 variants described in Table 3).

[0302] Table 3 shows the amino acid sequences of the CDRs, heavy chain variable regions (VH), and light chain variable regions (VL) of HBC34 and its engineered variants ("HBC34v7," "HBC34v23," "HBC34v31," "HBC34v32," "HBC34v33," "HBC34v34," and "HBC34v35"), as well as "HBC24." Also shown are the full-length heavy chain (HC) and light chain (LC) amino acid sequences of exemplary antibodies of the present disclosure. One or more amino acid residues are indicated in the name).

[0303] Table 3: Sequences of HBC34 and HBC24 antibodies

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319] In certain embodiments, the antibodies of the present disclosure are HBC34, or non-natural variants of the HBC34 antibody. Examples of non-natural variants of HBC34 include, for example, "HBC34v7," "HBC34v23," "HBC34v31," "HBC34v32," "HBC34v33," "HBC34v34," and "HBC34v35."

[0320] In certain embodiments, the anti-HBV antibody comprises one or more amino acid sequences listed in Table 3. In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure comprises the CDR sequences, V H Sequence, V L The present invention also provides an embodiment of the present invention wherein the antibody or antigen-binding fragment comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the CDR, VGFR, VEGF and / or FGF sequence, HC sequence and / or LC sequence. In any of the presently disclosed embodiments, the antibody or antigen-binding fragment may comprise the CDR, VGFR, VEGF and / or FGF sequence as shown in Table 3. H 、V L , HC and / or LC sequences.

[0321] In some embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise: (i) the CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 or 46, and 47, respectively; and (ii) the CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 or 50, and 51 or 52, respectively.

[0322] Thus, in some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 44, 45, and 47, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 44, 46, and 47, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are according to SEQ ID NOs: 48, 49, and 51, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are according to SEQ ID NOs: 48, 49, and 52, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are according to SEQ ID NOs: 48, 50, and 51, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are according to SEQ ID NOs: 48, 50, and 52, respectively.

[0323] It will be understood that the antibodies or antigen-binding fragments of the present disclosure may comprise any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 44-52.

[0324] In specific embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise: CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45, and 47, respectively; and CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49, and 51, respectively. In other embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise: CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45, and 47, respectively; and CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49, and 52, respectively.

[0325] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise: (a) a light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 55-69; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53 or 54.

[0326] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise:

[0327] (a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 55-63; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:53.

[0328] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise:

[0329] (a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 55-57 or 64-69; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:54.

[0330] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise:

[0331] (i) (a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 55; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0332] (ii) (a) light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 55; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54;

[0333] (iii) (a) light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 56; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0334] (iv) (a) light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 56; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54;

[0335] (v)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 57; and (b) a heavy chain variable domain (V H), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0336] (vi)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 57; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54;

[0337] (vii) (a) light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 58; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0338] (viii) (a) light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 59; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0339] (ix) (a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 60; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0340] (x)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 61; and (b) a heavy chain variable domain (V H), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0341] (xi)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 62; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0342] (xii)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 63; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53;

[0343] (xiii) (a) light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 64; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54;

[0344] (xiv)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 65; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54;

[0345] (xv)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 66; and (b) a heavy chain variable domain (V H), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54;

[0346] (xvi)(a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54;

[0347] (xvii) (a) Light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 68; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54; or

[0348] (xviii) (a) light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 69; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:54.

[0349] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise:

[0350] (a) a light chain comprising, or consisting of, an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 73; and (b) a heavy chain comprising, or consisting of, an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 70-72 and 97; or

[0351] (a) a light chain comprising, or consisting of, an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 74; and (b) a heavy chain comprising, or consisting of, an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 70-72 and 97; or

[0352] (a) a light chain comprising, or consisting of, an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 83-95; and (b) a heavy chain comprising, or consisting of, an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 70-72, 97 and 98; or

[0353] In a specific embodiment, the antibody or antigen-binding fragment of the present disclosure comprises (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:73, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:70.

[0354] In a specific embodiment, the antibody or antigen-binding fragment of the present disclosure comprises (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:73, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:71.

[0355] In other embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:73, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:72.

[0356] In other embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:73, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:97.

[0357] In yet other embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:74, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:70.

[0358] In yet other embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:74, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:71.

[0359] In another embodiment, the antibody or antigen-binding fragment of the present disclosure comprises (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:74, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:72.

[0360] In yet other embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:74, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:97.

[0361] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 having amino acid sequences according to SEQ ID NOs: 77-82, respectively. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain variable domain (V LLD) according to SEQ ID NO: 76; L ) amino acid sequence; and (b) a heavy chain variable domain (V H ) amino acid sequence.

[0362] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain variable domain (V L ), which has at least 90%, at least 95% or 100% identity with the amino acid sequence shown in SEQ ID NO: 76, and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:75.

[0363] d. Pharmaceutical Compositions

[0364] In some embodiments, the antibody or antigen-binding fragment thereof of the combination therapy is provided as a pharmaceutical composition comprising an anti-HBV antibody and an optional pharmaceutically acceptable carrier. In some embodiments, the composition may comprise an anti-HBV antibody, wherein the antibody may comprise at least 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of the total protein in the composition by mass. In such compositions, the antibody may be in a purified form.

[0365] Pharmaceutical compositions of anti-HBV antibodies may include an antimicrobial agent, particularly if packaged in multidose form. They may include a detergent, for example, a Tween (polysorbate), such as Tween 80. When present, the detergent is typically present at low levels (e.g., less than 0.01%). The composition may also include a sodium salt (e.g., sodium chloride) to increase tonicity. For example, in some embodiments, the pharmaceutical composition includes NaCl at a concentration of 10 ± 2 mg / ml.

[0366] In addition, the pharmaceutical composition may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose), e.g., at about 15-30 mg / ml (e.g., 25 mg / ml), particularly if it is lyophilized, or if it comprises material reconstituted from a lyophilized material. Prior to lyophilization, the pH of the composition for lyophilization may be adjusted to between 5 and 8, or between 5.5 and 7, or about 6.1.

[0367] The antibody compositions of the present disclosure may also comprise one or more immunomodulators. In some embodiments, the one or more immunomodulators comprise an adjuvant.

[0368] The method of preparing a pharmaceutical composition of an anti-HBV antibody may include the following steps: (i) preparing the antibody; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

[0369] IV. Methods of Treatment Using Combination Therapies

[0370] In some embodiments, the present disclosure provides methods for treating HBV infection or hepatitis B virus-related diseases.

[0371] As used herein, "subject" is an animal, such as a mammal, including any mammal that can be infected with HBV, for example, a primate (such as a human, non-human primate, for example, a monkey or a chimpanzee), or an animal that is considered to be an acceptable clinical model of HBV infection, an HBV-AAV mouse model (see, for example, Yang et al., Cell and Mol Immunol 11:71 (2014)) or an HBV1.3xfs transgenic mouse model (Guidotti et al., J. Virol. 69:6158 (1995)). In some embodiments, the subject has hepatitis B virus (HBV) infection. In some other embodiments, the subject has hepatitis B virus (HBV) infection and hepatitis D virus (HDV) infection. In some other embodiments, the subject is a human, such as a human with HBV infection, particularly chronic hepatitis B virus (CHBV) infection.

[0372] As used herein, the term "treating" or "treatment" refers to the following beneficial or desired results, including but not limited to: alleviation or improvement of one or more signs or symptoms associated with unwanted HBV gene expression or HBV replication, such as the presence of serum or liver HBV cccDNA, the presence of serum HBV DNA, presence of serum or liver HBV antigen (e.g., HBsAg or HBeAg), elevated ALT, elevated AST (normal range is generally considered to be approximately 10 to 34 U / L), absence or low levels of anti-HBV antibodies; liver damage; cirrhosis; hepatitis D; acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, low-grade fever; myalgia; fatigability; taste and smell disturbances (aversion to food and cigarettes); or right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; lethargy; disturbed sleep patterns; confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (mildly enlarged, soft liver); splenomegaly; palmar erythema; spider angiomas; muscle wasting; spider angiomas; vasculitis; varicose bleeding; peripheral edema; male breast disease developmental disorders; testicular atrophy; abdominal collateral veins (varicose veins around the abdomen); an ALT level higher than the AST level; elevated gamma-glutamyl transpeptidase (GGT) (normal range generally considered to be approximately 8 to 65 U / L) and alkaline phosphatase (ALP) levels (normal range generally considered to be approximately 44 to 147 IU / L (international units per liter) and not more than 3 times the ULN); a slightly low albumin level; an elevated serum iron level; leukopenia (i.e., granulocytopenia); lymphocytosis; an increased erythrocyte sedimentation rate (ESR); a decreased red blood cell survival time; hemolysis; thrombocytopenia; a prolonged international normalized ratio (INR); the presence of serum or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc), immunoglobulin M (IgM), hepatitis B surface antibody (anti-HB), hepatitis B e antibody (anti-HBe), or HBV DNA; increased bilirubin levels; hyperglobulinemia; the presence of tissue nonspecific antibodies, such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%); the presence of tissue specific antibodies, such as anti-thyroid antibodies (10-20%); elevated rheumatoid factor (RF) levels; decreased platelet and white blood cell counts; lobules with degenerative and regenerative hepatocellular changes, accompanied by inflammation; and major centrilobular necrosis, whether or not detectable. Reducing the likelihood of developing a process such as liver fibrosis, for example, when an individual has one or more risk factors for liver fibrosis (e.g., chronic hepatitis B infection), they will not develop liver fibrosis or will develop liver fibrosis with less severity than a group of people with the same risk factors but who do not receive treatment as described herein."Treatment" can also mean prolonging survival as compared to expected survival without treatment.

[0373] As used herein, the term "preventing" or "prevention" refers to not developing a disease, disorder, or condition, or reducing the development of signs or symptoms associated with such a disease, disorder, or condition (e.g., by a clinically relevant amount), or showing delayed signs or symptoms (e.g., days, weeks, months, or years). Prevention may require administration of more than one dose.

[0374] In some embodiments, treating HBV infection results in a "functional cure" of hepatitis B. As used herein, functional cure is understood to be the clearance of circulating HBsAg, and may be accompanied by a transition to a state in which HBsAg antibodies can be detected using a clinically relevant assay. For example, detectable antibodies may include a signal higher than 10mIU / ml, such as by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay. Functional cure does not require the elimination of all replicated forms of HBV (e.g., cccDNA from the liver). Approximately 0.2-1% of chronically infected patients spontaneously undergo anti-HB seroconversion each year. However, even after anti-HB seroconversion, low levels of HBV are often observed to persist for decades, indicating that a functional cure rather than a complete cure has occurred. Without being constrained by a specific mechanism, the immune system may be able to control HBV under conditions in which a functional cure has been achieved. Functional cure allows the cessation of any treatment for HBV infection. However, it should be understood that a "functional cure" of HBV infection may not be sufficient to prevent or treat a disease or condition caused by HBV infection, such as liver fibrosis, HCC, or cirrhosis. In some specific embodiments, "functional cure" may refer to a sustained decrease in serum HBsAg, such as <1 IU / mL, for at least 3 months, at least 6 months, or at least one year after the start of a treatment regimen or completion of a treatment regimen.

[0375] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" is a disease or condition caused by or associated with HBV infection or replication. The term "HBV-related disease" includes diseases, disorders, or conditions that would benefit from a reduction in HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0376] In some embodiments, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or hepatitis D virus (HDV) is a human pathogen. However, the virus is defective and relies on mandatory helper functions provided by hepatitis B virus (HBV) for transmission; in fact, HDV requires an associated or pre-existing HBV infection to become infectious and fertile, in particular the viral envelope containing the hepatitis B surface antigen. HDV can cause severe acute and chronic liver diseases associated with HBV. Hepatitis D infection or hepatitis D is highly prevalent in several African countries, the Amazon region and the Middle East, while its prevalence is lower in industrialized countries (except the Mediterranean).

[0377] HDV can be transmitted through concurrent infection with HBV (coinfection) or superimposed on chronic hepatitis B or the hepatitis B carrier state (superinfection). Dual infection and coinfection with HDV generally result in more severe complications than infection with HBV alone. These complications include a greater likelihood of liver failure with rapid progression to cirrhosis in acute infection and an increased risk of liver cancer in chronic infection. In combination with HBV, hepatitis D has the highest mortality rate of all hepatitis infections, at 20%.

[0378] In some embodiments, the HBV-related disease is acute hepatitis B. Acute hepatitis B includes inflammation of the liver that lasts less than six months. Typical symptoms of acute hepatitis B are fatigue, anorexia, nausea, and vomiting. Very high transaminase values (>1000 U / L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B may rapidly progress to acute liver failure, manifested by poor liver synthetic function. This is usually defined as a prothrombin time (PT) of 16 seconds or an international normalized ratio (INR) of 1.5 in the absence of pre-existing liver disease. Acute hepatitis B may evolve into chronic hepatitis B.

[0379] In some embodiments, the HBV-related disease is chronic hepatitis. Chronic hepatitis B (CHB) includes inflammation of the liver that persists for more than six months. Subjects with CHB are HBsAg positive and have high viremia (≥10 4 HBV-DNA copies / ml blood) or low viremia (<10 3HBV-DNA copies / ml blood). In certain embodiments, the subject has been infected with HBV for at least 5 years. In certain embodiments, the subject has been infected with HBV for at least 10 years. In certain embodiments, the subject was infected with HBV at birth. A subject with chronic hepatitis B disease may be immunotolerant or have an inactive chronic infection without any evidence of active disease, and they also have no symptoms. Patients with chronic active hepatitis, especially those in a replicating state, may present with symptoms similar to acute hepatitis. A subject with chronic hepatitis B disease may have an active chronic infection with necroinflammatory liver disease, with increased hepatocyte turnover in the absence of detectable necroinflammation, or an inactive chronic infection without any signs of active disease, and they also have no symptoms. The persistence of HBV infection in a subject with CHB is a result of cccHBV DNA. In some embodiments, a subject with CHB is HBeAg positive. In some other embodiments, a subject with CHB is HBeAg negative. A subject with CHB has a serum HBV DNA level of less than 10 5 , and transaminases (eg, ALT, AST, and gamma-glutamyl transferase) remain elevated. Subjects with CHB may have a liver biopsy score (eg, necroinflammatory score) of less than 4.

[0380] In some embodiments, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B have symptoms of acute hepatitis as well as other symptoms of confusion or coma (because the liver cannot detoxify chemicals) and bruising or bleeding (because of a lack of clotting factors).

[0381] Subjects with HBV infection (e.g., CHB) may develop liver fibrosis. Thus, in some embodiments, the HBV-related disease is liver fibrosis. The histological definition of liver fibrosis (or cirrhosis) is a diffuse liver process characterized by fibrosis (excess fibrous connective tissue) and the conversion of normal liver architecture into structurally abnormal nodules.

[0382] Subjects with HBV infection (e.g., CHB) may develop end-stage liver disease. Therefore, in some embodiments, the HBV-related disease is end-stage liver disease. For example, due to liver fibrosis, the body may not be able to compensate for the disease, such as decreased liver function causing liver fibrosis (i.e., decompensated liver), and leading to, for example, mental and neurological symptoms and liver failure.

[0383] Subjects with HBV infection (e.g., CHB) may develop hepatocellular carcinoma (HCC), also known as malignant liver cancer. Thus, in some embodiments, the HBV-related disease is HCC. HCC typically develops in individuals with CHB and may be fibrolamellar, pseudoglandular (adenoids), pleomorphic (giant cell), or clear cell.

[0384] An "HDV-associated disorder" or "hepatitis D virus-associated disorder" is a disease or disorder associated with the expression of HDV. Exemplary HDV-associated disorders include hepatitis B virus infection, acute hepatitis B, acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0385] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent or anti-HBV antibody that, when administered to a patient to treat an individual suffering from HBV infection and / or HBV-related disease, is sufficient to effectively treat the disease (e.g., by reducing or maintaining one or more symptoms of an existing disease or disease). A "therapeutically effective amount" may vary depending on the RNAi agent and / or anti-HBV antibody, its mode of administration, the disease and its severity, and the medical history, age, weight, family medical history, genetic makeup, stage of the pathological process mediated by HBV genes, type of previous or concomitant treatment (if any), and other individual characteristics of the patient being treated. A therapeutically effective amount may require administration of more than one dose.

[0386] "Therapeutically effective amount" also includes the amount of RNAi agent or anti-HBV antibody that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutic agent (e.g., RNAi agent, anti-HBV antibody) used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0387] As used herein, the term "sample" includes a collection of similar fluids, cells or tissues separated from an object, and fluids, cells or tissues present in the object body. The example of a biological fluid includes blood, serum and serosity, plasma, lymph, urine, saliva etc. Tissue sample can include the sample from tissue, organ or local area. For example, the sample may be from the fluid or cell in a part of a specific organ, an organ or these organs. In some embodiments, the sample can derive from the liver (for example, some cell types in the whole liver or some parts of the liver or the liver, such as, for example, hepatocytes). In some embodiments, "sample deriving from an object" refers to blood or plasma or serum obtained from a blood draw of an object. In other embodiments, "sample deriving from an object" refers to liver tissue (or its subfraction) or blood tissue (or its subfraction, for example, serum) deriving from an object.

[0388] Some embodiments of the present disclosure provide methods for treating chronic HBV infection or HBV-related diseases in subjects in need thereof, comprising: (i) administering to the subject an agent that reduces HBV antigen load; and (ii) administering to the subject an anti-HBV antibody. In certain embodiments, the agent that reduces HBV antigen load is administered before the anti-HBV antibody. In certain embodiments, the agent that reduces HBV antigen load is administered before the anti-HBV antibody so that the viral load is reduced when the anti-HBV antibody is administered. In certain embodiments, the therapeutically effective amount of the anti-HBV antibody of the combination therapy is lower than the therapeutically effective amount of the anti-HBV antibody delivered when the agent that reduces HBV antigen load has not yet been administered to the subject (e.g., when the anti-HBV antibody is administered alone as a monotherapy). In some embodiments, the agent that reduces HBV antigen load is an RNAi agent (e.g., siRNA) that inhibits the expression of HBV transcripts.

[0389] In certain embodiments, the present disclosure provides a method of treating chronic HBV infection or an HBV-related disease in a subject in need thereof, comprising: administering to the subject an agent that reduces HBV antigen load; and administering to the subject an anti-HBV antibody; and further comprising measuring the presence of HBsAg in a blood sample from the subject before and after administration of the agent that reduces HBV antigen load, wherein a decrease in HBsAg indicates a decrease in expression of at least one HBV gene.

[0390] In certain embodiments, the present disclosure provides an agent for reducing HBV antigen load for treating chronic HBV infection or HBV-related diseases in a subject, wherein an anti-HBV antibody is subsequently administered to the subject. In certain other embodiments, the present disclosure provides an anti-HBV antibody for treating chronic HBV infection or HBV-related diseases in a subject, and the subject has previously been administered an agent that reduces HBV antigen load. In other embodiments, the expression of at least one HBV gene is reduced after administration of an agent that reduces HBV antigen load, and the anti-HBV antibody is administered to the subject when the expression of the at least one HBV gene is reduced.

[0391] In certain embodiments, the present disclosure provides the use of an agent that reduces HBV antigen load and / or an anti-HBV antibody in the preparation of a medicament for treating chronic HBV infection or HBV-related diseases.

[0392] Some embodiments of the present disclosure provide methods for treating chronic HBV infection or HBV-related diseases in subjects in need thereof, comprising: (i) administering an HBV gene expression inhibitor to the subject; and (ii) administering an anti-HBV antibody to the subject. In certain embodiments, the HBV gene expression inhibitor is administered before the anti-HBV antibody is administered. In certain embodiments, the HBV gene expression inhibitor is administered before the anti-HBV antibody, resulting in a reduced viral load when the anti-HBV antibody is administered. In certain embodiments, the therapeutically effective amount of the anti-HBV antibody of the combination therapy is lower than the therapeutically effective amount of the anti-HBV antibody delivered when the HBV gene expression inhibitor has not yet been administered to the subject (e.g., when the anti-HBV antibody is administered alone as a monotherapy).

[0393] In certain embodiments, expression of at least one HBV gene is reduced after administration of an HBV gene expression inhibitor, and an anti-HBV antibody is administered to the subject when expression of the at least one HBV gene is reduced. In a specific embodiment, the at least one HBV gene is an HBV X gene and / or HBsAg.

[0394] In certain embodiments, the present disclosure provides a method for treating chronic HBV infection or HBV-related disease in a subject in need thereof, comprising: administering an HBV gene expression inhibitor to the subject; and administering an anti-HBV antibody to the subject; and further comprising measuring the presence of HBsAg in a blood sample from the subject before and after administration of the HBV expression inhibitor, wherein a reduction in HBsAg indicates a decrease in expression of at least one HBV gene.

[0395] In certain embodiments, the present disclosure provides an HBV gene expression inhibitor for treating chronic HBV infection or an HBV-related disease in a subject, wherein the subject is subsequently administered an anti-HBV antibody. In certain other embodiments, the present disclosure provides the use of an anti-HBV antibody for treating chronic HBV infection or an HBV-related disease in a subject, and the subject has previously been administered a gene expression inhibitor. In other embodiments, after administration of the HBV gene expression inhibitor, expression of at least one HBV gene is reduced, and the anti-HBV antibody is administered to the subject when expression of at least one HBV gene is reduced.

[0396] In certain embodiments, the present disclosure provides the use of an HBV gene expression inhibitor and / or an anti-HBV antibody in the preparation of a medicament for treating chronic HBV infection or HBV-related diseases.

[0397] In any of the above methods, compositions, uses in preparation, the methods and compositions can be used to treat chronic HBV infection.

[0398] In certain embodiments, the HBV gene expression inhibitor is administered in a single dose, two doses, three doses, four doses, or five doses. In certain specific embodiments, at least one dose of the HBV gene expression inhibitor is administered before the anti-HBV antibody is administered.

[0399] In certain embodiments, the HBV gene expression inhibitor is administered in a single dose, two doses, three doses, four doses, or five doses, six doses, seven doses, or eight doses. One or more doses can be administered, for example, twice a day, once a day, every two days, every three days, twice a week, once a week, every other week, every four weeks, or once a month.

[0400] In certain embodiments, administering the anti-HBV antibody comprises administering the anti-HBV antibody twice a week, once a week, every other week, every two weeks, or once a month.

[0401] In certain embodiments, administering the anti-HBV antibody comprises administering at least two doses of a therapeutically effective amount of the anti-HBV antibody. In certain other embodiments, at least two doses are administered twice a week, once a week, every other week, every two weeks, or once a month.

[0402] In certain embodiments, administration of the anti-HBV antibody is initiated at least 1 week after administration of the HBV gene expression inhibitor. In certain embodiments, administration of the anti-HBV antibody is initiated 2 weeks after administration of the HBV gene expression inhibitor. In certain embodiments, administration of the anti-HBV antibody is initiated 8 weeks after administration of the HBV gene expression inhibitor.

[0403] In certain embodiments, the anti-HBV antibody and the HBV gene expression inhibitor are administered separately subcutaneously.

[0404] In certain embodiments of the above methods, compositions, or uses in the preparation thereof, the anti-HBV antibodies can recognize HBV genotypes A, B, C, D, E, F, G, H, I, and J.

[0405] In certain embodiments of the above methods, compositions, or uses in the preparation, the anti-HBV antibody can be a human antibody; a monoclonal antibody; or a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating immune effectors (e.g., a second specificity for stimulating cytotoxicity or a vaccination effect). In certain other embodiments of the above methods, compositions, or uses in the preparation disclosed herein, the anti-HBV antibody is a monoclonal antibody.

[0406] In certain embodiments of the above methods, compositions, or uses in preparation, the anti-HBV antibody may be HBC34 or a non-natural variant of HBC34 as disclosed herein. For example, in certain embodiments, the anti-HBV antibody comprises CDRs having the following amino acid sequences: (i) according to SEQ ID NOs: 44, 45, 47-49, and 51; or (ii) according to SEQ ID NOs: 44, 45, 47-49, and 52. In certain embodiments, the anti-HBV antibody comprises CDRs having amino acid sequences according to SEQ ID NOs: 44, 45, 47, 48, 49, and 51. In certain embodiments, the anti-HBV antibody comprises CDRs having amino acid sequences according to SEQ ID NOs: 44, 45, 47, 48, 49, and 52. In certain embodiments, the anti-HBV antibody comprises: (1) (a) a light chain variable domain (V L ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 55-63; and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53; or (2) (a) a light chain variable domain (V L ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 55-57 and 64-69; and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:54.

[0407] In certain embodiments, the anti-HBV antibody comprises: (1) (a) a light chain variable domain (V L ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 55-69; and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 53; or (2) (a) a light chain variable domain (V L ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 55-69; and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:54.

[0408] In certain embodiments, the anti-HBV antibody comprises: (a) a light chain variable domain (V L ) sequence; and (b) a heavy chain variable domain (V H )sequence.

[0409] In certain embodiments, the anti-HBV antibody comprises: (a) a light chain variable domain (V L ) sequence; and (b) a heavy chain variable domain (V H )sequence.

[0410] In a specific embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:73, and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs:70-72 and 97.

[0411] In a specific embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:74, and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:70-72 and 97.

[0412] In a specific embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 83-95, and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 70-72, 97 and 98.

[0413] In a specific embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain according to the amino acid sequence of SEQ ID NO: 73, and (b) a heavy chain according to the amino acid sequence of SEQ ID NO: 70.

[0414] In a specific embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain according to the amino acid sequence of SEQ ID NO:73, and (b) a heavy chain according to the amino acid sequence of SEQ ID NO:71.

[0415] In a specific embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain according to the amino acid sequence of SEQ ID NO:74, and (b) a heavy chain according to the amino acid sequence of SEQ ID NO:70.

[0416] In certain other embodiments of the above methods, compositions, or uses in preparation, the anti-HBV antibody comprises a CDR having an amino acid sequence according to SEQ ID NOs: 77-82. In certain embodiments of the above methods, compositions, or uses in preparation, the anti-HBV antibody comprises (a) a light chain variable domain (V) having an amino acid sequence according to SEQ ID NO: 76; L ); and (b) a heavy chain variable domain (V H ).

[0417] In certain embodiments of the above methods, compositions, or uses in the preparation of the anti-HBV antibody, the anti-HBV antibody comprises (a) a light chain variable domain (V L ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO: 76, and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:75.

[0418] In certain embodiments, the therapeutically effective amount of the anti-HBV antibody is lower than the therapeutically effective amount of the anti-HBV antibody delivered when the HBV gene expression inhibitor is not administered to the subject. For example, the combination therapy can reduce the effective dose of the anti-HBV antibody as compared to administering the anti-HBV antibody alone.

[0419] In certain embodiments, the anti-HBV antibody is administered in at least two separate doses. In a specific embodiment, at least two doses are administered twice a week, once a week, every other week, every two weeks, or once a month.

[0420] In certain embodiments, the subject is a human, and the anti-HBV antibody is administered in a therapeutically effective amount; wherein the therapeutically effective amount is from about 3 mg / kg to about 30 mg / kg.

[0421] In certain embodiments of the above methods, compositions, or uses in the preparation, the inhibitor is an RNAi agent that inhibits expression of HBV transcripts. In some embodiments, inhibition of HBV transcript expression is measured by rtPCR. In some embodiments, inhibition of HBV transcript expression is measured by a reduction in protein levels as measured by ELISA.

[0422] In certain embodiments, the RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from nucleotides 1579-1597 of SEQ ID NO: 1. In certain embodiments, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotides 1579-1597 of SEQ ID NO: 1.

[0423] In certain embodiments of the above methods, compositions, or uses in the manufacture of the same, at least one strand of the RNAi agent may comprise a 3' overhang of at least 1 nucleotide or at least 2 nucleotides.

[0424] In specific embodiments of the above methods, compositions, or uses in the preparation, the double-stranded region of the RNAi agent can be 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs in length; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

[0425] In certain embodiments of the above methods, compositions, or uses in the manufacture of the same, each strand of the RNAi agent can be 15-30 nucleotides or 19-30 nucleotides.

[0426] In certain embodiments of the above methods, compositions, or uses in preparation, the RNAi agent is an siRNA. In certain embodiments, the siRNA inhibits expression of HBV transcripts encoding HBsAg, HBcAg, and HBx proteins, or HBV DNA polymerase protein. In certain embodiments, the siRNA binds to at least 15 contiguous nucleotides of a target encoded by: the P gene, nucleotides 2309-3182 and 1-1625 of NC_003977.2; the S gene (encoding L, M, and S proteins), nucleotides 2850-3182 and 1-837 of NC_003977.2; HBx, nucleotides 1376-1840 of NC_003977.2; or the C gene, nucleotides 1816-2454 of NC_003977.2.

[0427] In a specific embodiment of the above-mentioned method, composition or use in preparation, the RNAi agent is siRNA, and the antisense strand of the siRNA comprises at least 15 adjacent nucleotides or 19 adjacent nucleotides of the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4). In some embodiments, the antisense strand of the siRNA comprises the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4). In some embodiments, the antisense strand consists of the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4). In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3). In some embodiments, the sense strand of the siRNA consists of the nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

[0428] In a specific embodiment of the above-mentioned method, composition or use in preparation, the RNAi agent is siRNA, and the antisense strand of the siRNA comprises at least 15 adjacent nucleotides or 19 adjacent nucleotides of the nucleotide sequence of 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107). In some embodiments, the antisense strand of the siRNA comprises the nucleotide sequence of 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107). In some embodiments, the antisense strand consists of the nucleotide sequence of 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107). In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence of 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 106). In some embodiments, the sense strand of the siRNA consists of the nucleotide sequence of 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 106).

[0429] In certain embodiments of the above methods, compositions, or uses in the preparation, the RNAi agent is an siRNA, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end. In certain embodiments, the ligand is one or more GalNAc derivatives attached via a monovalent linker, a bivalent branched linker, or a trivalent branched linker. In certain embodiments, the GalNAc derivative attached via a linker is or comprises:

[0430]

[0431] In a specific embodiment, the siRNA is conjugated to the ligand as shown below (i.e., the GalNAc derivative attached via the linker is):

[0432]

[0433] Wherein X is O or S.

[0434] In certain embodiments of the above methods, compositions, or uses in the preparation, the RNAi agent is an siRNA, wherein at least one nucleotide of the siRNA is a modified nucleotide including deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl modified nucleotides, In some embodiments, the siRNA comprises a phosphate backbone modification, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a thiophosphate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, an adenosine-diol nucleic acid, or a nucleotide comprising a 5'-phosphate analog. In certain embodiments, the siRNA comprises a phosphate backbone modification, a 2' ribose modification, a 5' triphosphate modification, or a GalNAc conjugated modification. In certain embodiments, the phosphate backbone modification comprises a thiophosphate bond. In certain embodiments, the 2' ribose modification comprises a fluorine or -O-methyl substitution.

[0435] In a specific embodiment of the above methods, compositions, or uses in preparation, the RNAi agent is a siRNA having a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0436] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0437] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0438] s is a phosphorothioate linkage; and

[0439] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0440] In a specific embodiment of the above methods, compositions, or uses in preparation, the RNAi agent is a siRNA having a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 7) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 8),

[0441] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0442] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0443] (Agn) is adenosine diol nucleic acid (GNA);

[0444] s is a phosphorothioate linkage; and

[0445] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0446] In certain embodiments of the above methods, compositions, or uses in preparation, the RNAi agent is a siRNA having a sense strand comprising 5'-gsgsuggaCfuUfCfUfcucaAfUfuuuaL96-3' (SEQ ID NO: 108) and an antisense strand comprising 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 109),

[0447] where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;

[0448] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0449] s is a phosphorothioate linkage; and

[0450] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0451] In certain embodiments of the above methods, compositions, or uses in the manufacture of, the subject is a human, and the RNAi or siRNA is administered to the subject in a therapeutically effective amount; wherein the therapeutically effective amount of the RNAi or siRNA is from about 1 mg / kg to about 8 mg / kg.

[0452] In some embodiments of the methods, compositions, or uses disclosed herein, the siRNA is administered to the subject twice a day, once a day, every two days, every three days, twice a week, once a week, every other week, every four weeks, or once a month. In some embodiments, the siRNA is administered to the subject every four weeks.

[0453] In certain embodiments, the method includes administering two HBV gene expression inhibitors and anti-HBV antibodies. The two HBV gene expression inhibitors can be two siRNAs, such as two siRNAs targeting different HBV genes. The two different HBV genes can be, for example, HBsAg and HBV X. The two HBV gene expression inhibitors can be administered simultaneously. In certain embodiments, two siRNAs each directed to an HBV gene are administered, and the first siRNA has an antisense strand comprising SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and the second siRNA comprises an siRNA having a sense strand comprising at least 15 adjacent nucleotides from nucleotides 2850 to 3182 of SEQ ID NO: 1. In certain embodiments, two siRNAs each directed to an HBV gene are administered, and the first siRNA has an antisense strand comprising SEQ ID NO: 107 or SEQ ID NO: 109; and the second siRNA comprises an siRNA having a sense strand comprising at least 15 adjacent nucleotides from nucleotides 2850 to 3182 of SEQ ID NO: 1. In certain embodiments, two siRNAs are administered, each directed to an HBV gene, and the first siRNA has an antisense strand comprising SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and the second siRNA has an antisense strand comprising SEQ ID NO: 107 or SEQ ID NO: 109. In certain embodiments, the first siRNA has a sense strand comprising SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; and the second siRNA has a sense strand comprising SEQ ID NO: 106 or SEQ ID NO: 108.

[0454] In certain embodiments, anti-HBV antibodies and HBV gene expression inhibitors show synergistic therapeutic effects. The term "synergistic effect" is used to describe the combined effect of two or more active agents, which is greater than the sum of the individual effects of each corresponding active agent. Therefore, when the combined effect of two or more agents results in "synergistic inhibition" of an activity or process, it means that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each corresponding active agent. The term "synergistic therapeutic effect" refers to a therapeutic effect observed with a combination of two or more therapies, wherein the therapeutic effect (as measured by any one of a plurality of parameters) is greater than the sum of the individual therapeutic effects observed with each individual therapy.

[0455] In some embodiments, administration of a RNAi agent targeting HBV mRNA to a subject having HBV infection, and / or HBV-related disease results in a decrease in expression of one or more HBV genes, HBV ccc DNA levels, HBV antigen levels, HBV viral load levels, ALT, and / or AST (e.g., in the subject's cells, tissues, blood, or fluids) by at least about 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%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 1 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.

[0456] In some embodiments, a RNAi agent targeting HBV mRNA is administered to a subject having HBV infection, and / or HBV-related disease, and HBV gene expression is inhibited by at least about 5%, 6%, 7%, 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%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 11 %, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or about 100% (i.e., below the level of detection of the assay).

[0457] In some embodiments, the combination therapy according to the present disclosure comprises administering a nucleoside (acid) analog as a third component. As used herein, the term "nucleoside (acid) analog" (or "polymerase inhibitor" or "reverse transcriptase inhibitor") is a DNA replication inhibitor that is structurally similar to nucleotides or nucleosides and specifically inhibits the replication of HBV cccDNA without significantly inhibiting the replication of host (e.g., human) DNA. Such inhibitors include tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, disoproxil, lobcavir, famciclovir, N-acetyl-cysteine (NAC), PC1323, theradigm-HBV, thymosin-α, ganciclovir, besfovir (ANA-380 / LB-80380) and tenofovir-exaliades (TLX / CMX157). In certain embodiments, the nucleoside (acid) analog is entecavir (ETV). Nucleoside (acid) analogs are commercially available from many sources and are determined according to their label instructions (e.g., typically administered orally in a specific dose) or according to those skilled in the art for treating HBV and are used in the methods provided herein.

[0458] The anti-HBV antibody or HBV gene expression inhibitor may be present in the same pharmaceutical composition as the third active ingredient, or the anti-HBV antibody, HBV gene expression inhibitor, and the third active ingredient may be present in three different pharmaceutical compositions. Such different pharmaceutical compositions may be administered in combination / simultaneously or at different times or locations (e.g., different parts of the body).

[0459] V. Kits for HBV Combination Therapy

[0460] Provided herein are kits for components comprising HBV therapies. In some embodiments, the kit includes one or more anti-HBV antibodies, one or more HBV gene expression inhibitors, and an optional third component (e.g., nucleoside (acid) analogues) of an HBV combination therapy. The kit may additionally include instructions for preparing and / or administering components of the HBV combination therapy.

[0461] Example

[0462] Example 1

[0463] Combination therapy of antibodies and HBV-targeted siRNA reduces markers of HBV infection in AAV-HBV mice

[0464] To determine whether siRNA-antibody combination therapy might be effective in treating HBV infection, AAV / HBV-infected C57BL / 6 mice were administered one of fourteen different treatments: (1) HBV-specific siRNA (HBV02, having the antisense strand of SEQ ID NO: 8); (2)-(5) one of four doses of anti-HBV antibody (HBC34 antibody HBC34v7, a mouse chimeric form of HBC34-v7-mu-IgG2a); (6-7) HBV02 siRNA and one of two antibody doses of HBC34-v7-mu-IgG2a antibody; (8-11) HBV02 siRNA, one of four antibody doses of HBC34-v7-mu-IgG2a antibody, and entecavir (ETV); (12) control siRNA and control antibody; (13) entecavir alone; or (14) saline alone (see Table 4).

[0465] Table 4: Therapeutic Levels and Doses

[0466]

[0467] HBV02 is a chemically synthesized double-stranded oligonucleotide covalently linked to a ligand containing three GalNAc residues. All nucleotides are 2'-OMe or 2'-F modified, and one nucleotide of the antisense strand is replaced by (S)-1-(2,3-dihydroxypropyl)adenosine (Agn). The nucleotides of the sense and antisense strands are linked by 3'-5' phosphodiester or 3'-5' phosphorothioate linkages, forming the sugar-phosphate backbone of the oligonucleotide.

[0468] HBC34 is a highly neutralizing monoclonal antibody against HBV surface antigens (PreS1, PreS2, and S). The HBC34 antibody used in this experiment is a fully murinized HBC34v7, excluding the Fab fragment that binds to the HBV surface antigen. Human HBC34v7 has a V domain as shown in SEQ ID NO: 53. H Sequence and V shown in SEQ ID NO:56 L The mouse chimeric version of the HBC34v7 sequence in the HBC34-v7-mu-IgG2a antibody used in this experiment has the heavy and light chain amino acid sequences shown in SEQ ID NOs: 99 and 100, respectively.

[0469] siRNA targeting the human transthyretin gene was used as a control siRNA because it was not expected to result in a reduction of HBV markers of infection in serum.

[0470] The control monoclonal antibody (mAb) used in this example is a respiratory syncytial virus-specific antibody and is not expected to result in a reduction in HBV markers of infection in serum.

[0471] Mice (C57BL / 6 strain) were inoculated with the following amount of rAAV8-1.3HBV strain ayw, type D: 1.0×10 per mouse via tail vein injection. 11 Viral genomes in a volume of 200 μl. Treatment with test compounds was initiated four weeks after viral inoculation.

[0472] The dosing schedule is shown in Figure 1. Entecavir was administered orally once daily. HBV-specific siRNA was administered subcutaneously once at the start of the study, and anti-HBV antibodies were administered intraperitoneally twice weekly during the third and fourth weeks of the study. Some mice were sacrificed during the fourth week, and others during the sixth week.

[0473] Viral load, HBsAg, and free HBc34 antibodies were measured from serum samples twice weekly. Serum HBeAg, serum alanine aminotransferase (ALT), liver HBcAg, liver HBsAg, total HBV DNA in the liver (by qPCR), and serum anti-HBV antibodies were also measured. Liver lymphocytes, spleen cells, and lymph nodes (portal vein / peritoneal and inguinal) were analyzed for HBV-specific IFNg + CD4 + cells and IFNg + CD8 + The proportion of cells.

[0474] The mean HBsAg values of the treatment groups are shown in Table 5.

[0475]

[0476] Figures 2A and 2B show viral load as measured by HBV DNA copy number, and Figures 3A and 3B show serum HBsAg levels. Figures 2A and 3A show viral load and HBsAg levels, respectively, when HBV02 siRNA or HBC34 antibody (15 mg / kg) was administered alone. HBV02 siRNA reduced serum HBV DNA and HBsAg by approximately 0.5-log, respectively, relative to saline control. 10 and 1-log 10 The HBC34 antibody alone had no effect on HBV DNA and reduced serum HBsAg by <1-log relative to saline controls. 10 Figures 2B and 3B show that treatment with HBV02 siRNA and HBC34 antibody (15 mg / kg) reduced viral load and HBsAg levels by approximately 3-log compared with saline controls. 10. When HBV02 siRNA and HBC34 antibody were used in combination, reductions in serum HBV DNA and HBsAg were significantly greater than when either molecule was used alone, and the combined effect exceeded the sum of the effects of the monotherapy. The combination therapy also reduced viral load and HBsAg levels more than treatment with entecavir alone. The effects of the combination of HBV02 siRNA and HBC34 antibody were observed regardless of whether entecavir was also administered. These results suggest that HBV02 siRNA and HBC34 antibody have the potential to synergistically reduce viral load and HBsAg, an effect that is independent of entecavir treatment.

[0477] Figure 4 depicts free HBC34 antibody levels measured between 14 and 42 days after the start of the study on day 1 (day 1 = siRNA was administered to the selected treatment group). Treatment with the combination of HBV02 siRNA and HBC34 antibody resulted in higher initial free antibody levels relative to treatment with HBC34 alone, regardless of whether the treatment included entecavir, and these levels were maintained for more than 28 days. The results shown in Figure 4, along with viral load and serum HBsAg levels, suggest that the therapeutic effect depends on the amount of free circulating HBC34 antibody, and that lower doses of antibody may become effective as the HBsAg load decreases. For example, combination therapy may allow for effective treatment with fewer doses of antibody, lower doses of antibody, and / or less invasive routes of administration (e.g., subcutaneous rather than intravenous), based at least in part on reducing the HBsAg load prior to antibody treatment.

[0478] In summary, this study demonstrates that administering siRNA targeting HBV followed by an antibody targeting HBV effectively reduces serum HBV DNA and HBsAg. Furthermore, the individual components appear to interact synergistically, such that the effect of this combination therapy is greater than that of each component alone and greater than would be expected if the effects were simply additive. Finally, the results suggest that administration of siRNA reduces serum HBsAg, thereby enabling the antibody to be more effective.

[0479] Example 2

[0480] Combination therapy with one of two anti-HBV antibodies and HBV-targeted siRNA

[0481] To determine whether siRNA-antibody combination therapy using siRNA and the anti-HBV antibody HBC24 is effective in treating HBV infection, AAV / HBV-infected C57BL / 6 mice were administered one of the following eleven different treatments: (1) HBV-specific siRNA (HBV02, having the antisense strand of SEQ ID NO: 8; see description in Example 1); (2)-(3) anti-HBV antibody (fully murinized HBC24) at one of two doses; (4)-(5) HBV02 siRNA (at one dose) and fully murine HBC24 (at one of two doses); (6-9) HBV02 siRNA (at one of two doses) and fully murine anti-HBV antibody HBC34 (HBC34-v35-mu-IgG2a) (at one of three antibody doses); (10) control siRNA and control antibody; or (11) PBS alone, administered intraperitoneally (see Table 6).

[0482] Table 6: Therapeutic Levels and Doses for Example 2

[0483]

[0484] The HBC24 and HBC34 antibodies used in this experiment are fully murinized except for the Fab fragment portion that binds to the HBV surface antigen. Human HBC24 has a V domain as shown in SEQ ID NO: 75. H The amino acid sequence is as shown in SEQ ID NO: 76. L Amino acid sequences. The murinized HBC24 sequence of the antibody used in this experiment has a heavy chain and light chain comprising the amino acid sequences set forth in SEQ ID NOs: 103 and 104, respectively. The HBC34 antibody is a murinized HBC34v35 variant, HBC34-v35-mu-IgG2a. Human HBC34v35 has a heavy chain amino acid sequence set forth in SEQ ID NO: 70 and a light chain amino acid sequence set forth in SEQ ID NO: 73. The murinized HBC34v35 sequence of the HBC34-v35-mu-IgG2a antibody used in this experiment has a heavy chain and light chain comprising the amino acid sequences set forth in SEQ ID NOs: 101 and 102, respectively.

[0485] Control siRNA targets the human transthyretin gene and is not expected to result in a reduction in markers of HBV infection in serum.

[0486] The control monoclonal antibody (mAb) is a specific antibody against respiratory syncytial virus and is not expected to result in a reduction in HBV infection markers in serum.

[0487] Treatment was performed on WuXi AppTec's immunocompromised HBV mouse. This mouse model is generated by transducing hepatocytes with an adeno-associated virus containing the HBV genome in immunocompetent mice. Using this model, HBV protein production is controlled by the endogenous HBV promoter, and the mice develop HBV-specific cellular and humoral T cell responses. However, HBV infection does not occur, cccDNA is not produced, replication is transient, and the immune response is hampered by vector-driven interference.

[0488] Mice (C57BL / 6 strain) were inoculated with the following amount of rAAV8-1.3HBV strain ayw, type D: 1.0×10 per mouse via tail vein injection. 11 viral genomes in a 200 μl volume.

[0489] Each treatment group consisted of five mice. HBV-specific siRNA was administered subcutaneously once at the start of the study, and anti-HBV antibodies were administered intraperitoneally twice weekly during the second and third weeks of the study. Mice were sacrificed on the sixth week of the study.

[0490] Serum samples were collected regularly throughout the study to measure viral load, HBsAg, and free HBC34 antibodies. Serum HBeAg, serum alanine aminotransferase (ALT), liver HBcAg, liver HBsAg, total HBV DNA in the liver (by qPCR), and serum anti-HBV antibodies were also measured. Liver lymphocytes, spleen cells, and lymph nodes (portal vein / peritoneal and inguinal) were analyzed to determine HBV-specific IFNg + CD4 + cells and IFNg + CD8 + The proportion of cells.

[0491] The experimental results are shown in Figures 5A and 5B (serum HBV DNA concentration), Figures 6A and 6B (serum HBsAg concentration), and Figures 7A and 7B (serum HBeAg concentration). Mice treated with HBV02 and one of the anti-HBV antibodies had lower serum HBV DNA concentrations, HBsAg concentrations, and HBeAg concentrations compared to mice treated with siRNA or control alone. This effect was observed for both the HBC34 and HBC24 antibodies. Furthermore, the effect was greater at higher doses of HBV02, and when higher doses of HBV02 were used, HBsAg reductions were achieved at lower antibody doses. These results provide further evidence that combination therapy with HBV02 and an HBV-targeting monoclonal antibody can provide greater HBsAg and HBeAg reductions than HBV02 monotherapy. These results also suggest that higher doses of siRNA prior to antibody administration can provide similar HBsAg reductions at lower antibody doses.

[0492] Example 3

[0493] HBsAg seroclearance and viral entry inhibition in a mouse model

[0494] Immunodeficient mice transplanted with human hepatocytes were used to test the effectiveness of combined treatment with HBV-specific siRNA and anti-HBV antibodies in clearing HBsAg. The model (PhoenixBio, Japan) uses uPA / SCID mice to generate mice with ≥70% human hepatocytes in their livers (Ohshita H and Tateno C, Methods Mol Biol. 1506:91-100, (2017)). Unlike the AAV-HBV model, cccDNA has been established and intrahepatic dissemination of HBV is possible.

[0495] Primary human hepatocytes are transplanted into SCID mice whose mouse hepatocytes have been previously enzymatically destroyed. The mice lack T cells and B cells. This model can be used to study HBV infection, including entry, spread, cccDNA regulation, hepatocyte intrinsic immune responses, and viral integration into the host genome. This model can also be used to study the effects of human IFNα on infection. However, this model does not include the induction of an adaptive immune response. Mice are inoculated via the tail vein with 1.0X10 7 The patients were treated with HBV genotype C. Treatment was started at week eight after infection.

[0496] HBV-infected mice (n=4 per treatment group) were administered one of seven different treatments: (1) PBS alone; (2-4) anti-HBV antibody (fully murinized HBC34v35 antibody, HBC34-v35-mu-IgG2a) administered intraperitoneally twice weekly at one of three doses during weeks 2 and 3; or (5-7) HBV-specific siRNA (HBV02, antisense strand having SEQ ID NO: 8; see description in Example 1) administered subcutaneously once at the start of the study, and fully murinized HBC34v35 administered intraperitoneally twice weekly at one of three doses during weeks 2 and 3 (see Table 7). Mice were sacrificed at week 6. See also Figure 8 for the study design.

[0497] Table 7: Therapeutic Levels and Doses

[0498]

[0499] The HBC34 antibody HBC34v35 used in this experiment is fully murinized, except for the Fab fragment that binds to the HBV surface antigen. Human HBC34v35 has a heavy chain amino acid sequence as shown in SEQ ID NO:70 and a light chain amino acid sequence as shown in SEQ ID NO:73. The murinized version of the HBC34-v35-mu-IgG2a antibody used in this experiment, HBC34v35, has heavy and light chains comprising the amino acid sequences set forth in SEQ ID NOs:101 and 102, respectively.

[0500] Serum samples were collected regularly throughout the study to measure viral load, HBsAg, and free HBC34 antibodies. Serum HBeAg, serum alanine aminotransferase (ALT), liver HBcAg, liver HBsAg, total HBV DNA in the liver (by qPCR), and serum anti-HBV antibodies were also measured. Liver lymphocytes, spleen cells, and lymph nodes (portal vein / peritoneal and inguinal) were analyzed to determine HBV-specific IFNg + CD4 + cells and IFNg + CD8 + The proportion of cells.

[0501] The combination of siRNA and anti-HBV antibody reduced serum HBV DNA concentration ( FIG. 9 ) and serum HBsAg concentration ( FIG. 10 ) compared to the same dose of antibody. Figure 11Similar trends were observed for serum HBV DNA concentration (Figure 9) and serum HBsAg concentration (Figure 12). Furthermore, in this model system, the antibody can also act as an inhibitor of viral entry into hepatocytes; when the HBC34 antibody was administered as a monotherapy (i.e., not in combination with siRNA) at 15 mg / kg, serum HBV DNA concentration (Figure 9) and serum HBsAg concentration (Figure 10) were also lower.

[0502] This study provides experimental support in a real infection model that when HBV02 siRNA and HBC34 antibody are used in combination, HBV DNA and HBsAg levels are reduced to a greater extent than when HBC34 is used alone.

[0503] Example 4

[0504] Clinical evaluation of siRNA-antibody combination therapy for chronic HBV infection

[0505] Phase 2 clinical study of siRNA-antibody combination therapy was conducted to evaluate the efficacy of combination therapy in human patients with chronic HBV infection. Table 8 shows the treatment regimen of this study. This study may include additional cohorts to test the impact of additional treatments on combination therapy (e.g., 9 cohorts, if two additional treatments are tested). Each group / cohort includes 15 patients.

[0506] Table 8: Therapeutic levels and doses in clinical trials

[0507]

[0508] Figure 13 shows the treatment regimen designed for the Phase 2 study. The study consisted of 24 weeks of treatment and 24 weeks of follow-up. All patients were free of cirrhosis and had NUC suppressed (using nucleos(t)ide analogs) at study entry. Throughout the study, all patient groups were eligible for NUC treatment (e.g., daily oral tenofovir or entecavir). The study began with all cohorts receiving an eight-week lead-in treatment with HBV02 siRNA. For example, the HBV02 dose could be two 400 mg doses administered subcutaneously four weeks apart. However, the appropriate dose could be determined in a monotherapy trial prior to the Phase 2 trial. After eight weeks of study, all cohorts continued treatment with HBV02; cohorts 2, 4, and 6 began treatment with a low dose of the HBC34 antibody (HBC34v35); cohorts 3, 5, and 7 began treatment with a higher dose of the HBC34 antibody; and cohort 1 did not receive the HBC34 antibody. A low dose of HBC34v35 can be, for example, 0.5 grams intravenously every two weeks; a higher dose can be, for example, 2 grams intravenously every two weeks. Prior to the Phase 2 trial, the appropriate dose can be verified by a monotherapy trial. During week 12 of the study, groups 1-3 may receive additional treatment once a week. In addition, some cohorts (e.g., cohorts 6 and 7) may receive another treatment. After 24 weeks of treatment, patients are monitored and evaluated to determine whether a functional cure has been achieved, which is indicated by the disappearance of detectable serum HBsAg and / or anti-HB seroconversion.

[0509] While specific embodiments have been illustrated and described, it will be readily appreciated that the various embodiments described above can be combined to provide further embodiments and that various changes can be made without departing from the spirit and scope of the invention.

[0510] Unless otherwise indicated, all U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 62 / 782,896 filed on December 20, 2018, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.

[0511] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> Well Biotechnology Co., Ltd. Hummerbos Biomedical A. Bakargiyev P.S. Pang D. Coty <120> Combination HBV therapy <130> 930485.401WO <140> PCT <141> 2019-12-19 <150> US 62 / 782,896 <151> 2018-12-20 <160> 109 <170> FastSEQ Windows version 4.0 <210> 1 <211> 3182 <212> DNA <213> Hepatitis B virus <220> <223> Hepatitis B virus genome, NC_003977.2 <400> 1 aattccacaa ccttccacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60 gctggtggct ccagttcagg aacagtaaac cctgttctga ctactgcctc tcccttatcg 120 tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180 ctaggacccc ttctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240 ccgcagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac taccgtgtgt 300 cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaact 360 tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttctctt catcctgctg 420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480 ctaattccag gatcctcaac aaccagcacg ggaccatgcc ggacctgcat gactactgct 540 caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac cttcggacgg aaattgcacc 600 tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660 cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttcccc 720 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780 ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 840 ctaaaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 900 atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 960 ctttaacag gcctattgat tggaagtat gtcaacgaat tgtgggtctt ttgggttttg 1020 ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 1080 ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 1140 acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 1200 ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 1260 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 1320 acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 1380 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 1440 cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 1500 gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 1560 cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 1620 cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 1680 aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 1740 gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 1800 ctgcgcacca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct 1860 actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat cgacccttat 1920 aaagaatttg gagctactgt ggagttactc tcgtttttgc cttctgactt ctttccttca 1980 gtacgagatc ttctagatac cgcctcagct ctgtatcggg aagccttaga gtctcctgag 2040 cattgttcac ctcaccatac tgcactcagg caagcaattc tttgctgggg ggaactaatg 2100 actctagcta cctgggtggg tgttaatttg gaagatccag cgtctagaga cctagtagtc 2160 agttatgtca acactaatat gggcctaaag ttcaggcaac tcttgtggtt tcacatttct 2220 tgtctcactt ttggaagaga aacagttata gagtatttgg tgtctttcgg agtgtggatt 2280 cgcactcctc cagcttatag accaccaaat gcccctatcc tatcaacact tccggagact 2340 actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400 aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc 2460 ttggactcat aaggtgggga actttactgg gctttattct tctactgtac ctgtctttaa 2520 tcctcattgg aaaacaccat cttttcctaa tatacattta caccaagaca tttcaaaaa 2580 atgtgaacag tttgtaggcc cactcacagt taatgagaaa agaagattgc aattgattat 2640 gcctgccagg tttatccaa aggttaccaa atattacca ttggataagg gtattaaacc 2700 ttattatcca gaacatctag ttaatcatta cttccaaact agacactatt tacacactct 2760 atggaaggcg ggtatattat ataagagaga aacaacacat agcgcctcat ttgtggggtc 2820 accatattct tgggaacaag atctacagca tggggcagaa tctttccacc agcaatcctc 2880 tgggattct tcccgaccac cagttggatc cagccttcag agcaaacacc gcaaatccag 2940 attgggactt caatcccaac aaggacacct ggccagacgc caacaaggta ggagctggag 3000 cattcgggct gggtttcacc ccaccgcacg gaggcctttt ggggtggagc cctcaggctc 3060 agggcatact acaaactttg ccagcaaatc cgcctcctgc ctccaccaat cgccagtcag 3120 gaaggcagcc taccccgctg tctccacctt tgagaaacac tcatcctcag gccatgcagt 3180 gg 3182 <210> 2 <211> 18 <212> DNA <213> Hepatitis B virus <220> <223> Hepatitis B virus genome, nucleotides 1579-1597 of NC_003977.2 <400> 2 gtgtgcactt cgcttcac 18 <210> 3 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic sequence HBV02, positive strand <400> 3 gugugcacuu cgcuucaca 19 <210> 4 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic sequence HBV02, antisense strand <400> 4 ugugaagcga agugcacacu u 21 <210> 5 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide (HBV01, positive strand) <400> 5 gugugcacuu cgcuucaca 19 <210> 6 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide (HBV01, antisense strand) <400> 6 ugugaagcga agugcacacu u 21 <210> 7 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide (HBV02, positive strand) <400> 7 gugugcacuu cgcuucaca 19 <210> 8 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide (HBV02, antisense strand) <400> 8 ugugaagcga agugcacacu u 21 <210> 9 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Contains the membrane translocation sequence of the peptide RFGF <400> 9 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Contains a membrane translocation sequence of a peptide RFGF analog <400> 10 Ala Ala Leu Leu Pro Val Leu Leu Ala Ala Pro 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial sequence <220> <223> HIV Tat protein <400> 11 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 12 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Drosophila antennae protein <400> 12 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys 1 5 10 15 <210> 13 <211> 226 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg S domain <400> 13 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 Island 225 <210> 14 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> J02203 (D, ayw3) HBsAg antigen loop sequence <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 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> 15 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> FJ899792 (D, adw2) HBsAg antigen loop sequence <400> 15 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> 16 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> AM282986 (A) HBsAg antigen loop sequence <400> 16 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> 17 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> D23678 (B1) HBsAg antigen loop sequence <400> 17 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> 18 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> AB117758 (C1) HBsAg antigen loop sequence <400> 18 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> 19 <211> 74 <212> PRT <213> Hepatitis B virus <220> <223> AB205192(E) HBsAg antigen loop sequence <400> 19 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 Leu Ser 65 70 <210> 20 <211> 71 <212> PRT <213> Hepatitis B virus <220> <223> X69798 (F4) HBsAg antigen loop sequence <400> 20 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 Thr Cys Thr Thr Leu Ala Gln Gly Thr Ser Met 20 25 30 Phe Pro Ser Cys Cys Cys Ser Lys Pro Ser Asp Gly Asn Cys Thr Cys 35 40 45 Ile Pro Ile Pro Ser Ser Trp Ala Leu Gly Lys Tyr Leu Trp Glu Trp 50 55 60 Ala Ser Ala Arg Phe Ser Trp 65 70 <210> twenty one <211> 70 <212> PRT <213> Hepatitis B virus <220> <223> AF1 60501 (G) HBsAg antigen loop sequence <400> twenty one 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 Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met 20 25 30 Tyr Pro Ser Cys Cys Cys Thr Pro Ser Asp Gly Asn Cys Thr Cys Ile 35 40 45 Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala 50 55 60 Ser Val Arg Phe Ser Trp 65 70 <210> twenty two <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> AY090454 (H) HBsAg antigen loop sequence <400> twenty two 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> twenty three <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> AF241409 (I) HBsAg antigen loop sequence <400> twenty three 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> twenty four <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> AB486012 (J) HBsAg antigen loop sequence <400> twenty four 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> 25 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg Y100C / P120T HBsAg antigen loop sequence <400> 25 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 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> <223> HBsAg P120T HBsAg antigenic loop sequence <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 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> 27 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg P120T / S143L HBsAg antigenic loop sequence <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 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> 28 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg C121 S HBsAg antigenic loop sequence <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 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> 29 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg R122D HBsAg antigenic loop sequence <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 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> 30 <211> 71 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg R122I HBsAg antigenic loop sequence <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 Ile Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser 20 25 30 Met Tyr Pro Ser Cys Cys Cys Thr Pro Ser Asp Gly Asn Cys Thr Cys 35 40 45 Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp 50 55 60 Ala Ser Ala Arg Phe Ser Trp 65 70 <210> 31 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg T123N HBsAg antigenic loop sequence <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 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> 32 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg Q129H HBsAg antigen loop sequence <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 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> 33 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg Q129L HBsAg antigenic loop sequence <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 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> 34 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg M1 33H HBsAg antigen loop sequence <400> 34 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> 35 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg M1 33L HBsAg antigenic loop sequence <400> 35 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> 36 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg M133T HBsAg antigenic loop sequence <400> 36 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> 37 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg K141 E HBsAg antigenic loop sequence <400> 37 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> 38 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg P142S HBsAg antigenic loop sequence <400> 38 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> 39 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg S143K HBsAg antigenic loop sequence <400> 39 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> 40 <211> 71 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg D144A HBsAg antigenic loop sequence <400> 40 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 Pro Ser Ala Gly Asn Cys Thr Cys 35 40 45 Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp 50 55 60 Ala Ser Ala Arg Phe Ser Trp 65 70 <210> 41 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg G145R HBsAg antigenic loop sequence <400> 41 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> 42 <211> 72 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg N146A HBsAg antigenic loop sequence <400> 42 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> 43 <211> 226 <212> PRT <213> Hepatitis B virus <220> <223> HBsAg genotype DS domain (Genbank accession no. FJ899792) <400&g...

Claims

1. Use of siRNA and anti-HBV antibodies that inhibit the expression of HBV transcripts in the preparation of a medicament for treating chronic HBV infection or hepatitis D virus (HDV) infection, wherein: (a) the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and (ii) the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively; and (b) the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3', wherein: (i) a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively; (ii) Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (iii) (Agn) is adenosine glycol nucleic acid (GNA); (iv) s is a phosphorothioate linkage; and (v) L96 is 2. The use according to claim 1, wherein the anti-HBV antibody comprises: (a) Light chain variable domain (V L ), which has at least 90%, at least 95% or 100% identity with the amino acid sequence shown in SEQ ID NO: 59; and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:

53.

3. The use according to claim 1, wherein the anti-HBV antibody comprises: (a) light chain variable domain according to SEQ ID NO: 59 (V L ) amino acid sequence; and (b) a heavy chain variable domain (V H ) amino acid sequence.

4. The method according to claim 1, wherein the anti-HBV antibody comprises: (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:73, and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:

71.

5. The use according to claim 1, wherein the anti-HBV antibody comprises: (a) the light chain amino acid sequence according to SEQ ID NO: 73, and (b) the heavy chain amino acid sequence according to SEQ ID NO:

71. The use according to claim 1 , wherein the anti-HBV antibody is a monoclonal antibody.

7. The use according to claim 1, wherein the anti-HBV antibody is a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating immune effectors.

8. The use according to claim 7, wherein the second specificity stimulates cytotoxicity or a vaccine effect.

9. The use according to claim 1, wherein the siRNA is conjugated to the L96 as shown in the following structure: Where X is O.

10. Use of siRNA, anti-HBV antibodies and nucleoside (acid) analogs that inhibit the expression of HBV transcripts in the preparation of a medicament for treating chronic HBV infection or hepatitis D virus (HDV) infection, wherein: (a) the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and (ii) the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively; and (b) the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3', wherein: (i) a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively; (ii) Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (iii) (Agn) is adenosine glycol nucleic acid (GNA); (iv) s is a phosphorothioate linkage; and (v) L96 is 11. The method according to claim 10, wherein the nucleoside (acid) analog is tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir, telbivudine, AGX-1009, emtricitabine, clavudine, lobcavir, famciclovir, ganciclovir, besfovir or tenofovir-exaliades.

12. A kit comprising: (a) A pharmaceutical composition comprising an RNAi agent targeting mRNA encoded by an HBV gene and a pharmaceutically acceptable excipient, wherein the RNAi agent is an siRNA having a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3', wherein: (i) a, c, g, and u are 2′-O-methyladenosine-3′-phosphate, 2′-O-methylcytidine-3′-phosphate, 2′-O-methylguanosine-3′-phosphate, and 2′-O-methyluridine-3′-phosphate, respectively; (ii) Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (iii) (Agn) is adenosine glycol nucleic acid (GNA); (iv) s is a phosphorothioate linkage; and (v) L96 is and (b) A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and (ii) the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

13. The kit according to claim 12, wherein the anti-HBV antibody comprises: (a) Light chain variable domain (V L ), which has at least 90%, at least 95% or 100% identity with the amino acid sequence shown in SEQ ID NO: 59; and (b) a heavy chain variable domain (V H ), which is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:

53.

14. The kit according to claim 12, wherein the anti-HBV antibody comprises: (a) light chain variable domain according to SEQ ID NO: 59 (V L ) amino acid sequence; and (b) a heavy chain variable domain (V H ) amino acid sequence.

15. The kit according to claim 12, wherein the anti-HBV antibody comprises: (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:73; and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:

71.

16. The kit according to claim 12, wherein the anti-HBV antibody comprises: (a) the light chain amino acid sequence according to SEQ ID NO: 73; and (b) the heavy chain amino acid sequence according to SEQ ID NO:

71. The kit according to claim 12 , wherein the HBV antibody is a monoclonal antibody.

18. The kit of claim 12, wherein the anti-HBV antibody is a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating immune effectors.

19. The kit of claim 18, wherein the second specificity stimulates cytotoxicity or a vaccine effect.

20. The kit according to claim 12, wherein the siRNA is conjugated to the L96 as shown in the following structure: Where X is O.

21. The kit according to claim 12, further comprising a pharmaceutical composition comprising a nucleoside (acid) analog.

22. The kit according to claim 21, wherein the nucleoside (acid) analog is tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir, telbivudine, AGX-1009, emtricitabine, clavudine, lobcavir, famciclovir, ganciclovir, besfovir or tenofovir-exaliades.

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