Combination HBV therapy

Abstract

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

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980092568.1, filed on December 19, 2019, entitled "Combined HBV Therapy". Chinese Patent Application No. 201980092568.1 is a Chinese national phase application with international application No. PCT / US2019 / 067643, which claims priority to U.S. Provisional Patent Application No. 62 / 782,896, filed on December 20, 2018, the entire contents of which are incorporated herein by reference.

[0002] Statements about sequence lists

[0003] The sequence listings related to this application are provided in text format instead of a paper copy and are incorporated herein by reference. The name of the text file containing the sequence listings is 930485.401WO_SEQUENCE_LISTING.txt. This text file is 111KB in size, created on December 17, 2019, and is being submitted electronically via EFS-Web. Background Technology

[0004] More than 400 million people worldwide have chronic HBV infection (CHB), thus increasing their risk of 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 CHB patients have shown a 5-year cumulative incidence of 8% to 20% in developing cirrhosis and approximately 20% in developing decompensated liver function. The global incidence of HCC has increased and it is now the third leading cause of cancer-related deaths worldwide (El-Serag HB and Rudolph KL, Gastroenterology 132:2557–76 (2007)).

[0005] HBV is a DNA virus with a lipid envelope and an icosahedral nucleocapsid that encapsulates the viral DNA genome and DNA polymerase. The HBV capsid is formed in the cytosol of infected cells during the packaging of the pre-RNA genome replication complex and consists of a core protein (also known as HBcAg) and its cleavage variant, HBeAg. When the viral DNA separates from the capsid upon entering a new cell, it can be converted into covalently closed circular DNA (cccDNA), which may remain in hepatocytes after HBV treatment and potentially reactivate the infection. The lipid envelope contains hepatitis B surface antigen (HBsAg), which refers to three distinct proteins: S-HBsAg (small antigen), M-HBsAg (medium antigen), and L-HBsAg (large antigen), encoded by the same open reading frame but using 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.

[0006] In addition to producing viral particles, HBV also leads to the production of subviral particles (SVPs), which contain the lipid envelope of HBV viral particles but lack replication capacity and typically lack a nucleocapsid. SVPs can produce amounts exceeding 3-4 logs of replicable virions. The pre-set high levels of HBsAg on SVPs deplete HBsAg-specific T-cell responses, which may be a significant factor contributing to the immune system's inability to clear HBV infection during chronic hepatitis B (Chisari, FV, et al., Pathologie Biologie 58:258-66 (2010)).

[0007] The natural evolution of CHB infection comprises four consecutive phases: (1) an early “immune tolerance” phase, associated with high levels of viral replication and minimal liver inflammation; (2) an immune-responsive phase, associated with significant liver inflammation and elevated serum transaminases; some patients progress to (3) a “non-replicating” or “inactive” phase, associated with: seroconversion to anti-HBe; undetectable or low-level viremia (less than 2000 IU / ml by PCR-based assays); and recovery 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 the production of HBeAg but do not impede viral replication, 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.

[0008] The primary goal of current HBV treatments is to permanently suppress HBV replication and improve liver disease. Important short-term goals include achieving HBeAg seroconversion, normalizing serum ALT and AST levels, 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, current HBV treatments cannot completely eliminate the virus. However, treatment-induced serum HBsAg clearance is a marker of termination of chronic HBV infection and is associated with optimal long-term outcomes.

[0009] Although all three major HBV proteins (HBsAg, HBeAg, and HBcAg) have immunosuppressive properties, HBsAg constitutes the vast majority of circulating HBV proteins in HBV-infected individuals. Furthermore, while HBeAg removal (through seroconversion) or a reduction in seroviremia is not associated with the development of sustained control of HBV infection after treatment, the removal of serum HBsAg (and seroconversion) from the blood in HBV infection is a recognized prognostic indicator of antiviral response during treatment, which leads to control of HBV infection after treatment (although this occurs only in a small percentage of patients receiving immunotherapy). Therefore, HBsAg removal may be an important strategy for overcoming viral immunosuppression in HBV-infected individuals.

[0010] Current standard treatments for HBV include interferon- or thymosin alpha 1-based immunotherapy and suppression of viral production by inhibiting HBV polymerase. HBV polymerase inhibitors are effective in reducing viral production but have little effect on rapidly reducing HBsAg, or can only slowly reduce HBsAg in a limited number of patients with long-term treatment (as in the case of tenofovir disoproxil fumarate). Interferon-based immunotherapy can reduce viral production and clear HBsAg from the blood early, but is only effective in a small percentage of treated subjects. The generally accepted role of HBsAg in the blood is to isolate anti-HBsAg antibodies and allow infectious viral particles to evade immune detection, which may be one reason why HBV infection remains chronic. 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 on preventing patients from achieving immune control of their HBV infection.

[0011] Currently available treatments have not restored immune control against HBV in most patients. Therefore, there remains a need for effective treatments for HBV infection that can suppress viral replication and restore immune control in most patients. Summary of the Invention

[0012] In some embodiments, this disclosure provides an HBV gene expression inhibitor for treating chronic HBV infection in a subject, wherein an anti-HBV antibody is subsequently administered to the subject.

[0013] This disclosure also provides a medicament for treating chronic HBV infection in a subject by reducing HBV antigen load, wherein an anti-HBV antibody is subsequently administered to the subject.

[0014] This disclosure also provides a composition for treating chronic HBV infection in a subject, wherein (a) the composition comprises an anti-HBV antibody and the subject has previously been administered an inhibitor of gene expression; or (b) the composition comprises an anti-HBV antibody and the subject has previously been administered an agent that reduces HBV antigen load.

[0015] In some embodiments, this disclosure provides the use of HBV gene expression inhibitors and anti-HBV antibodies in the preparation of medicaments for treating chronic HBV infection. This disclosure also provides the use of agents for reducing HBV antigen load and anti-HBV antibodies in the preparation of medicaments for treating chronic HBV infection.

[0016] In some embodiments, this disclosure provides a method for treating chronic HBV infection in a subject in need, comprising: administering an agent to the subject to reduce HBV antigen load; and administering an anti-HBV antibody to the subject. This disclosure also provides a method for treating chronic HBV infection in a subject in need, comprising: administering an HBV gene expression inhibitor to the subject; and administering an anti-HBV antibody to the subject.

[0017] In some of the methods, compositions or uses described herein, the anti-HBV antibody is HBC34 or a non-natural variant of HBC34.

[0018] In some of the methods, compositions or uses described herein, the agent for reducing HBV antigen load or the HBV gene expression inhibitor is an RNAi agent (e.g., siRNA, such as HBV02).

[0019] In some embodiments, this disclosure also provides kits comprising RNAi agents and anti-HBV antibodies as disclosed herein, and optionally instructions for carrying out the methods described herein. Attached Figure Description

[0020] Figure 1The dosing regimen for the combination therapy study in a mouse HBV model (AAV-HBV model) is described. Entecavir was administered orally once daily. At the start of the study, HBV-specific siRNA was injected subcutaneously once, followed by intraperitoneal administration of chimeric anti-HBV antibodies twice weekly during weeks three and four. A subset of mice was sacrificed at week four, and another subset was sacrificed at week six.

[0021] Figure 2A The results of HBV viral load determination in mouse serum samples using HBV DNA copy number as an indicator are described. Mice were treated with HBV02 siRNA (square, solid line); mouse chimeric HBC34 antibody (HBC34v7; 15 mg / kg) (circle, solid line); or saline (triangle, solid line).

[0022] Figure 2B The results of HBV viral load assay in mouse serum samples using HBV DNA copy number as an indicator are described, with mice treated using control siRNA and control antibody (square, dashed line); entecavir alone (“ETV”, diamond, dashed line); HBV02 siRNA and mouse chimeric HBC34v7 antibody (15 mg / kg) (circle, dashed line); or HBV02 siRNA, HBC34v7 antibody (15 mg / kg) and entecavir (triangle, solid line).

[0023] Figure 3A The study describes the HBsAg levels measured from mouse serum after treatment with HBV02 siRNA (square, solid line); mouse chimeric HBC34v7 antibody (15 mg / kg) (circle, solid line); or saline (triangle, solid line).

[0024] Figure 3B The study describes HBsAg levels measured from mouse serum after treatment with control siRNA and control antibody (squares, dashed lines); entecavir monotherapy (“ETV”, diamonds, dashed lines); HBV02 siRNA and mouse chimeric HBC34v7 antibody (15 mg / kg) (circles, dashed lines); or HBV02 siRNA, mouse chimeric HBC34v7 antibody (15 mg / kg) and entecavir (triangles, dashed lines).

[0025] Figure 4 The levels of free antibodies measured in mouse serum between days 14 and 42 following siRNA administration are described. The following treatment groups are described: mouse chimeric HBC34v7 antibody alone (circle); HBV02 siRNA, mouse chimeric HBC34v7 antibody, and entecavir (square); and HBV02 siRNA and mouse chimeric HBC34v7 antibody (triangle).

[0026] Figure 5A The results of HBV viral load determination in mouse serum samples, using HBV DNA copy number as an indicator, following treatment with siRNA, antibody, and / or control are described. Mice were injected with AAV / HBV virus on day -28. On day 0, AAV / HBV-infected C57Bl / 6 mice were administered one of 11 different treatments: (1) HBV-specific siRNA (HBV02, with 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 doses of whole-mouse anti-HBV antibody HBC34 (HBC34v35); (10) control siRNA and control antibody; or (11) PBS alone, administered intraperitoneally. The results of treatments 1-5, 10, and 11 are shown.

[0027] Figure 5B The results of HBV viral load determination in mouse serum samples, using HBV DNA copy number as an indicator, following treatment with siRNA, antibody, and / or control are described. Mice were injected with AAV / HBV virus on day -28. On day 0, AAV / HBV-infected C57Bl / 6 mice were administered one of 11 different treatments: (1) HBV-specific siRNA (HBV02, with 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 doses of whole-mouse anti-HBV antibody HBC34 (HBC34v35); (10) control siRNA and control antibody; or (11) PBS alone, administered intraperitoneally. The results of treatments 1 and 6-11 are shown.

[0028] Figure 6A The levels of HBsAg measured from mouse serum after treatment with siRNA, antibodies, and / or controls are described above. Figure 5A As described in [the text].

[0029] Figure 6B The levels of HBsAg measured from mouse serum after treatment with siRNA, antibodies, and / or controls are described above. Figure 5B As described in [the text].

[0030] Figure 7A HBeAg levels measured from mouse serum after treatment with siRNA, antibodies, and / or controls are described above. Figure 5A As described in [the text].

[0031] Figure 7B HBeAg levels measured from mouse serum after treatment with siRNA, antibodies, and / or controls are described above. Figure 5B As described in [the text].

[0032] Figure 8 The experimental design for the study described in Example 3 is shown, including the dosing regimen used to assess HBsAG serum clearance and viral entry inhibition in a mouse model after treatment with anti-HBV antibody 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-rat HBC34v35) administered intraperitoneally twice weekly for two to three weeks; or (5–7) a single subcutaneous administration of HBV-specific siRNA (HBV02, with the antisense strand of SEQ ID NO:8; see description in Example 1) at the start of the study, and two intraperitoneal administrations of whole-rat HBC34v35 twice weekly for two to three weeks at one of the three antibody doses. Mice were sacrificed at week 6.

[0033] Figure 9 The study showed the serum HBV DNA concentration in SCID mice that had undergone primary human hepatocyte transplantation, following treatment with PBS (control); HBC34v35 antibody; or HBV34v35 antibody and HBV02 siRNA.

[0034] Figure 10 The study showed the serum HBsAg concentrations in SCID mice that had undergone primary human hepatocyte transplantation, following treatment with PBS (control); HBC34v35 antibody; or HBV34v35 antibody and HBV02 siRNA.

[0035] Figure 11 The study showed serum HBeAg concentrations in SCID mice that had undergone primary human hepatocyte transplantation, following treatment with PBS (control); HBC34v35 antibody; or HBV34v35 antibody and HBV02 siRNA.

[0036] Figure 12The study showed the serum HBcrAg concentration in SCID mice that had undergone primary human hepatocyte transplantation, following treatment with PBS (control); HBC34v35 antibody; or HBV34v35 antibody and HBV02 siRNA.

[0037] Figure 13 The treatment regimen designed for a phase 2 study to evaluate the efficacy of siRNA antibody combination therapy in the treatment of HBV is described. Detailed Implementation

[0038] This disclosure provides methods and compositions, as well as related kits, for treating hepatitis B virus (HBV) infection using HBV protein expression inhibitors and anti-HBV antibodies. This combination therapy can be used to treat chronic hepatitis B (CHB).

[0039] In some embodiments, the method includes treating HBV infection in a subject in need by: (i) administering an HBV gene expression inhibitor to the subject; and (ii) administering an anti-HBV antibody to the subject. In a particular embodiment, after administration of the HBV gene expression inhibitor, the expression of at least one HBV gene is reduced, and when the expression of at least one HBV gene is reduced, the anti-HBV antibody is administered to the subject.

[0040] In some embodiments, the HBV gene expression inhibitor is an RNAi agent that inhibits the expression of HBV transcripts. In a particular embodiment, the RNAi agent is siRNA (also referred to herein as "double-stranded RNA" or "dsRNA") that targets and inhibits the expression of mRNA encoded by the HBV X gene.

[0041] In some embodiments, the anti-HBV antibody recognizes HBV genotypes A, B, C, D, E, F, G, H, I, and J; and / or a human antibody. In a particular embodiment, the anti-HBV antibody is selected from: wild-type HBC34 antibody, a non-natural variant of HBC34 antibody, and / or HBC24 antibody.

[0042] In some embodiments described herein, HBV gene expression inhibitors and anti-HBV antibodies work synergistically to reduce viral load and circulating HBsAg. This combination therapy can provide a functional cure for chronic HBV and allows for the administration of lower doses of antibodies, thereby reducing the likelihood of antibody-induced toxicity.

[0043] I. Glossary

[0044] The following sections provide a detailed description of HBV combination therapies, including: HBV protein expression inhibitors; anti-HBV antibodies; methods for treating subjects using a combination of HBV protein expression inhibitors and anti-HBV antibodies; and kits associated with the combination therapies. Before elaborating on this disclosure in more detail, definitions of certain terms used herein may aid in understanding. Other definitions are set forth throughout this disclosure.

[0045] In this specification, unless otherwise stated, the term "about" means ±20% of the range, value, or structure shown.

[0046] The term "comprising" means the presence of the features, elements, steps, or components mentioned in the claims, but does not exclude the presence or addition of one or more other features, elements, steps, components, or groups thereof. The term "consistent essentially of" limits the scope of the claims to specific materials or steps, and those specific materials or steps that do not materially affect the basis and novel features of the claimed invention.

[0047] 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 may be used synonymously with “and / or”. As used herein, the terms “comprising” and “having” are used synonymously, and these terms and their variations are intended to be interpreted as non-limiting.

[0048] The word “substantially” does not exclude “completely”; for example, a composition that is “substantially free” of Y may be completely free of Y. The word “substantially” may be omitted from the definitions provided herein if necessary.

[0049] As used herein, the term “disease” is intended to be generally synonymous with and interchangeable with the terms “symptom” and “condition” (as in medical condition) because they all reflect an abnormal condition of the human or animal body or one of the parts that impairs normal function, usually manifested as distinguishable signs and symptoms, and resulting in a duration or decline in quality of life in humans or animals.

[0050] As used herein, the terms “peptide,” “polypeptide,” and “protein,” and variations thereof, refer to molecules, particularly peptides, oligopeptides, polypeptides, or proteins, including fusion proteins, that comprise at least two amino acids linked together by normal peptide bonds or by modified peptide bonds, for example, in the case of isotropic peptides. For example, a peptide, polypeptide, or protein may consist of amino acids selected from the 20 amino acids defined by the genetic code, linked together by normal peptide bonds (“classical” polypeptides). Peptides, polypeptides, or proteins may consist 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 mimic or antagonize the biological effects of the natural parent peptide. Peptide mimics lack classic peptide characteristics, such as enzymatic cleavage of peptide bonds. In particular, in addition to these amino acids, a peptide, polypeptide, or protein may contain amino acids other than the 20 amino acids defined by the genetic code, or it may consist of amino acids other than the 20 amino acids defined by the genetic code. In particular, peptides, polypeptides, or proteins in the context of this disclosure can also be composed of amino acids modified by natural processes, such as post-translational maturation or chemical processes, as is well known to those skilled in the art. Such modifications are described in detail in the literature. These modifications can occur anywhere on the polypeptide: the peptide backbone, the amino acid chain, and even the carboxyl or amino terminus. Specifically, peptides or polypeptides can branch or form rings with or without branching after ubiquitination. This type of modification can be the result of natural or synthetic post-translational processes known to those skilled in the art. In the context of this disclosure, the terms "peptide," "polypeptide," or "protein" also include, in particular, modified peptides, polypeptides, and proteins. For example, peptide, polypeptide, or protein modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of nucleotides or nucleotide derivatives, covalent fixation of lipids or lipid derivatives, covalent fixation of phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, glycosylation including polyethylene glycolation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processes, phosphorylation, isopreneation, racemization, seneloylation, sulfation, amino acid addition such as arginination, or ubiquitination.Such modifications are well described in the literature (Proteins Structure and Molecular Properties, 2nd Ed., TECreighton, New York (1993); Post-translational Covalent Modifications of Proteins, BC Johnson, Ed., Academic Press, New York (1983); Seifter et al., Analysis for protein modification and nonprotein cofactors, Meth. Enzymol. 182:626-46 (1990); and Rattan et al., Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci.

[0051] 663:48-62 (1992)). Therefore, the terms “peptide,” “polypeptide,” and “protein” include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, etc.

[0052] 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., chains of one or more amino acid monomers linked by peptide bonds as explained above. In a particular embodiment, a protein according to this disclosure comprises 1, 2, 3, or 4 polypeptides.

[0053] As used herein, the term "recombinant" (e.g., recombinant antibody, recombinant protein, recombinant nucleic acid, etc.) refers to any molecule (antibody, protein, nucleic acid, siRNA, etc.) prepared, expressed, formed, or isolated by recombination and which is not naturally occurring. As used herein, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably and are intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded. In a particular embodiment, the nucleic acid molecule is double-stranded RNA.

[0054] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all of these names include progeny. Therefore, the terms “transformant” and “transformed cell” include the primary test cells and the cultures derived from them, regardless of the number of transductions. It should also be understood that the DNA content of all progeny may not be exactly the same due to intentional or unintentional mutations. This includes variant progeny with the same function or biological activity as those selected from the initially transformed cells. Where different names are intended to be used, the context will be clear.

[0055] As used herein, the term "sequence variant" refers to any sequence that has one or more alterations 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. Therefore, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. For sequence variants in a nucleotide sequence context, the reference sequence is also a nucleotide sequence, and for sequence variants in an amino acid sequence context, 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 the reference sequence. Unless otherwise stated, sequence identity is generally calculated based on the full length of the reference sequence (i.e., the sequence described in the application). Percentage identity as referred to herein may, for example, be determined using BLAST with the default parameters specified by NCBI (National Center for Biotechnology Information).

[0056] [http: / / www.ncbi.nlm.nih.gov / ][Blosum 62 matrix; gap opening penalty = 11 and gap extension penalty = 1]. A “sequence variant” in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more nucleotides in the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by standard single-letter names (A, C, G, or T). Due to the degeneracy of the genetic code, a “sequence variant” of a nucleotide sequence can result in a change to the corresponding reference amino acid sequence, i.e., an amino acid “sequence variant” or no change. In some embodiments, a nucleotide sequence variant is a variant that does not result in a variant that produces an amino acid sequence variant (i.e., a silent mutation). However, nucleotide sequence variants that result in “non-silent” mutations are also within the scope, particularly such nucleotide sequence variants that produce 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. A “sequence variant” in the context of an amino acid sequence has an altered sequence in which one or more amino acids are deleted, substituted, or inserted compared to the reference amino acid sequence. 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, for every 100 amino acids of the reference sequence, the variant sequence has no more than 10 alterations (i.e., any combination of deletions, insertions, or substitutions) that are “at least 90% identical” to the reference sequence.

[0057] In some embodiments, while non-conservative amino acid substitutions may be possible, the substitutions are conservative amino acid substitutions, wherein the substituted amino acid has a similar structure or chemical properties to the corresponding amino acid in the reference sequence. For example, conservative amino acid substitution involves replacing an aliphatic or hydrophobic amino acid (e.g., alanine, valine, leucine, and isoleucine) with another; replacing a hydroxyl-containing amino acid (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.

[0058] Amino acid sequence insertions include the fusion of amino and / or carboxyl ends of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion of the N-terminus or C-terminus of an amino acid sequence with a reporter molecule or enzyme.

[0059] Unless otherwise stated, alterations in sequence variants do not eliminate the function of the corresponding reference sequence. For example, in this case, the sequence of an anti-HBV antibody or an HBV gene expression inhibitor (e.g., siRNA) is functionally sufficient to neutralize HBV infection or reduce HBV protein expression, respectively. Guidelines for determining which nucleotide and amino acid residues can be substituted, inserted, or deleted without eliminating this function can be found using computer programs known in the art.

[0060] As used herein, “derived from” refers to the source of a nucleic acid, peptide, polypeptide, or protein nucleic acid, or amino acid sequence. In some embodiments, a nucleic acid or amino acid sequence derived from a particular sequence has a substantially identical amino acid sequence to the sequence from which it is derived, or a portion thereof, wherein “substantially identical” includes sequence variants as defined above. In some embodiments, a nucleic acid or amino acid sequence derived from a particular peptide or protein is derived from a corresponding domain in that particular peptide or protein. Therefore, “corresponds to” specifically refers to the same function. For example, “extracellular domain” corresponds to another “extracellular domain” of another (another protein), or “transmembrane domain” corresponds to another “transmembrane domain” of another (another protein). Therefore, the portion “corresponding to” a peptide, protein, or nucleic acid is identifiable by one of ordinary skill in the art. Similarly, sequences “derived from” other sequences are generally identifiable by one of ordinary skill in the art because they contain their source in the sequence.

[0061] In some embodiments, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the original nucleic acid, peptide, polypeptide, or protein from which it is derived. However, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations relative to the original nucleic acid, peptide, polypeptide, or protein from which it is derived. In particular, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a variant of the functional sequence of the original nucleic acid, peptide, polypeptide, or protein from which it is derived, as described above. For example, in a peptide / protein, one or more amino acid residues may be substituted by other amino acid residues or one or more amino acid residues may be inserted or deleted.

[0062] As used herein, the term “mutation” refers to an alteration in a nucleic acid sequence and / or amino acid sequence compared to a reference sequence (e.g., the corresponding genomic sequence). For example, a mutation compared to a genomic sequence can be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation (e.g., induced by enzymes, chemicals, or radiation), or a mutation obtained through site-directed mutagenesis (a molecular biological method for preparing specific and deliberate alterations in nucleic acid and / or amino acid sequences). Therefore, the terms “mutation” or “mutating” should be understood to also include, for example, the physical creation of mutations in a nucleic acid or 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 a mutation in an amino acid sequence, the mutation can be introduced into the nucleotide sequence encoding that amino acid sequence to express a (recombinant) mutated polypeptide. Mutations can be achieved, for example, by altering (e.g., by site-directed mutagenesis) the codons of a nucleic acid molecule that encodes an amino acid to produce codons that encode a different amino acid, or by synthesizing sequence variants (e.g., by knowing the nucleotide sequence of a nucleic acid molecule that encodes a polypeptide and by designing the synthesis of a nucleic acid molecule that contains the nucleic acid sequence encoding that polypeptide variant) without mutating one or more nucleotides of the nucleic acid molecule.

[0063] 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 a coding sequence are typically determined by an open reading frame, which usually begins with the ATG start codon.

[0064] As used in this article, the term “expression” refers to any step involved in the production of a polypeptide, including transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0065] In some aspects, this disclosure relates to the use of HBV gene expression inhibitors. As used herein, an "HBV gene expression inhibitor" is any agent that causes 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 (where the HBV gene is transcribed and has been treated with an HBV gene expression inhibitor such that the expression of the HBV gene has been maintained) compared to a second cell or cell population (which is substantially the same as the first cell or cell population but has not been treated in this way (control cells)). In some embodiments, the HBV gene expression inhibitor is an RNAi agent (e.g., siRNA). HBV gene expression can be measured by methods known in the art. Unless otherwise stated, "HBV gene expression" as used herein refers to protein expression determined using rtPCR or by measuring protein expression using enzyme-linked immunosorbent assay (ELISA) or immunohistochemistry.

[0066] In some aspects, this disclosure relates to the use of agents for reducing HBV antigen load. As used herein, "agent for reducing HBV antigen load" means any agent that results in a reduction in the amount of HBV antigen that can be isolated or detected from a first cell or cell population (treated with said agent) compared to a second cell or cell population (substantially identical to the first cell or cell population but not treated in this way (control cells)). In some embodiments, the agent for reducing HBV antigen load is an RNAi agent (e.g., siRNA). Antigen load can be measured by methods known in the art. Unless otherwise stated, "HBV antigen complex" as used herein refers to determination by measuring the amount of antigen (e.g., HBsAg) using an ELISA.

[0067] This disclosure provides combination therapies for treating HBV, including anti-HBV antibodies. In some embodiments, the anti-HBV antibody or its antigen-binding fragment binds to the antigenic loop region of HBsAg and neutralizes hepatitis B virus infection.

[0068] As used herein, the term "antibody" encompasses a wide range of 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 they retain the characteristic properties of the antibody. In some embodiments, the antibody is a human antibody and / or a monoclonal antibody. In certain embodiments, the antibody is a human monoclonal antibody. In some specific embodiments, the antibody is a recombinant human monoclonal antibody. As used herein, the terms "antigen-binding fragment," "fragment," and "antibody fragment" are used interchangeably to refer to any fragment of an antibody in 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 the antibody and its antigen-binding fragment.

[0069] As used herein, a “neutralizing antibody” is an antibody that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of a pathogen to initiate and / or persist 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 following appropriate formulations, in conjunction with active vaccination, as diagnostic tools, or as manufacturing tools as described herein.

[0070] Human antibodies are well known in the existing field of 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 library or selected human antibodies after immunization in the absence of endogenous immunoglobulin production. Transferring human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies after antigen challenge (see, for example, 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)). Techniques by 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 using modified EBV-B cell immortalization as described in Traggiai, E. et al. (Nat Med. 10(8):871-5 (2004)). As used herein, the term "human antibody" also includes, for example, antibodies that are modified in variable regions to produce properties as described herein.

[0071] The antibody in the combination therapy can be any isotype (e.g., IgA, IgG, IgM, i.e., κ, γ, or μ heavy chains), but in certain specific embodiments, the antibody is IgG. Within the IgG isotype, the antibody can be an IgG1, IgG2, IgG3, or IgG4 subclass. In a specific embodiment, the antibody is IgG1. The antibody in the combination therapy can have a κ or λ light chain. Based on the antigen-independent uptake of IgG into hepatocytes via 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 viral particles and HBsAg.

[0072] As used in this article, the term "variable region" (light chain variable region (V)) L ), heavy chain variable region (V H The term "complementarity-determining region" ("CDR") refers to a portion of the antibody light chain (LC) or heavy chain (HC) (typically around the 105-120th amino acid terminus of the mature antibody heavy or light chain), containing a complementarity-determining region ("CDR") and a framework region ("FR"), and directly involved in antibody-antigen binding. The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariant region" or "HVR" and refer, as is known in the art, to a non-adjacent sequence of amino acids within the variable region of an antibody that confers antigen specificity and / or binding affinity. Typically, there are three CDRs in each variable region of an immunoglobulin-binding protein; for example, for antibodies, 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 Immunogenetic Information System (IMGT), and Aho), which allow for the identification of equivalent positions and comparison of different molecules using Antigen Receptor Numbering and Receptor Classification (ANARCI) software tools (Bioinformatics 15:298-300 (2016)). It should be understood that in some embodiments, the antibody or antigen-binding fragment of this 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.

[0073] In some embodiments, the anti-HBV antibody, or its antigen-binding fragment, of the combination therapy according to this disclosure is a purified antibody, single-chain antibody, Fab, Fab', F(ab')2, Fv, or scFv. Therefore, the antibody for combination therapy can be a human antibody, monoclonal antibody, human monoclonal antibody, recombinant antibody, and / or purified antibody. This disclosure also provides fragments of antibodies, particularly 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 this disclosure may explicitly refer to one or more antigen-binding fragments of an antibody, one or more antibody fragments, one or more variants, and / or one or more derivatives, as used herein, the term "antibody" or "antibody for combination therapy" includes all classes of antibodies, i.e., one or more antigen-binding fragments of an antibody, one or more antibody fragments, one or more variants, and one or more derivatives.

[0074] Antibody fragments can be obtained from antibodies by methods including digestion with enzymes (such as pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Alternatively, antibody fragments can be obtained by cloning and expressing partial sequences of the heavy or light chain. This disclosure also covers single-chain Fv fragments (scFvs) of the heavy and light chains of antibodies derived from this disclosure. For example, this disclosure includes scFvs containing a CDR of an antibody derived from this 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, such as single-chain Fvs, in which the variable domains of the heavy and light chains are linked by peptide linkers.

[0075] The antibody fragments disclosed herein can be conferred with monovalent or multivalent interactions and are contained in various structures as described above. For example, scFv molecules can be synthesized to produce trivalent “trivalent” or tetravalent “tetravalent” antibodies. scFv molecules may contain domains of the Fc region that result in the production of bivalent microantibodies. Furthermore, the sequence of the antibody / antibody fragment can be a component of a multispecific molecule, wherein said sequence targets expression as described herein, and other regions of said multispecific molecule bind to other targets. Exemplary multispecific molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and dimeric antibodies (Holliger and Hudson, Nature Biotechnology 9:1126-36 (2005)).

[0076] The antibodies according to this disclosure may be provided in purified form. Typically, the antibodies will be present in a composition that substantially does not contain other peptides, for example, in which less than 90% (by weight), typically less than 60%, and more typically less than 50% of the composition is composed of other peptides.

[0077] In embodiments, the antibody and antigen-binding fragments of this disclosure may be multispecific (e.g., bispecific, trispecific, tetraspecific, etc.) and may be provided in any multispecific form as disclosed herein. In some embodiments, the antibody or antigen-binding fragment of this disclosure is a multispecific antibody, such as a bispecific or trispecific antibody. Bispecific antibodies are disclosed, for example, in Spiess et al. (Mol. Immunol. 67(2):95(2015)) and Brinkmann and Kontermann (mAbs In 9(2):182-212(2017)), the bispecific form and the method of preparing it are incorporated herein by reference and include, for example, bispecific T cell conjugates (BiTE), DART, Knobs-Into-Holes (KIH) assembly, scFv-CH3-KIH assembly, KIH common light chain antibody, TandAb, triplet, TriBi microsome, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCab, endosome, 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 combination of the HBV and / or HDV-specific binding domains of this disclosure with another such binding domain of this 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.

[0078] As used herein, the term "vaccine" is generally understood to be a prophylactic or therapeutic substance that provides at least one antigen or immunogen. An antigen or immunogen can be derived from any substance suitable for vaccination. For example, an antigen or immunogen can be derived from pathogens, such as bacterial or viral particles, or from tumor or cancerous tissue. An antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response. Specifically, an "antigen" or "immunogen" generally refers to a substance that can be recognized by the immune system (e.g., the 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 may contain a peptide or protein presented to T cells by the MHC.

[0079] Dosage is usually expressed relative to body weight. Therefore, a dosage expressed as [g, mg or other units] / kg (or g, mg, etc.) usually refers to [g, mg or other units] "per kg (or g, mg, etc.) of body weight", even if the term "body weight" is not explicitly mentioned.

[0080] As used herein, "hepatitis B virus," used interchangeably with the term "HBV," refers to the well-known non-cytopathic, hepatic DNA virus of the Hepatoviridae family. 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 "transcriptions"): C, X, P, and S. The core protein (HBcAg) is encoded by gene C. Hepatitis B e antigen (HBeAg) is produced through the proteolytic hydrolysis 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 within the frame, producing three polypeptides of different sizes, referred to as large, medium, and small S antigens (pre-S1+pre-S2+S, pre-S2+S, or S). In addition to decorating the HBV envelope, the surface antigen is also part of the subviral particle, which is produced in excess compared to the viral particle and plays a role in immune tolerance and chelation of anti-HBsAg antibodies, thus allowing infectious particles to evade immune detection. The function of the non-structural 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 (designated A through H) have been identified, and two additional genotypes, I and J, have been proposed, each with different geographical distributions. The term "HBV" includes any genotype of HBV (A through J). The complete coding sequence of the reference sequence of the HBV genome can be found, for example, in GenBank 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 accessions 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). Further examples of HBV mRNA sequences are readily available from publicly available databases such as GenBank, UniProt, and OMIM. The International Hepatitis B Virus Database can be accessed at http: / / www.hpa-bioinformatics.org.uk / HepSEQ / main.php. As used in this article, the term "HBV" also refers to naturally occurring DNA sequence variants of the HBV genome, namely genotypes A through J and their variants.

[0081] II. HBV protein expression inhibitors and delivery systems

[0082] This disclosure provides HBV protein expression inhibitors for combination therapies to treat HBV. In some embodiments, the HBV gene expression inhibitor is an RNAi agent. As used herein, the terms "RNA interfering agent" or "RNAi agent" refer to an RNA-containing agent containing the terms defined herein, which mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agents described herein affect the inhibition of HBV gene expression.

[0083] In one aspect, RNA interference agents comprise single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of that target RNA. Not wishing to be bound by any particular theory, long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is cleaved into siRNA by a type III endonuclease (called Dicer) (Sharp et al., Genes Dev. 15:485 (2001)). Dicer is a ribonuclease III-like enzyme that processes dsRNA into short interfering RNA (siRNA) of 19 to 23 base pairs with a characteristic two-base 3' overhang (Bernstein et al., Nature 409:363 (2001)). This siRNA is then introduced into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to guide target recognition (Nykanen et al., Cell 107:309 (2001)). When bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target site to induce silencing (Elbashir et al., Genes Dev. 15:188 (2001)). Therefore, in one aspect, the techniques described herein relate to a single-stranded RNA that promotes the formation of the RISC complex to influence the silencing of target genes.

[0084] Wherever it refers to the HBV gene, the terms “silencing,” “inhibiting its expression,” “downregulating its expression,” “suppressing its expression,” etc., in this document refer 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 (where 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 suppressed) compared to a second cell or cell population (which is substantially the same as the first cell or cell population but has not undergone such treatment (control cells)). The degree of inhibition can be measured, for example, by subtracting the level of mRNA expression in the treated cells from the level of mRNA expression in the control cells. Alternatively, the degree of inhibition can be given by a reduction in parameters that are functionally related to HBV gene expression, such as the amount of protein edited by the HBV gene, or data from cells exhibiting certain phenotypes, such as HBV infection phenotypes (e.g., HBV infection), HBV protein expression (e.g., hepatitis B surface antigen, HBsAg), or changes in cellular gene expression reflecting HBV gene expression (e.g., Smc5 / 6 expression and localization). The degree of suppression can also be measured using cells engineered to express a reporter gene that reflects HBV RNA expression. Theoretically, HBV gene silencing can occur in any cell expressing the HBV gene, such as HBV-infected cells or cells engineered to express the HBV gene, and can be detected by any appropriate assay.

[0085] The level of HBV RNA expressed by cells or cell populations, or the level of circulating HBV RNA, can be determined using any method known in the art for assessing mRNA expression, such as Example 2 provided in International Application Publication No. WO2016 / 077321A1 and the rtPCR method provided in U.S. Patent Application No. US2017 / 0349900A1, which are incorporated herein by reference. In some embodiments, the HBV gene expression level in a sample (e.g., total HBV RNA, HBV transcripts, e.g., 3.5 kb transcripts) is determined by detecting transcribed polynucleotides or portions thereof (e.g., HBV gene RNA). RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using phenol / guanidine isothiocyanate (RNAzol B; Biogenesis) and the RNeasy RNA preparation kit. Alternatively, PAXgene (PreAnalytix, Switzerland). Common detection methods 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 detected using the methods described in International Application Publication No. WO 2012 / 177906A1 and US Patent Application No. US2014 / 0275211A1, which are incorporated herein by reference.

[0086] As used herein, “target sequence” refers to an adjacent portion of the nucleotide sequence of the mRNA molecule formed during transcription of the HBV gene, including mRNA of the RNA processing product of the primary transcription product. The target portion of the sequence will be at least long enough to serve as a receptor for targeted RNAi cleavage at or near that portion. For example, the target sequence will typically be 9–36 nucleotides in length, e.g., 15–30 nucleotides in length, including all sub-ranges therein. As a non-limiting example, the target sequence can be 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.

[0087] As used herein, the term "chain containing a sequence" refers to an oligonucleotide containing a nucleotide chain, which is described using the sequence referred to by standard nucleotide nomenclature.

[0088] As used herein, and unless otherwise indicated, the term "complementarity" when used to describe the association of a first nucleotide sequence with a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double helix structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which 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 vivo. Those skilled in the art will be able to determine the optimal conditions for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides.

[0089] Complementary sequences within RNAi agents (e.g., within siRNA as described herein) comprise base pairings of an oligonucleotide or polynucleotide containing the first nucleotide sequence with an oligonucleotide or polynucleotide containing the second nucleotide sequence over a length of one or two nucleotide sequences. Such sequences may be referred to herein as “perfectly complementary” relative to each other. However, when the first sequence is referred to herein as “substantially complementary” relative to the second sequence, the two sequences may be perfectly complementary, or may hybridize with a double helix of up to 30 base pairs to form one or more, but typically no more than 5, 4, 3, or 2 mismatched base pairs, while retaining hybridization capacity under conditions most relevant to their final application (e.g., repression of gene expression via RISC pathways). However, when two oligonucleotides are engineered to form one or more single-stranded overhangs after hybridization, such overhangs should not be considered mismatches for the purpose of determining complementarity. For example, an siRNA comprising an oligonucleotide of length 21 and another oligonucleotide of length 23, wherein the longer oligonucleotide contains a sequence of 21 nucleotides that are perfectly complementary to the shorter oligonucleotide, and for the purposes described herein, the siRNA may still be referred to as “perfectly complementary”.

[0090] As used herein, “complementary” sequences may also include base pairs formed entirely from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides, provided that the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein pairings.

[0091] The terms “complementary,” “fully complementary,” and “substantially complementary” are used herein to refer to base pairing between the meaningful strand and the antisense strand of siRNA, or base pairing between the antisense strand and the target sequence of an RNAi agent, as will be understood from the context in which they are used.

[0092] As used herein, a polynucleotide that is “substantially complementary” to at least a portion of messenger RNA (mRNA) is a polynucleotide that is substantially complementary to an adjacent portion of the mRNA of interest (e.g., the mRNA encoding the 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.

[0093] a.siRNA

[0094] 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 the specific degradation of the desired target RNA via the RISC pathway, but will typically be in the range of 9 to 36 base pairs, for example, 15–30 splice pairs. Given that the double helix ranges from 9 to 36 splice pairs, the double helix can be of 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 subrange thereof, 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 produced in cells by treatment with Dicer and similar enzymes are typically in the range of 19 to 22 base pairs in length. The terms “double-stranded RNA” or “dsRNA” are used herein synonymously to refer to siRNAs as described above.

[0095] One strand of the double helix region of siRNA contains a sequence substantially complementary to the region of the target RNA. The two strands forming the double helix can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more different RNA molecules. When the double helix region is a double helix formed from a single molecule, that molecule may have a double helix region separated by a single nucleotide chain (referred to herein as a "hairpin loop") between the 3' end of one strand and the 5' end of the corresponding other strand forming the double helix. The hairpin loop may contain at least one unpaired nucleotide; in some embodiments, the hairpin loop may contain 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 strands of siRNA are composed of different RNA molecules, these molecules do not need to be, but may be, covalently linked. When the two strands are covalently linked in a manner other than a hairpin loop, the linking structure is referred to as a "connector."

[0096] The term “antisense strand” or “guide strand” refers to the strand in an RNAi agent, such as siRNA, that includes a region substantially complementary to the target sequence. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence) as defined herein. When the complementary region is not perfectly complementary to the target sequence, mismatches can occur in internal or terminal regions of the molecule.

[0097] Typically, the most tolerable mismatch is in the terminal region, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0098] As used herein, the term “sense chain” or “follower chain” refers to a chain of RNAi that includes regions substantially complementary to regions of the antisense chain (as defined herein).

[0099] In another aspect, the agent is a single-stranded antisense RNA molecule. This antisense RNA molecule may have 15 to 30 nucleotides complementary to the target. For example, the antisense RNA molecule may have a sequence of at least 15, 16, 17, 18, 19, 20, 21 or more adjacent nucleotides from an antisense sequence disclosed herein.

[0100] Those skilled in the art will recognize that the terms "RNA molecule" or "ribonucleic acid molecule" encompass not only RNA molecules expressed or found in nature, but also RNA analogs and derivatives comprising one or more ribonucleotide / ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, "ribonucleoside" includes a nucleoside base and ribose, while "ribonucleotide" refers to a ribonucleoside having one, two, or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleoside" as used herein are considered equivalent. RNA can be modified in its nucleobase structure or in its ribophosphate backbone structure, as described in more detail below. However, siRNA molecules containing ribonucleoside analogs or derivatives retain the ability to form a double helix. As a non-limiting example, the RNA molecule may also include at least one modified ribonucleotide, including but not limited to 2'-O-methyl modified ribonucleotides, ribonucleotides containing a 5'-thiophosphate group, terminal ribonucleotides linked to a cholesterol derivative or a dodecanoic acid bis(decanoic acid) group, locked ribonucleotides, baseless ribonucleotides, 2'-deoxy-2'-fluoro modified ribonucleotides, 2'-amino-modified ribonucleotides, 2'-alkyl-modified ribonucleotides, morpholino ribonucleotides, aminophosphates, or non-natural bases containing the ribonucleotide, or any combination thereof. Alternatively, the RNA molecule may contain at least two modified ribonucleotides, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, or more, up to the entire length of the siRNA molecule. For each of such multiple modified ribonucleotides in the RNA molecule, the modification need not be identical. In some embodiments, the modified RNA supplied for use in the methods and compositions described herein is considered to be a peptide nucleic acid (PNA) that has the ability to form the desired double helix structure and allows or mediates the specific degradation of the target RNA via the RISC pathway.

[0101] In some embodiments, the modified ribonucleosides include deoxyribonucleosides. For example, an RNAi agent may contain one or more deoxyribonucleosides, including, for example, one or more deoxyribonucleosides overhangs, or one or more deoxyribonucleosides within the double-stranded portion of siRNA. However, as used herein, the term "RNAi agent" does not include a complete DNA molecule.

[0102] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double helix structure of an RNAi agent (e.g., siRNA). For example, a nucleotide overhang exists when the 3' end of one strand of siRNA extends beyond the 5' end of the other strand, or vice versa. siRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two, three, four, five, or more nucleotides. The nucleotide overhang may contain or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. One or more overhangs may be located on the sense strand, antisense strand, or any combination thereof. Additionally, one or more nucleotides of the overhang may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the siRNA.

[0103] In some embodiments, the antisense strand of the siRNA has a 1-10 nucleotide overhang at the 3' and / or 5' ends. In some embodiments, the sense strand of the siRNA has a 1-10 nucleotide overhang at the 3' and / or 5' ends. In some other embodiments, one or more nucleotides in the overhang are replaced by nucleoside phosphate thioesters.

[0104] In some embodiments, the siRNA has a dangling overhang of 1 to 4 single-stranded nucleotides at at least one end, typically 1 or 2 nucleotides. siRNAs with a dangling overhang of at least one nucleotide can exhibit unexpectedly superior repressive properties relative to their blunt-terminal counterparts.

[0105] As used in this article with reference to siRNA, the term "blunt-ended" or "knuckle-end" 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 an siRNA can be blunt-ended. When both ends of an siRNA are blunt-ended, the siRNA is called "blunt-ended." A "blunt-ended" siRNA is an siRNA with blunt ends, meaning there are no nucleotide overhangs at either end of the molecule. Most commonly, such a molecule will be double-stranded throughout its entire length.

[0106] In some embodiments, the combination therapies described herein comprise one or more RNAi agents that inhibit HBV gene expression. In some embodiments, an RNAi agent comprising short interfering ribonucleic acid (siRNA) is used to inhibit HBV gene expression in mammals (e.g., humans infected with HBV), wherein the siRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during HBV gene expression, and wherein the complementary region is 30 nucleotides long or less, typically 19 to 24 nucleotides long, and, as detected by, for example, PCR or branched DNA (bDNA)-based methods, or by, for example, protein-based methods such as, Western blotting, when the siRNA comes into contact with cells expressing the HBV gene, inhibits HBV gene expression by at least 10%. The expression of HBV genes in cells that express them in cell culture or 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 biological samples from the 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).

[0107] siRNA consists of two complementary RNA strands that hybridize under the conditions used to form a double helix. One strand of the siRNA (the antisense strand) includes a complementary region that is substantially complementary to, and usually perfectly complementary to, the target sequence. The target sequence may be derived from the sequence of mRNA formed during the expression of the HBV gene. The other strand (the sense strand) includes a region complementary to the antisense strand, such that when the two strands are combined under suitable conditions, they hybridize and form a double helix. Typically, the double helix has between 15 and 30 base pairs (including the end values), more commonly between 18 and 25 base pairs (including the end values), and even more commonly between 19 and 24 base pairs (including the end values), and is typically between 19 and 21 base pairs in length (including the end values). Similarly, the complementary region of the target sequence is between 15 and 30 nucleotides (including the end value), more typically between 18 and 25 nucleotides (including the end value), and even more typically between 19 and 24 nucleotides (including the end value), and is generally between 19 and 21 nucleotides in length (including the end value). In some embodiments, the siRNA is between 15 and 20 nucleotides in length (including the end value), and in other embodiments, the siRNA is between 25 and 30 nucleotides in length (including the end value). As those skilled in the art will appreciate, the target region of the RNA being targeted for cleavage is most commonly a portion of a larger RNA molecule (typically an mRNA molecule). Accordingly, a “portion” of the mRNA target is an adjacent sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). siRNAs with double helices as short as 9 base pairs can mediate RNAi-directed RNA cleavage in some environments. The most common target will be at least 15 nucleotides in length. In some embodiments, the target is 15-30 nucleotides in length.

[0108] Those skilled in the art will recognize that the double-helix region is the major functional portion of the siRNA, for example, a double-helix region of 9 to 36 (e.g., 15 to 30) base pairs. Therefore, in some embodiments, an RNA molecule or complex of RNA molecules having a double-helix region of more than 30 base pairs is siRNA in order to achieve a functional double helix (e.g., 15 to 30 base pairs) that has been processed to target the desired RNA cleavage. Therefore, those skilled in the art will recognize that in some embodiments, the miRNA is siRNA. In some other embodiments, the siRNA is not a naturally occurring miRNA. In some embodiments, the RNAi agent that can be used to target HBV gene expression is not produced in target cells by cleaving a larger double-stranded RNA.

[0109] The siRNA described herein can be synthesized using standard methods known in the art, for example, by using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0110] In some embodiments, the RNAi agent comprises siRNA that targets and inhibits HBV mRNA expression. In some embodiments, the RNAi agent comprises siRNA that targets and inhibits the 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, siRNAs in combination therapies targeting a single HBV gene can lead to significant inhibition of the expression of most or all HBV transcripts. In some embodiments, the mRNA target of siRNA may 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.

[0111] In some embodiments, siRNA targets and inhibits the expression of mRNA encoded by the X gene of HBV. In some embodiments, the RNAi agent or siRNA targets mRNA encoded by a portion of the HBV genome containing the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO:2), which corresponds to nucleotides 1579-1597 of NC_003977.2 (GenBank accession number GI:21326584) (SEQ ID NO:1).

[0112] In yet another embodiment, the siRNA has a sense strand containing 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO:3) and an antisense strand containing 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:4).

[0113] In some embodiments, the HBV gene expression inhibitor comprises siRNA containing a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO:3, or a sequence differing from SEQ ID NO:3 by no more than 4, 3, 2, or 1 nucleotide; and wherein the antisense strand comprises SEQ ID NO:4, or a sequence differing from SEQ ID NO:4 by no more than 4, 3, 2, or 1 nucleotide.

[0114] In one aspect, the siRNA will comprise at least two nucleotide sequences, a sense sequence and an antisense sequence, wherein 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 in HBV gene expression. Therefore, 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 known in the art, in contrast to those located on individual oligonucleotides, the complementary sequence of the siRNA may also comprise a self-complementary region as a single nucleic acid molecule.

[0115] In 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).

[0116] In some embodiments, the HBV gene expression inhibitor comprises siRNA containing a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO:106, or a sequence differing from SEQ ID NO:106 by no more than 4, 3, 2, or 1 nucleotide; and wherein the antisense strand comprises SEQ ID NO:107, or a sequence differing from SEQ ID NO:107 by no more than 4, 3, 2, or 1 nucleotide.

[0117] In one aspect, the siRNA will comprise at least two nucleotide sequences, a sense sequence and an antisense sequence, wherein 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 the mRNA produced in HBV gene expression. Therefore, 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 known in the art, in contrast to those located on individual oligonucleotides, the complementary sequence of the siRNA may also comprise a self-complementary region as a single nucleic acid molecule.

[0118] Those skilled in the art will appreciate that siRNAs having a double helix structure of about 20 to 23, but particularly 21, base pairs have been recognized as particularly effective in inducing RNA interference (Elbashir et al., EMBO 20:6877-88 (2001)). However, others have found that shorter or longer RNA double helical structures may also be effective. In the embodiments described above, the siRNAs described herein may comprise at least one chain with a minimum length of 21 nucleotides. In some embodiments, shorter double helices having a sequence of one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:106, or SEQ ID NO:107, with only a few nucleotides subtracted from one or both ends, are similarly effective compared to the siRNAs described above. Therefore, the siRNA has a partial sequence of at least 15, 16, 17, 18, 19, 20 or more adjacent nucleotides from one or two of SEQ ID NO:3 or SEQ ID NO:4, and according to the techniques described herein, its ability to inhibit HBV gene expression is expected to differ from that of siRNA containing the full-length sequence by no more than 5, 10, 15, 20, 25 or 30%. Also included in this disclosure is an siRNA having a partial sequence of at least 15, 16, 17, 18, 19, 20 or more adjacent nucleotides from one or two of SEQ ID NO:106 and SEQ ID NO:107, and according to the techniques described herein, its ability to inhibit HBV gene expression is expected to differ from that of siRNA containing the full-length sequence by no more than 5, 10, 15, 20, 25 or 30%.

[0119] Furthermore, the siRNAs provided herein identify sites in the HBV gene transcript susceptible to RISC-mediated cleavage. Thus, the techniques described herein further characterize RNAi agents targeting one of these sequences. As used herein, an RNAi agent is said to target a specific site within the RNA transcript if RNAi promotes cleavage anywhere within a specific site. In some embodiments, the RNAi agent comprises at least 15 adjacent nucleotides from one or both of the sequences SEQ ID NO:3 and SEQ ID NO:4, said adjacent nucleotides being coupled to additional nucleotide sequences derived from a region adjacent to a selected sequence in the HBV gene. In some embodiments, the RNAi agent comprises at least 15 adjacent nucleotides from one or both of the sequences SEQ ID NO:106 and SEQ ID NO:107, said adjacent nucleotides being coupled to additional nucleotide sequences derived from a region adjacent to a selected sequence in the HBV gene.

[0120] While target sequences are typically 15–30 nucleotides in length, the applicability of specific sequences within this range for targeted cleavage of any given target RNA varies widely. This document lists different software packages and guidelines to provide guidance for identifying optimal target sequences for any given gene target, but empirical methods can also be employed, where a “window” or “mask” of a given size (21 nucleotides as a non-limiting example) is placed literally or symbolically (including, for example, computer simulations) onto the target RNA sequence to identify sequences within a size range that can serve as target sequences. The next potential target sequence can be identified by progressively moving the sequence “window” one nucleotide upstream or downstream of the initial target sequence position until a complete set of possible sequences for any chosen target size is identified. This process, coupled with the systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that best perform, identifies those RNA sequences that provide optimal repression of target gene expression when targeted with RNAi agents. It is anticipated that further optimization of inhibition efficiency can be achieved by progressively “windowing” one nucleotide upstream or downstream of a given sequence to identify sequences with equal or better inhibitory properties.

[0121] Furthermore, it is anticipated that any identified sequence (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 produce longer or shorter sequences, and by testing these, as well as those generated by moving a longer or shorter sequence window up or down along the target RNA from that point. Again, combining this method of generating new candidate targets with efficacy testing 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. Furthermore, such optimized sequences can be tuned, for example, by introducing modified nucleotides as described herein or as known in the art, adding or altering overhangs, or other modifications known in the art and / or discussed herein, to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting specific sites or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0122] RNAi agents as described herein may contain one or more mismatches with the target sequence. In some embodiments, RNAi agents as described herein contain no more than three mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatched region is not located at the center of the complementary region. In a particular embodiment, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch is confined to the last five nucleotides from the 5' or 3' end of the complementary region. For example, for a 23-nucleotide RNAi agent complementary to a region of the HBV gene, the RNA strand must not contain any mismatches within the central 13 nucleotides. The effectiveness of an RNAi agent containing a mismatch with the target sequence in inhibiting HBV gene expression can be determined using the methods described herein or methods known in the art. It is important to consider the efficacy of RNAi agents with mismatches in inhibiting HBV gene expression, especially if polymorphic sequence variations are known in a specific complementary region of HBV.

[0123] b. Chemically modified RNAi agents

[0124] 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 techniques 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, whose methods are incorporated herein by reference.

[0125] For example, modifications include (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, inversion, etc.), 3' terminal modifications (conjugation, DNA nucleotides, inversion, etc.), (b) base modifications, such as substitutions for stabilizing bases, destabilizing bases, or bases that pair with an expanded pairing body library, removed bases (base-free nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) backbone modifications, including modifications or substitutions of phosphodiester linkages. Specific examples of RNA compounds used in the embodiments described herein include, but are not limited to, RNAs containing a modified backbone or without native internucleotide linkages. RNAs with a modified backbone particularly include those whose backbone does not contain a phosphorus atom. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not contain a phosphorus atom in their internucleotide backbone may also be considered oligonucleotides. In certain embodiments, the modified RNA will have a phosphorus atom in its internucleotide backbone.

[0126] Not all positions in a given compound need to be modified, and in fact, more than one of the aforementioned modifications can be introduced in a single compound or even at a single nucleotide within an RNAi agent. The techniques described herein also include RNAi agent compounds that are chimeric compounds. In the context of this disclosure, a “chimeric” RNAi agent compound or “chimera” is an RNAi compound, such as siRNA, containing two or more chemically distinct regions, each consisting 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 where the RNA is modified to confer increased nuclease degradation resistance, increased cellular uptake, and / or increased binding affinity to target nucleic acids. Additional regions of the RNAi agent can be used to cleave RNA:DNA or...

[0127] RNA:RNA hybrids use enzymes as substrates. For example, RNase H is a cellular endonuclease that cleaves the RNA:DNA double helix. Therefore, activation of RNase H leads to cleavage of the RNA target, thereby significantly enhancing the efficiency of RNAi inhibitor gene expression. Thus, when using chimeric siRNAs, shorter RNAi agents can often achieve comparable results compared to phosphate-thioester deoxysiRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0128] Modified RNA backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkyl phosphates (including 3'-alkylene phosphates and chiral phosphates), phosphites, phosphatases (including 3'-aminophosphatases and aminoalkylphosphatases), thiophosphatases, thioalkyl phosphates, thioalkyl phosphate triesters, and boran phosphates, 2'-5' linked analogs of these esters, and those esters with antipolarity, 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.

[0129] Representative U.S. patents teaching the preparation of the aforementioned phosphorus-containing linkages include, but are not limited to, U.S. patent numbers 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; and 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.

[0130] Modified RNA backbones excluding phosphorus atoms, wherein the backbone is formed by inter-nucleoside linkages via short-bonded alkyl or cycloalkyl groups, inter-nucleoside linkages between mixed heteroatoms and alkyl or cycloalkyl groups, or inter-nucleoside linkages between one or more short-bonded heteroatoms or heterocycles. These include backbones with morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; alkylene-containing backbones; aminosulfonate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones having mixed N, O, S, and CH2 component moieties.

[0131] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. patent numbers 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; and 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 the purpose of teaching in relation to such preparation methods.

[0132] In other embodiments, it is suitable to consider using a suitable RNAi mimic in an RNAi agent, wherein both the sugar and nucleoside linkages (i.e., the backbone) of the nucleotide unit are replaced with novel groups. The base units are retained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound, which is an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and the aza-nitrogen atoms are directly or indirectly bonded to the amide moiety of the backbone. Representative U.S. patents teaching 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 relating to such preparation methods. Other teachings on PNA compounds can be found, for example, in Nielsen et al. (Science, 254:1497-1500 (1991)).

[0133] Some embodiments characterized in the techniques described herein include RNAs having a phosphate thioester backbone and oligonucleotides having a heteroatom backbone, and in particular, the backbone is -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as -OPO-CH2-] as in U.S. Patent No. 5,602,240, and an amide backbone as in U.S. Patent No. 5,602,240. In some embodiments, the RNA characterized herein has a morpholine backbone structure as in U.S. Patent No. 5,034,506.

[0134] Modified RNA may also contain one or more substituted sugar moieties. The RNAi agents characterized herein (e.g., siRNA) 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 the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. 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, where n and m range from 1 to approximately 10. In other embodiments, the siRNA at the 2' position comprises one of the following: C1 to C 10 Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl groups, O-alkylaryl or O-aryl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups for improving the pharmacokinetic properties of RNAi agents, or groups for improving the pharmacodynamic properties of RNAi agents, 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 groups. Another exemplary modification is the 2'-dimethylaminoethoxy group, namely the 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), namely 2*-O-CH2-O-CH2-N(CH2)2.

[0135] Other exemplary modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluorine (2'-F). Similar modifications can also be prepared at other positions on the RNA of the RNAi agent, particularly at the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in the 2'-5' linked siRNA. The RNAi agent may also have a sugar mimic, such as replacing the cyclobutyl moiety of pentofuranose.

[0136] Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. patent numbers 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 teachings relating to such preparation methods.

[0137] RNAi agents may also include nucleobase (generally referred to as "bases" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and 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-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine Pyridine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Other nucleosides include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine (edited by Herdewijn, P., Wiley-VCH, (2008)); those disclosed in The Concise Encyclopedia Of Polymer Science and Engineering (pp. 858-859, edited by Kroschwitz, JL, 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, edited by Crooke, ST. and Lebleu, B., CRC Press (1993)). Some of these nucleosides are particularly suitable for increasing the binding affinity of the oligomeric compounds characterized in the techniques 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 substitution has been shown to increase the stability of nucleic acid double helixes by 0.6–1.2 °C (Sanghvi, YS, Crooke, ST, and Lebleu, B., edsRNA Research and Applications, CRC Press, Boca Raton, pp. 276–278 (1993)) and is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0138] Representative U.S. patents teaching the preparation of certain modified nucleosides and other modified nucleosides described above 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; and 5,59 4,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088; each of which is incorporated herein by reference for the purpose of teaching in relation to such preparation methods.

[0139] RNA in RNAi agents can also be modified to include one or more locked nucleic acids (LANs). A locked nucleic acid is a nucleotide with a modified ribose moiety containing an additional bridge connecting the 2' and 4' carbons. This effectively "locks" the nucleic acid in a 3'-inner conformation. 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(l):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)).

[0140] Representative U.S. patents teaching 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 teaching in relation to such preparation methods.

[0141] In some implementations, the combination therapy includes siRNA modified to contain one or more adenosine-GNA nucleic acids (“GNA”). A description of adenosine-GNA can be found, for example, by Zhang et al. (JACS127(12):4174–75(2005)).

[0142] In some embodiments, this disclosure provides methods and related compositions wherein RNAi is 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.

[0143] Table 1: Abbreviations for nucleotide monomers used in the modified nucleic acid sequence representation. It will be understood that, unless otherwise indicated, these monomers are interconnected by 5'-3'-phosphodiester bonds when present in oligonucleotides.

[0144]

[0145]

[0146] In some embodiments, the HBV gene expression inhibitor comprises siRNA, wherein the siRNA has a sense strand containing 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO:5) and an antisense strand containing 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:6).

[0147] 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).

[0148] In some embodiments, the HBV gene expression inhibitor comprises siRNA, which comprises a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO:5 or SEQ ID NO:7, or a sequence differing from SEQ ID NO:5 or SEQ ID NO:7 by no more than 4, 3, 2, or 1 nucleotides, respectively.

[0149] In some embodiments, the HBV gene expression inhibitor comprises siRNA, which comprises a sense strand and an antisense strand, wherein the antisense strand comprises SEQ ID NO:6 or SEQ ID NO:8, or a sequence differing from SEQ ID NO:6 or SEQ ID NO:8 by no more than 4, 3, 2, or 2 nucleotides, respectively.

[0150] In some embodiments, the HBV gene expression inhibitor comprises siRNA, wherein the siRNA has a sense strand containing 5'-gsgsuggaCfuUfCfUfcucaAfUfuuuaL96-3' (SEQ ID NO:108) and an antisense strand containing 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO:109).

[0151] In some embodiments, the HBV gene expression inhibitor comprises siRNA, which comprises a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO:108, or a sequence differing from SEQ ID NO:108 by no more than 4, 3, 2, or 1 nucleotides.

[0152] c. RNAi agents conjugated with ligands

[0153] In some embodiments, the RNAi agent includes modifications involving the chemical linking of 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 cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA 86:6553-56 (1989)), bile acids (Manoharan et al., Biorg. Med. Chem. Let. 4:1053-60 (1994)), thioethers, for example, beryl-S-triphenylmethylthiol (Manoharan et al., Ann. NYAcad. Sci. 660:306-9 (1992); Manoharan et al., Biorg. Med. Chem. Let. 3:2765-70 (1993)), and thiocholesterols (Oberhauser et al., Nucl. Acids). Res. 20:533-38 (1992)), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J10:1111-18 (1991); Kabanov et al., FEBS Lett. 259:327-30 (1990); Svinarchuk et al., Biochimie 75:49-54 (1993)), phospholipids, such as 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)), palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1264:229-37(1995)) or octadecylamine or hexylamino-carbonyl-cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 277:923-37(1996)).

[0154] In some embodiments, the ligand alters the distribution, targeting, or lifetime of the RNAi agent to which it is introduced. In some embodiments, such as compared to substances without such ligands, the ligand provides enhanced affinity for selected targets such as molecules, cells, cell types, compartments (e.g., compartments of cells or organs, tissues, organs, or regions of the body). In such embodiments, the ligand will not participate in double-helix pairing within the double-stranded nucleic acid.

[0155] Ligands may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Lipids may 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-glycolic acid) copolymer, diethylene ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide mimicry polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0156] Ligands may also contain targeting groups, such as cell or tissue targets that bind to specific cell types (e.g., hepatocytes), such as lectins, glycoproteins, lipids, or proteins, such as antibodies. Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimics. 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 acids, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl group, palmitic acid, myristic acid, etc.). O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine), peptide conjugates (e.g., antennal peptides, Tat peptides), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled substances, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraaza macrocyclic Eu3+ complexes), dinitrophenyl, HRP, and AP.

[0157] Ligands can be proteins (e.g., glycoproteins), peptides (e.g., molecules with specific affinity for the coligand), or antibodies (e.g., antibodies that bind to specific cell types, such as hepatocytes). Ligands can also include hormones and hormone receptors. They can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, and polyfucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or NF-κB activators.

[0158] The ligand can be a substance (e.g., a drug) that can increase the uptake of RNAi agents into cells, for example, by disrupting the cell's cytoskeleton (e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments). Drugs can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0159] In another aspect, the ligand is a portion taken up by a target molecule (e.g., hepatocytes), such as a vitamin. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other microorganisms or nutrients taken up by target cells (such as hepatocytes). HSA and low-density lipoprotein (LDL) are also included.

[0160] In some embodiments, the ligand attached to the RNAi agent described herein acts as a pharmacokinetic (PK) modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acids, lithocholic acids, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing multiple thiophosphate linkages are also known to bind to serum proteins; therefore, short oligonucleotides (e.g., about 5, 10, 15, or 20 bases) containing multiple thiophosphate linkages in their backbone are also suitable as ligands (e.g., as PK-regulating ligands) for the techniques described herein. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable as PK-regulating ligands in the embodiments described herein.

[0161] (i) Lipid conjugates. In some embodiments, the ligand or conjugate is a lipid or 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 modulate binding to serum proteins (e.g., HSA). Such lipid or lipid-based molecules can bind to serum proteins, such as human serum albumin (HSA). A ligand binding to HSA allows the conjugate to distribute to target tissues, such as non-renal target tissues of the body. For example, the target tissue could be the liver, including hepatic parenchymal cells. Other molecules that bind HAS can also be used as ligands. For example, naproxen or aspirin can be used.

[0162] Lipid-based ligands can be used to inhibit (e.g., control) the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidney.

[0163] In some embodiments, lipid-based ligands bind to HSA. Lipid-based ligands have sufficient affinity to bind to HSA, allowing the conjugate to distribute to non-renal tissues. In certain specific embodiments, HSA-ligand binding is irreversible.

[0164] In some other embodiments, the lipid-based ligand binds weakly to HSA or not to HSA at all, allowing the conjugate to distribute to the kidney. Other portions targeting renal cells may also be used instead of the lipid-based ligand, or ligands other than the lipid-based ligand.

[0165] (ii) Cell-penetrating peptides and cell-penetrating agents. In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In some embodiments, the agent is amphiphilic. Exemplary agents are peptides, such as tat or tentacle peptides. If the agent is a peptide, it may be modified, including peptide mimics, inverted isomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. In some embodiments, the helical agent is an α-helical agent. In certain specific embodiments, the helical agent has a lipophilic phase and a lipophobic phase.

[0166] "Cell-permeable peptides" are capable of penetrating cells, such as microbial cells, like bacterial or fungal cells, or mammalian cells, like human cells. Permeable microbial cell peptides can be, for example, α-helical linear peptides (e.g., LL-37 or CeropinPI), disulfide-containing peptides (e.g., α-defensins, β-defensins, or antimicrobial peptides), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidin).

[0167] The ligand can be a peptide or a peptide mimic. A peptide mimic (also referred to herein as an oligopeptide mimic) is a molecule capable of folding into a defined three-dimensional structure similar to that of a natural peptide. The attachment of peptides and peptide mimics to RNAi agents can influence the pharmacokinetic distribution of RNAi, such as by enhancing cellular recognition and uptake. The peptide or peptide mimic moiety can be approximately 5–50 amino acids long, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0168] The peptide or peptide mimic can be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety can be a dendritic peptide, a restricted peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary peptide containing a hydrophobic MTS is an RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:9). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO:10)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry large polar molecules comprising peptides, oligonucleotides, and proteins across the cell membrane. For example, sequences from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:11)) and Drosophila antennal foot protein (RQIKIWFQNRRMKWK (SEQ ID NO:12)) have been found to function as delivery peptides. Peptides or peptide mimics can be encoded by random DNA sequences, such as peptides identified from phage display libraries or one-to-one compounds (OBOCs) (Lam et al., Nature 354:82-84 (1991)).

[0169] Cellular permeable peptides can also include nuclear localization signals (NLS). For example, a cellular permeable peptide can be a dual amphiphilic peptide, such as MPG, which is an NLS derived from the fusion protein domain of HIV-1 gp41 and the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-24 (2003)).

[0170] (iii) Carbohydrate conjugates. In some embodiments, the RNAi drug oligonucleotides described herein also include carbohydrate conjugates. Carbohydrate conjugates can be advantageous for the delivery of nucleic acids in vivo and for compositions suitable for in vivo therapeutic use. As used herein, “carbohydrate” means a compound that is itself composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), wherein an oxygen, nitrogen, or sulfur atom is bonded to each carbon atom; or a compound having carbohydrates as part thereof that are composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), wherein an oxygen, nitrogen, or sulfur atom is bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing from about 4 to 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars with 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).

[0171] In some embodiments, the carbohydrate conjugate is selected from the following:

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] Another representative carbohydrate conjugate used in the embodiments described herein includes, but is not limited to,

[0179] (Formula XXII), where one of X or Y is an oligonucleotide and the other is hydrogen.

[0180] In some embodiments, the carbohydrate conjugate also contains another ligand, such as, but not limited to, a PK regulator, an endosomal lysis ligand, or a cell-permeable peptide.

[0181] (iv) Connectors. In some embodiments, the conjugates described herein can be attached to RNAi drug oligonucleotides having various connectors, which may be cleavable or non-cleavable.

[0182] The term "connector" or "linking group" refers to the organic part that connects two parts of a compound. Connectors typically contain direct bonds or atoms (such as oxygen or sulfur), units (such as NR8, C(O), C(O)NH, SO, SO2, SO2NH), or atomic chains, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalenyl, arylalkynyl, heteroarylalkyl, heteroarylalenyl, heteroarylalkynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alkenyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalenyl, alkenylarylalkyl, alkenylarylalenyl, alkenylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalenyl, alkylheteroarylalkynyl Alkynyl, alkenylheteroarylalkyl, alkenylheteroarylalyl, alkenylheteroarylynyl, alkenylheteroarylalkyl, alkenylheteroarylalyl, alkenylheteroarylynyl, alkylheterocyclic alkyl, alkylheterocyclic alkenyl, alkylheterocyclic ynyl, alkenylheterocyclic alkenyl, alkenylheterocyclic ynyl, alkenylheterocyclic alkenyl, alkenylheterocyclic ynyl, alkylaryl, alkenylaryl, alkenylaryl, alkylheteroaryl, alkenylheteroaryl and alkenylheteroaryl, wherein one or more methylene groups 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; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In some implementations, the connector is between 1-24 atoms, between 4-24 atoms, between 6-18 atoms, between 8-18 atoms, or between 8-16 atoms.

[0183] A cleavable linker is a linker that is sufficiently stable outside the cell but is cleaved upon entry into a target cell to release the two parts of the linker that remain together. In some embodiments, the cleavable linker is cleaved at least 10 times or at least 100 times faster in the target cell or under a first reference condition (which may be selected, for example, to simulate or represent intracellular conditions) than in the blood of the subject or under a second reference condition (which may be selected, for example, to simulate or represent conditions found in blood or serum).

[0184] Cleavable linker groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degrading molecules. Typically, cleaving agents are more prevalent or found at higher levels or with higher activity intracellularly than in serum or blood. Examples of such cleaving agents include: substrate-selective or non-substrate-specific redox agents, including, for example, oxidases or reductases, such as thiols present in cells, which can degrade redox-cleavable linker groups through reduction; endosomes or agents that can create an acidic environment, for example, those resulting in a pH of 5 or lower; and enzymes that can hydrolyze or degrade acid-cleavable linker groups by acting as general acids, peptidases (which can be substrate-specific), and phosphatases. Cleavable linker groups, such as disulfide bonds, may be pH-sensitive. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, around 7.1–7.3. Endosomes have more acidic pH values, ranging from 5.5 to 6.0, and lysosomes have even more acidic pH values, around 5.0. Some connectors will have a cleavable linker group that is cleaved at a specific pH value, thereby releasing cationic lipids from intracellular ligands or into desired cell compartments.

[0185] Linkers can include cleavable linker groups that can be cleaved by specific enzymes. The type of cleavable linker group introduced into the linker can depend on the cell to be targeted. For example, liver-targeting ligands can be linked to cationic lipids via linkers containing ester groups. Hepatocytes are rich in esterases, so linkers in hepatocytes will be cleaved more efficiently compared to cell types that are not rich in esterases. Other cell types rich in esterases include lung cells, renal cortical cells, and testicular cells.

[0186] When targeting cell types rich in peptidase (such as hepatocytes and synovial cells), a linker containing peptide bonds can be used.

[0187] Typically, the suitability of a candidate cleavable linker can be assessed by detecting the ability of a degrading agent (or condition) to cleave the candidate linker. It may also be necessary to test the ability of the candidate cleavable linker to resist cleavage in blood or in contact with other non-target tissues. Thus, a relative sensitivity to cleavage can be determined between a first and a second condition, where the first is selected to indicate cleavage in target cells, and the second to indicate cleavage in other tissues or biological fluids (e.g., blood or serum). Evaluation can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. Preliminary evaluation under cell-free or culture conditions, followed by confirmation through further evaluation in whole animals, can be useful. In some embodiments, the useful candidate compound is cleaved at least 2-fold, at least 4-fold, at least 10-fold, or at least 100-fold faster in cells (or under selected in vitro conditions to simulate intracellular conditions) compared to blood or serum (or under selected in vitro conditions to simulate extracellular conditions).

[0188] One class of cleavable linkers is a redox-cleavable linker that is cleaved upon reduction or oxidation. An example of a reducible cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable “reducible cleavable linker,” or, for example, whether it is suitable for use with a specific RNAi moiety and a specific target, reference can be made to the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that simulate the cleavage rate to be observed in cells (e.g., target cells). Candidates can also be evaluated under conditions simulating blood or serum. In some embodiments, the candidate compound is cleaved by up to 10% in blood. In some embodiments, the useful candidate compound degrades at least 2-fold, at least 4-fold, at least 10-fold, or at least 100-fold faster in cells (or under selected in vitro conditions simulating intracellular conditions) compared to blood (or under selected in vitro conditions simulating extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic analysis under selected intracellular media and compared to conditions simulating extracellular media.

[0189] Phosphate-based cleavable linkers are cleaved by reagents that degrade or hydrolyze phosphate groups. Examples of reagents that cleave phosphate groups intracellularly are enzymes, such as intracellular phosphatases. Examples of phosphate-based linkers 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 some embodiments, the phosphate-based linking group is selected from: -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 particular embodiment, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0190] Acid-cleavable linkers are linkers that are cleaved under acidic conditions. In some embodiments, acid-cleavable linkers are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or by reagents that can be used as general acids (such as enzymes). In cells, specific low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linkers. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable linkers have the general formula -C=N-, C(O)O, or -OC(O). In some embodiments, the carbon atom attached to the oxygen (alkoxy) of the ester is an aryl, substituted alkyl, or tertiary alkyl group, such as dimethylpentyl or tert-butyl. These candidates can be evaluated using methods similar to those described above.

[0191] Ester-based cleavable linkers are cleaved by cellular enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, alkylene, alkenene, and yntylide esters. Ester-based cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0192] Peptide-based cleavable linkers are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linkers are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linkers do not include amide groups (-C(O)NH-). Amide groups can form between any alkylene, alkenylene, and alkyne groups. Peptide bonds are a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleavable linkers are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins and do not include the entire amide functional group. Peptide-based cleavable linkers have 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.

[0193] Representative carbohydrate conjugates with joints include, but are not limited to,

[0194]

[0195]

[0196] When one of X or Y is an oligonucleotide, the other is hydrogen.

[0197] In some embodiments of the composition and method, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker. For example, in some embodiments, siRNA is conjugated to a GalNAc ligand as shown in the following structure:

[0198]

[0199] Where X is O or S.

[0200] In some embodiments, the positive strand of the siRNA is conjugated to a ligand attached to the 3' end of the positive strand via a linker, as shown in the following structure:

[0201]

[0202] Where X is O or S.

[0203] In some embodiments, the combination therapy comprises siRNA conjugated to a bivalent or trivalent branched adapter selected from the group consisting of structures represented by any of formulas (XXXI)–(XXXIV):

[0204]

[0205] in:

[0206] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each appear independently to represent 0-20, and the repeating units can be the same or different;

[0207] 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 independent of the following: non-existent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O;

[0208] Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B and Q 5C Each occurrence is independently of absent, alkylene, or substituted alkylene, wherein one or more methylene groups can be interrupted or terminated by one or more of the following: O, S, S(O), SO2, N(R) N ), C(R')=C(R”), C≡C or C(O);

[0209] R 2A R 2B R 3A R 3B R 4A R 4B R 5A R 5B and R 5CEach occurrence is independent of the following: 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 heterocyclic group;

[0210] L 2A L 2B L 3A L 3B L 4A L 4B L 5A L 5B and L 5C The term "ligand" indicates that each instance of its ligand is independently a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R... a It is an H or 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):

[0211]

[0212] Where L 5A L 5B and L 5C Represents monosaccharides, such as GalNAc derivatives.

[0213] 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.

[0214] Representative U.S. patents teaching the preparation of RNA conjugates include U.S. patent numbers 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; and 5,486,600. 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;5,567 810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; and 7,037,646; each of which is incorporated herein by reference for the purpose of teaching in relation to such preparation methods.

[0215] In some cases, the RNA of an RNAi agent can be modified with non-ligand groups. Various non-ligand molecules have been conjugated to RNAi agents to enhance their activity, cellular distribution, or cellular uptake, and procedures for 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)), bile acids (Manoharan et al., Bioorg. Med. Chem. Lett. 4:1053(1994)), thioethers, for example, hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci. 660:306(1992); Manoharan et al., Bioorg. Med. Chem. Let. 3:2765(1993)), and thiocholesterol (Oberhauser et al., Nucl. Acids Res. 20:533 (1992)), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J. 10:111 (1991); Kabanov et al., FEBS Lett. 259:327 (1990); Svinarchuk et al., Biochimie 75:49 (1993)), phospholipids, such as 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)), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides) 14:969(1995)) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 36:3651(1195)), palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1264:229(1995)) or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 277:923(1996)).

[0216] A typical conjugation scheme involves the synthesis of RNA with amino linkers at one or more positions in the sequence. The amino group is then reacted with the conjugated molecule using a suitable conjugating agent or activator. The conjugation reaction can be carried out in solution while the RNA is still bound to a solid support or after RNA cleavage. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0217] d. Delivery of RNAi drugs

[0218] "Introduced into cells" when referring to RNAi agents means to promote or effectively take up or absorb into cells, as understood by those skilled in the art.

[0219] Absorption or uptake of RNAi agents can occur via unaided diffusion or active cellular processes, or via an auxiliary agent or device. The term is not limited to in vitro cells; RNAi agents can also be “introduced into cells,” where the cells are part of a living organism. In this case, introduction into cells will include delivery to the organism. For example, for in vivo delivery, the RNAi agent can be injected into a tissue site or administered systemically. In vivo delivery can also be performed using β-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 teaching purposes in relation to such delivery systems. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Other methods are described below or are known in the art.

[0220] RNAi agents can be delivered to the recipient in a variety of ways. In vivo delivery can be achieved by directly administering a composition containing an RNAi agent (e.g., siRNA) to the recipient. Alternatively, delivery can be achieved indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These alternatives will be discussed further below.

[0221] In general, any method of delivering nucleic acid molecules can be applied to RNAi agents (see, for example, Akhtar S. and Julian RL., Trends Cell. Biol. 2(5):139-44(1992) and WO94 / 02595, which are incorporated herein by reference for teaching purposes relating to such delivery methods). Three factors are particularly important for the successful delivery of RNAi agents in vivo: (a) the biostability 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 application, for example, by direct injection or implantation into tissue (as a non-limiting example, tumor) or by topical application of the formulation. Local application to the treatment site maximizes the local concentration of the agent, limits the exposure of the agent to systemic tissues that might otherwise be damaged by the agent or may degrade the agent, and allows for the administration of a lower total dose of RNAi agent. Several studies have shown that gene products are successfully knocked out when RNAi agents are applied locally. For example, intraocular delivery of VEGF siRNA via 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 angiogenesis in experimental models of age-related macular degeneration. Furthermore, direct intratumoral injection of siRNA in mice reduced tumor volume (Pille, J. et al., Mol. Ther. 11:267-74 (2005)) and increased 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 via direct injection (Dorn, G. et al., Nucleic Acids 32:e49 (2004); Tan, PH et al., Gene Ther. 12:59-66 (2005);

[0222] 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 via 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 diseases, RNA can be modified or delivered using alternative drug delivery systems; both methods work to prevent rapid degradation of siRNA by endonucleases and exonucleases in vivo. Modification of the RNA or drug carrier can also allow the RNAi agent composition to target tissues and avoid unwanted off-target effects. RNAi agents can be modified by chemically conjugating them to lipophilic groups (such as cholesterol) to enhance cellular uptake and prevent degradation. For example, systemic injection of RNAi agents targeting ApoB conjugated to a lipophilic cholesterol moiety into mice resulted in the knockout of apoB mRNA in the liver and jejunum (Soutschek, J. et al., Nature 432:173-78 (2004)). In some other embodiments, RNAi agents can be delivered using delivery systems such as nanoparticles, dendritic polymers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems generally promote the binding of negatively charged RNAi agents and enhance interactions at negatively charged cell membranes to allow for efficient cellular uptake of RNAi agents. Cationic lipids, dendritic polymers, or polymers can bind to RNAi or be induced to form vesicles or micelles that encapsulate RNAi agents (see, for example, Kim, S, H. et al., Journal of Controlled Release 129(2):107-16(2008)). When administered systemically, the formation of vesicles or micelles further inhibits the degradation of RNAi agents.Methods for preparing and administering cationic-RNAi drug complexes are within the capabilities of those skilled in the art (see, for example, 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), ibid.; Verma, UN et al., (2003), ibid.), 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. Oncol. 26:1087-91 (2005)), polyethyleneimine (Bonnet, ME 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 polyamide (Tomalia, DA et al., Biochem. Soc. Trans. 35:61-7 (2007); Yoo, H. et al., Pharm. Res. 16:1799-1804 (1999)).

[0223] As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. SNALP represents a lipid vesicle enclosing a shrunken, aqueous interior containing nucleic acids such as RNAi agents or plasmids derived from transcribed RNAi agents. SNALPs are described, for example, 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 teaching purposes relating to SNALPs.

[0224] In some embodiments, RNAi forms a complex with cyclodextrin for systemic administration. Methods of administering RNAi and cyclodextrin, and pharmaceutical compositions thereof, can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference for teaching purposes relating 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.

[0225] e. RNAi drug compositions and formulations

[0226] In some embodiments, this document provides pharmaceutical compositions comprising an RNAi agent as described herein and a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions comprising an RNAi agent can be used in combination therapy to treat HBV infection or reduce HBV viral load in a subject. Such pharmaceutical compositions are formulated based on the delivery method. For example, the composition can be formulated for systemic administration via parenteral delivery, such as intravenous (IV) delivery, or for direct delivery into the brain parenchyma, such as by infusion into the brain, such as via a continuous pump infusion.

[0227] A “pharmaceutically acceptable carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert carrier used to deliver one or more nucleic acids to animals. Excipients can be liquid or solid and are selected based on the planned administration method to provide the desired volume, consistency, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition. Typically, pharmaceutically acceptable carriers or excipients include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium glycolate); and wetting agents (e.g., sodium dodecyl sulfate).

[0228] Pharmaceutically acceptable organic or inorganic excipients that do not react harmfully with nucleic acids and are suitable for non-parenteral administration may also be used to formulate the compositions of this disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0229] Formulations for topical application of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or nucleic acid solutions in liquid or solid oil bases. Solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral application that do not react adversely with nucleic acids may be used.

[0230] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0231] In some embodiments, the pharmaceutical composition containing the RNAi agent described herein is administered at a dose sufficient to inhibit HBV gene expression. Typically, a suitable dose range for the RNAi agent is 0.001 to 200.0 mg 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 at single doses of 0.01 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg. The pharmaceutical composition can be administered once daily, or the RNAi agent can be administered at appropriate intervals throughout the day in two, three, or more sub-dose doses, or even delivered using continuous infusion or a controlled-release formulation. In this case, the amount of RNAi agent contained in each sub-dose must be correspondingly smaller to achieve the total daily dose. Dosage units can also be combined 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 sites, such as those that can be used with the techniques described herein. In this embodiment, the dosage unit contains a corresponding plurality of daily doses.

[0232] The effect of a single dose on HBV gene expression levels can be persistent, therefore subsequent doses should be administered at intervals of no more than 3, 4, or 5 days, or no more than 1, 2, 3, or 4 weeks.

[0233] Those skilled in the art will understand that certain factors can influence the dosage and duration required for effective treatment of a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the composition may comprise a single treatment or a series of treatments. As described elsewhere herein, the effective dose and in vivo half-life of a single RNAi agent covered by the techniques described herein can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0234] Mouse models can be used to study HBV infection, and these models can be used for in vivo testing of RNAi, as well as to determine the dosage that can effectively reduce HBV gene expression.

[0235] In some embodiments, the pharmaceutical compositions and formulations described herein may be administered topically (e.g., via a transdermal patch), pulmonaryly (e.g., by inhalation or blowing of powder or aerosol, including via a nebulizer); intratracheally; intranasally; epidermally and transdermally; or orally; or parenterally. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subcutaneous administration (e.g., via an implanted device); or intracranial administration (e.g., via intraparenchymal, intrathecal, or intraventricular administration).

[0236] In some embodiments, the RNAi agent in the combination therapy for treating HBV disclosed herein is delivered subcutaneously.

[0237] In some implementations, RNAi agents can be delivered in a manner that targets a specific tissue, such as the liver (e.g., hepatocytes of the liver).

[0238] Pharmaceutical compositions and formulations for topical application may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily matrices, 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 techniques described herein are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine, dimyristoylphosphatidylcholine DMPC, distearylphosphatidylcholine), anionizing (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropylDOTAP and dioleoylphosphatidylethanolamine DOTMA). RNAi agents may be encapsulated in liposomes or may form complexes with them, particularly with cationic liposomes. Alternatively, RNAi agents can be conjugated with lipids, particularly cationic lipids. 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, didecanoic acid, tridecanoic acid, monooleic acid, dilaurate, 1-monodecanoic acid glyceride, 1-dodecylazine-2-one, acylcarnitine, acylcholine, or C. 1-20Alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Examples of topical formulations are detailed in U.S. Patent No. 6,747,014, which is incorporated herein by reference for the purpose of teaching relating to such topical formulations.

[0239] Vesicles, such as liposomes, can be used as formulations for delivering the RNAi agents disclosed herein; such formulations may possess 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 bilayer.

[0240] Liposomes are monolayered or multilayered vesicles with a membrane formed by 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 the cell wall. Non-cationic liposomes, while not effectively fusing with the cell wall, can be taken up by macrophages in vivo. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and water volume of the liposome.

[0241] In some embodiments, liposome delivery may have the following advantageous properties: high deformability and the ability to penetrate pores in the skin; biocompatibility and biodegradability; the ability to integrate multiple water-soluble and lipid-soluble drugs; the ability to protect the drug encapsulated 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 local application, reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer multiple hydrophilic and hydrophobic drugs to the skin; and the ability to deliver agents including high molecular weight nucleic acids, analgesics, antibodies and hormones to the skin.

[0242] Liposomes are divided into two main categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. Positively charged DNA / liposome complexes bind to the negatively charged cell surface and are endosomed. Due to the acidic pH in the endosome, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun. 147, 980-985 (1987)).

[0243] pH-sensitive or negatively charged liposomes capture nucleic acids rather than complex with them. Since DNA and lipids carry similar charges, repulsion occurs rather than complex formation. However, some DNA is encapsulated within 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)).

[0244] In some embodiments, the liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. In some embodiments, the liposome composition includes phospholipids other than naturally derived phosphatidylcholine. For example, a neutral liposome composition may be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions may be formed from dimyristoyl phosphatidylglycerol, while anionic fused liposomes may be formed from dioleoyl phosphatidylethanolamine (DOPE). In still other embodiments, the liposome composition is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0245] In some embodiments, the liposomal drug formulation is delivered topically to the skin.

[0246] In some embodiments, the RNAi agent used in the combination therapies described herein is completely encapsulated in a lipid formulation, for example, to form SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle containing plasmid DNA encapsulated within lipid vesicles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALP and SPLP can be used for systemic application because they exhibit prolonged circulation life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLP includes “pSPLP,” which comprises an encapsulated condenser-nucleic acid complex 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. Furthermore, in some embodiments, when present in nucleic acid-lipid particles, the nucleic acid resists degradation by nucleases in aqueous solutions. 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.

[0247] In some embodiments, RNAi agents are delivered via liposomes or other lipid formulations, wherein the ratio (mass / mass ratio) of lipid to drug (e.g., the ratio of lipid to siRNA) is in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0248] III. Anti-HBV antibodies

[0249] This disclosure provides anti-HBV antibodies for use in combination therapies to treat HBV.

[0250] a. Antibodies that bind to HBV protein

[0251] In some implementations, the anti-HBV antibody or its antigen-binding fragment of the combination therapy binds to the antigen loop region of HBsAg. The envelope of the hepatitis B virus contains three “HBV envelope proteins” (also known as “HBsAg,” “hepatitis B surface antigen”): the S protein (for the “small,” also called S-HBsAg), the M protein (for the “medium,” also called M-HBsAg), and the L protein (for the “large,” also called L-HBsAg). S-HBsAg, M-HBsAg, and L-HBsAg share the same C-terminal (also called the “S domain,” 226 amino acids) corresponding to the S protein (S-HBsAg) and involved in viral assembly and infectivity. S-HBsAg, M-HBsAg, and L-HBsAg are synthesized, assembled, and secreted as particles via the Golgi apparatus in the endoplasmic reticulum (ER). The S domain contains four predicted transmembrane (TM) domains, with both the N-terminus and C-terminus of the S domain exposed to the lumen. Transmembrane domains TM1 and TM2 are essential for co-translation of proteins to integrate into the ER membrane, while transmembrane domains TM3 and TM4 are located at the C-terminal third of the S domain. The "antigen loop region" of HBsAg lies between the predicted transmembrane domains TM3 and TM4 of the HBsAg S domain, containing 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 antigen loop region of the HBV envelope protein. Specifically, 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, GA and Sureau, C., Journal of Virology 79:10460-6 (2005)).

[0252] As used herein, the S domain of HBsAg refers to the amino acid sequence shown in SEQ ID NO:13 (as shown below) or its natural or artificial sequence variants.

[0253] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTA QGTSMYPSCCCTKPSDGN CTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0254] (SEQ ID NO:13; Amino acids 101-172 are underlined)

[0255] For example, the expression "amino acids 101-172 of the S domain" refers to amino acid residues at positions 101-172 of the polypeptide according to SEQ ID NO:13. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions, e.g., different genotypes of HBsAg or different HBsAg mutants described herein) can occur naturally 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, including, for example, the polypeptide according to SEQ ID NO:13 and its natural or artificial mutants. Furthermore, when sequence fragments of the HBsAg S domain are described herein (e.g., amino acids 101-172 or 120-130 of the HBsAg S domain), it includes not only the corresponding sequence fragment of SEQ ID NO:13 but also the corresponding sequence fragments of its natural or artificial mutants. For example, the expression "amino acid residues at positions 101-172 of the S domain of HBsAg" includes amino acid residues at positions 101-172 of SEQ ID NO:13 and corresponding fragments of its mutants (natural or artificial mutants).

[0256] As used herein, the terms "corresponding sequence fragment" or "corresponding fragment" refer to fragments located at the same position in the sequence during optimized alignment, i.e., aligning sequences to obtain the highest percentage of identity. The M protein (M-HBsAg) corresponds to the S protein, which is 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 viral particle (on the cytoplasmic side of the ER), playing a crucial role in viral assembly, or on the outer surface (on the luminal side of the ER), used for interaction with target cells and essential for viral infectivity. Furthermore, the HBV surface protein (HBsAg) not only binds to the virion envelope but also spontaneously buds from the ER-Golgi interseptal membrane to form empty "subviral particles" (SVPs), which are released from the cell through secretion.

[0257] Since all three HBV envelope proteins—S-HBsAg, M-HBsAg, and L-HBsAg—contain an S domain, they also contain an "antigen loop region." Therefore, antibodies or their antigen-binding fragments that bind to the HBsAg antigen loop region bind to all three HBV envelope proteins: S-HBsAg, M-HBsAg, and L-HBsAg.

[0258] Furthermore, in some embodiments, the combination therapy uses an anti-HBV antibody or its antigen-binding fragment to neutralize hepatitis B virus infection. In other words, the antibody or its antigen-binding fragment can reduce the infectivity of hepatitis B virus.

[0259] To study and quantify viral infectivity (or “neutralization”) in the laboratory, those skilled in the art are familiar with various standard “neutralization assays.” For neutralization assays, animal viruses are typically propagated in cells and / or cell lines. In the context of this disclosure, for a neutralization assay, cultured cells may be incubated with a fixed amount of HBV in the presence (or absence) of the test antibody. As a reading, 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, particularly differentiated HepaRG cells, are incubated with a fixed amount of HBV in the presence or absence of the test antibody, for example, at 37°C for 16 hours. Incubation may be performed in a culture medium (e.g., supplemented with 4% PEG 8000). After incubation, the cells may be washed and further cultured. To measure viral infectivity, the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the culture supernatant, for example, from day 7 to day 11 post-infection, can be determined by enzyme-linked immunosorbent assay (ELISA). Additionally, HBcAg staining can be assessed by immunofluorescence assay.

[0260] In some embodiments, the antibody and antigen-binding fragment have strong neutralizing capacity. The antibody concentration of this disclosure required for 50% neutralization of hepatitis B virus (HBV) is, for example, about 10 pg / ml or less. In some embodiments, the antibody concentration of this disclosure required for 50% neutralization of HBV is about 5 pg / ml, about 1 pg / ml, or about 750 ng / ml. In some embodiments, the antibody concentration of this disclosure required for 50% neutralization of HBV is 500 ng / ml or less, for example, 450, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, or about 50 ng / ml or less. This means that 50% HBV neutralization requires only a low concentration of antibody. Specificity and potency can be measured using standard assays known to those skilled in the art.

[0261] In some implementations, anti-HBV antibodies are used as part of combination therapy for the prevention and / or treatment of hepatitis B.

[0262] In some embodiments, antibodies or antigen-binding fragments thereof according to this disclosure promote the clearance of HBsAg and HBV. Specifically, antibodies or antigen-binding fragments thereof according to this 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 HBV clearance rate can be assessed by measuring, for example, the HBV level in a blood sample from a hepatitis B patient.

[0263] In the serum of patients infected with HBV, in addition to infectious particles (HBV), there is usually an excess (typically 1,000 to 100,000 times) of empty subviral particles (SVPs) consisting solely of HBV envelope proteins (HBsAg), which are relatively small spheres and filaments of variable length. These subviral particles exhibit a strong enhancement of 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 infectivity in HBV-containing serum, as infectivity depends not only on the viral load but also on the number of SVPs (Bruns, M. et al., J Virol 72(2):1462-8(1998)). Furthermore, the excess subviral particles can act as decoys 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 therapeutic endpoint and the closest possible outcome to curing chronic hepatitis B (CHB). Therefore, in some embodiments, the antibody or antigen-binding fragment thereof according to this disclosure promotes the clearance of HBsAg, particularly the clearance of hepatitis B virus and HBV subviral particles, thus improving the treatment of hepatitis B, especially in cases of chronic hepatitis B. Thus, the antibody or antigen-binding fragment thereof according to this disclosure can effectively neutralize HBV because less antibody is absorbed by SVP, which is used as a decoy. Furthermore, in some embodiments, the antibody or antigen-binding fragment thereof according to this disclosure promotes the clearance of hepatitis B virus subviral particles and reduces the infectivity of HBV in serum.

[0264] Based on genomic sequences, HBV can be classified 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)). Genotypes are known to affect disease progression, and differences in response to antiviral therapy between genotypes have been identified. For example, genotype A tends to become chronic, while genotype C frequently encounters viral mutations. Genotype D is both chronic and has a high mutation frequency. Moreover, the distribution of HBV genotypes varies worldwide (Sunbul, M., 2014, ibid.). In some embodiments, antibodies or antigen-binding fragments thereof according to this disclosure bind to at least six, at least eight, or all ten of the HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In some embodiments, the antibody or its antigen-binding fragment according to this disclosure binds to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I and J. Examples of different HBsAg genotypes include the following: GenBank accession number J02203 (HBV-D, ayw3), GenBank accession number FJ899792.1 (HBV-D, adw2), GenBank accession number AM282986 (HBV-A), GenBank accession number D23678 (HBV-B1 Japan), GenBank accession number AB1 1 7758 (HBV-C1 Cambodia), GenBank accession number AB205192 (HBV-E Ghana), GenBank accession number X69798 (HBV-F4 Brazil), GenBank accession number AF160501 (HBV-GUSA), GenBank accession number AY090454 (HBV-H Nicaragua), GenBank accession number AF241409 (HBV-I Vietnam), and GenBank accession number AB486012 (HBV-J Borneo). The amino acid sequences of the antigenic loop region of the HBsAg S domain of different genotypes are shown in Table 2 (SEQ ID NO:14-42).

[0265] Table 2: Antigen loop sequences from different HBV genotypes

[0266]

[0267]

[0268] In some embodiments, the antibody or its antigen-binding fragment according to this disclosure binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the HBsAg mutants, said mutants having mutations in the following antigen loop regions: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. These mutants are naturally occurring mutants of the S domain of HBsAg genotype D (SEQ ID NO:43, Genbank accession number FJ899792), (where one or more mutated amino acid residues are indicated in the name).

[0269] MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSD GNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI(SEQ ID NO:43) (Antigen loop region, i.e., amino acids 101-172 are underlined)

[0270] In a particular embodiment, the antibody or its antigen-binding fragment according to the present disclosure binds to at least 12, at least 15, or all 18 of an infectious HBsAg mutant, said mutant having a mutation in the following antigen loop regions: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121 S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M1 33H, HBsAg M133L, HBsAg M1 33T, HBsAg K141 E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A.

[0271] In some embodiments, the antibody or its antigen-binding fragment according to this disclosure binds to at least one, at least two, at least three, or at least four amino acid epitopes comprising 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 115-133, 120-133, or 120-130 of the S domain of HBsAg. It is noteworthy that 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.

[0272] In a particular embodiment, an antibody or its antigen-binding fragment according to the present disclosure binds to an epitope in the antigenic loop region of HBsAg, wherein the epitope is formed by one or more amino acids located at positions 115-133, 120-133, or 120-130 of the S domain of HBsAg.

[0273] As used in the context of epitopes herein, the term "formed by" means that the epitope bound to the antibody or its antigen-binding fragment of this disclosure can be linear (continuous) or conformational (discontinuous). A linear or continuous epitope is one that an antibody recognizes through its linear amino acid sequence or primary structure. Conversely, a conformational epitope has a specific three-dimensional shape and protein structure. Therefore, if an epitope is linear and contains more than one amino acid located at positions 115-133 or 120-133 of the HBsAg S domain, the amino acids contained in the epitope can be located in adjacent positions in the primary structure (i.e., consecutive 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 the epitope; therefore, the amino acid forming the epitope (or the amino acid "contained" by the epitope) may or may not be located in adjacent positions in the primary structure (i.e., may or may not be consecutive amino acids in the amino acid sequence). In some embodiments, the epitope binding to the antibody or antigen-binding fragment of this disclosure is formed only by one or more amino acids selected from amino acid positions 115-133, 120-133, or 120-130 of the S domain of HBsAg. In certain embodiments, it is not necessary to use amino acids other than those located at positions 115-133, 120-133, or 120-130 to form the epitope binding to the antibody or antigen-binding fragment of this disclosure.

[0274] In some embodiments, the epitope in the antigenic ring of HBsAg bound by the antibody or its antigen-binding fragment of this disclosure is formed by two or more amino acids located at amino acid positions 115-133, 120-133, or 120-130 of the S domain of HBsAg. In some embodiments, the epitope in the antigenic ring of HBsAg bound by the antibody or its antigen-binding fragment of this disclosure is formed by three or more amino acids located at amino acid positions 115-133, 120-133, or 120-130 of the S domain of HBsAg. In some embodiments, the epitope in the antigenic ring of HBsAg bound by the antibody or its antigen-binding fragment of this disclosure is formed by four or more amino acids located at amino acid positions 115-133, 120-133, or 120-130 of the S domain of HBsAg. Therefore, the antibody or antigen-binding fragment thereof according to this disclosure can bind to at least one, at least two, at least three, or at least four amino acids of the antigenic loop region of HBsAg, selected from amino acids 115-133, 120-133, or 120-130 of the S domain of HBsAg. In a particular embodiment, the antibody or antigen-binding fragment thereof according to this disclosure binds to an epitope containing at least two, at least three, or at least four amino acids of 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 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., consecutive sequences in the amino acid sequence / primary structure).

[0275] In some embodiments, the epitope that binds to the antibody or antigen-binding fragment thereof according to this disclosure is a conformational epitope. Therefore, the antibody or antigen-binding fragment thereof according to this disclosure may bind to at least two, at least three, or at least four amino acids comprising 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 120-130 of the S domain of HBsAg, and wherein the at least two, at least three, or at least four amino acids are not located in adjacent positions (in the primary structure).

[0276] In certain embodiments, the antibodies of this disclosure are bispecific antibodies that have a first specificity for HBsAg and a second specificity for stimulating immune effector cells (e.g., by targeting T cell surface proteins, such as, for example, the extracellular portion of the CD3 protein). The second specificity can induce, for example, cytotoxic effects or vaccination effects.

[0277] b.Fc section

[0278] In some embodiments, the binding portion (e.g., an antibody or its antigen-binding fragment) comprises an Fc portion. In some embodiments, the Fc portion may be derived from a human source, such as human IgG1, IgG2, IgG3, and / or IgG4. In certain embodiments, the antibody or antigen-binding fragment may comprise an Fc portion derived from human IgG1.

[0279] As used herein, the term "Fc moiety" refers to a sequence that includes or is derived from a portion of the immunoglobulin heavy chain located upstream of the papain cleavage site (e.g., residue 216 in native IgG, where the first residue of the heavy chain constant region is 114) and terminates at the C-terminus of the immunoglobulin heavy chain. Thus, an Fc moiety can be the entire Fc moiety or a portion thereof (e.g., a domain). In some embodiments, the entire Fc moiety includes a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). Additional lysine residues (K) are sometimes present at the extreme C-terminus of the Fc moiety, but are typically cleaved from the mature antibody. The amino acid positions within the Fc moiety have been numbered according to Kabat's EU numbering system (see, for example, Kabat et al., "Sequences of Proteins of Immunological Interest", USDept. Health and Human Services, 1983 and 1987). The amino acid positions of the Fc moiety can also be numbered according to the IMGT numbering system (including unique numbering for the C domain and exon numbering) and the Kabat numbering system.

[0280] 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 or a portion of the hinge domain, CH2 domain, or CH3 domain may be deleted. For example, the Fc part may include or consist of: (i) a hinged structural domain (or a portion thereof) fused to the CH2 structural domain (or a portion thereof), (ii) a hinged structural domain (or a portion thereof) fused to the CH3 structural domain (or a portion thereof), (iii) a CH2 structural domain (or a portion thereof) fused to the CH3 structural domain (or a portion thereof), (iv) a hinged structural domain (or a portion thereof), (v) a CH2 structural domain (or a portion thereof), or (vi) a CH3 structural domain (or a portion thereof).

[0281] The Fc portion of this disclosure can be modified such that its amino acid sequence differs from the intact Fc portion of naturally occurring immunoglobulin molecules, 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 regulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Parts of the naturally occurring Fc portion associated with these functions have been described in the art.

[0282] For example, to activate the complement cascade, the C1q protein complex can bind to at least two molecules of IgG1 or one molecule of IgM when immunoglobulin molecules are attached to an antigen target (Ward, ES and Ghetie, V., Ther. Immunol. 277-94 (1995)). Heavy chain regions containing amino acid residues 318 to 337 are involved in complement fixation (Burton, DR, Mol. Immunol. 22:161-206 (1985)). Duncan, AR and Winter, G. (Nature 332:738-40 (1988)) reported, using site-directed mutagenesis, that Glu318, Lys320, and Lys322 form binding sites for C1q. The role of Glu318, Lys320, and Lys322 residues in C1q binding was demonstrated by the ability of short synthetic peptides containing these residues to inhibit complement-mediated cleavage.

[0283] For example, FcR binding can be mediated by the interaction of the (antibody's) Fc moiety with the Fc receptor (FcR), 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 via phagocytosis of immune complexes, as well as the cytotoxic lysis of antibody-coated erythrocytes and various other cellular targets (e.g., tumor cells) via 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 to immunoglobulin classes; the Fc receptor for IgG antibodies is called FcγR, for IgE FcεR, for IgA FcαR, and so on, with the neonatal Fc receptor being called FcRn. For example, Fc receptor binding is described in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9:457-92 (1991); Capel, PJ et al., Immunomethods 4:25-34 (1994); de Haas, M. et al., J Lab. Clin. Med. 126:330-41 (1995); and Gessner, JE et al., Ann. Hematol. 76:231-48 (1998).

[0284] Cross-linking of the Fc domain of a natural IgG antibody (FcγR) to its receptor triggers a variety of effector functions, including phagocytosis, antibody-dependent cytotoxicity and release of inflammatory mediators, as well as the clearance of immune complexes and the regulation of antibody production. This paper considers providing the Fc portion of a receptor (e.g., FcγR) cross-link. In humans, three types of FcγR 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 complex IgG with medium to low affinity and 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 to IgG with medium to low 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.

[0285] 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 inhibitory process and is present on B cells, macrophages, as well as 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 highly expressed in hepatic sinusoidal endothelium (called LSEC) and hepatic Kupffer cells, LSEC being a major site for the clearance of small immune complexes (Ganesan, LP et al., 2012, ibid.).

[0286] In some embodiments, the antibodies and antigen-binding fragments disclosed herein include an Fc moiety for binding FcγRIIb, particularly the Fc region, such as IgG type antibodies. Furthermore, the Fc moiety can be engineered to enhance FcγRIIb binding by introducing the mutations S267E and L328F, as described by Chu, SY, et al. (Molecular Immunology 45:3926–33 (2008)). Thus, the 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 of this disclosure include engineered Fc moieties having the mutations S267E and L328F, particularly as described by Chu, SY, et al. (2008, ibid.).

[0287] On B cells, FcγRIIB appears to inhibit further immunoglobulin production and isotype switching, such as IgE. On macrophages, FcγRIIB is thought to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the b form may contribute to the inhibition of these cell activations by binding to IgE with its independent receptor.

[0288] For FcγRI binding, modifications of at least one of E233-G236, P238, D265, N297, A327, and P329 in native IgG reduce binding to FcγRI. Replacing IgG2 residues at positions 233-236 with the corresponding IgG1 and IgG4 positions reduces the binding of IgG1 and IgG4 to FcγRI by 10%. 3It doubled and eliminated the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29: 2613-2624 (1999)).

[0289] Regarding FcγRII binding, it was found that IgG mutations, for example, for at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414, reduced binding to FcγRII.

[0290] Regarding FcγRIII binding, mutations in at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 were found to reduce binding to FcγRIIIA.

[0291] The mapping of the Fc receptor binding site on human IgG1, the above-mentioned mutation sites, and the method for measuring binding to FcγRI and FcγRIIA were described in Shields, RL et al. (J. Biol. Chem. 276:6591-6604 (2001)).

[0292] For binding to FcγRII, two regions of native IgG Fc appear to be involved in the interaction between FcγRII and IgG: (i) the low hinge site of IgG Fc, particularly amino acid residues L, L, G, and G (234–237, EU numbers), and (ii) the adjacent region of the CH2 domain of IgG Fc, particularly the loops and chains in the upper CH2 domain adjacent to the lower hinge region, e.g., in the region of P331 (Wines, BD, et al., J. Immunol. 164:5313–8 (2000)). Furthermore, FcγRI appears to bind to the same site on IgG Fc, while FcRn and protein A bind to different sites on IgG Fc, seemingly at the CH2-CH3 interface (Wines, BD, et al., 2000, ibid.).

[0293] It is also considered to add mutations to the binding affinity of the Fc portion of this disclosure to (i.e., one or more) Fcγ receptors (e.g., compared to a reference Fc portion or antibody that does not contain the mutation). See, for example, Delillo and Ravetch, Cell 161(5):1035-45 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016), the Fc mutation and the technique thereof are incorporated herein by reference. In any of the embodiments disclosed herein, the binding protein may comprise an Fc moiety containing a mutation selected from the following: G236A; S239D; A330L; and I332E; or a combination thereof, such as S239D / I332E; S239D / A330L / I332E; G236A / S239D / I332E; G236A / A330L / I332E; and G236A / S239D / A330L / I332E.

[0294] In some embodiments, the Fc moiety may comprise or consist of at least a portion of the Fc moiety involved in binding to FcRn. In some embodiments, the Fc moiety comprises one or more amino acid modifications that improve binding affinity to FcRn and, in some embodiments, thereby prolonging the in vivo half-life of the molecule comprising the Fc moiety (e.g., compared to a reference Fc moiety or antibody without modifications). In some embodiments, the Fc moiety comprises or is derived from IgG and a half-life-prolonging 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 encoded). In some embodiments, the half-life-prolonging mutation comprises M428L / N434S. In some embodiments, the mutation extending half-life includes M252Y / S254T / T256E. In some embodiments, the mutation extending half-life includes T250Q / M428L. In some embodiments, the mutation extending half-life includes P257I / Q311I. In some embodiments, the mutation extending half-life includes P257I / N434H. In some embodiments, the mutation extending half-life includes D376V / N434H. In some embodiments, the mutation extending half-life includes T307A / E380A / N434A.

[0295] In certain embodiments, the binding protein includes an Fc portion containing substitution mutations: M428L / N434S and G236A / A330L / I332E. In some embodiments, the antibody or antigen-binding fragment includes an Fc portion containing substitution mutations: M428L / N434S and G236A / S239D / A330L / I332E.

[0296] In certain embodiments, the binding protein includes an Fc moiety containing a substitution mutation: G236A / A330L / I332E. In some embodiments, the antibody or antigen-binding fragment includes an Fc moiety containing a substitution mutation:

[0297] G236A / S239D / A330L / I332E.

[0298] Alternatively, the Fc portion of the binding protein of this disclosure may contain at least a portion of the protein A binding known in the art; and / or the Fe portion of the antibody of this disclosure may contain at least a portion of the Fe molecule known in the art required for protein G binding. In some embodiments, the retained function includes scavenging HBsAg and HBVg. Therefore, in some embodiments, the Fc portion contains at least a portion of the FcγR binding known in the art. As described above, the Fc portion may therefore contain at least (i) the lower hinge site of the native IgG Fc, particularly amino acid residues L, L, G, and G (234–237, EU-encoded), and (ii) the adjacent region of the CH2 domain of the native IgG Fc, particularly loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example, in the P331 region, such as the region of at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upper CH2 domain of the native IgG Fc around P331 (e.g., between amino acids 320 and 340 (EU number) of the native IgG Fc).

[0299] In some embodiments, the binding protein according to this disclosure includes 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, an Fc region can be a monomeric or "single-chain" Fc region (i.e., a scFc region). A single-chain Fc region consists of an Fc portion linked within a single polypeptide chain (e.g., encoded in a single continuous nucleic acid sequence). An exemplary scFc region is disclosed in WO 2008 / 143954 A2 and is incorporated herein by reference. An Fc region can be or includes a dimerized Fc region. A "dimerized Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. The dimerized Fc region can be a homodimer of two identical Fc moieties (e.g., the Fc region of a naturally occurring immunoglobulin) or a heterodimer of two different Fc moieties (e.g., one Fc monomer of the dimerized Fc region contains at least one amino acid modification (e.g., substitution, deletion, insertion, or chemical modification) that is not present in the other Fc monomers, or one Fc monomer may be truncated compared to the other).

[0300] The currently disclosed Fc portion can contain Fc sequences or regions of the same or different classes and / or subclasses. For example, the Fc portion can be derived from immunoglobulins of the IgG1, IgG2, IgG3, or IgG4 subclasses (e.g., human immunoglobulins), or from any combination thereof. In some 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) can also be chimeric, whereby the chimeric Fc region can contain 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 can be derived from different immunoglobulin classes and / or subclasses. In some embodiments, the dimer Fc region can contain sequences from two or more different types or subclasses; for example, the SEED body (“chain exchange engineered domain”) (see Davis et al., Protein Eng. Des. Sel. 23(4):195(2010)).

[0301] Alternatively, the chimeric Fc region may comprise one or more chimeric Fc portions. For example, a chimeric Fc region or portion may comprise portions of one or more immunoglobulins 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 may comprise CH2 and / or CH3 domains of immunoglobulins derived from a first subclass (e.g., IgG1, IgG2, or IgG3 subclass) and a hinge region of immunoglobulins derived from a second subclass (e.g., IgG3 subclass). For example, the Fc region or Fc portion may comprise a hinge and / or CH2 domain of immunoglobulins derived from a first subclass (e.g., IgG4 subclass) and a CH2 domain of immunoglobulins derived from a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). For example, a chimeric Fc region may comprise an Fc portion (e.g., an intact Fc portion) of an immunoglobulin from a first subclass (e.g., the IgG4 subclass) and an Fc portion of an immunoglobulin from a second subclass (e.g., the IgG1, IgG2, or IgG3 subclass). For example, an Fc region or Fc portion may comprise a CH2 domain from the IgG4 immunoglobulin and a CH3 domain from the IgG1 immunoglobulin. For example, an Fc region or Fc portion may comprise a CH1 and CH2 domain from the IgG4 molecule and a CH3 domain from the IgG1 molecule. For example, an Fc region or Fc portion may comprise a portion of the CH2 domain from a specific subclass of the antibody, e.g., EU positions 292-340 of the CH2 domain. For example, an Fc region or Fc portion may comprise amino acid positions 292-340 of the CH2 from the IgG4 portion and the remainder of the CH2 from the IgG1 portion (or, CH2 from 292-340 may be derived from the IgG1 portion and the remainder of the CH2 from the IgG4 portion).

[0302] Furthermore, the Fc region or Fc portion may (alternatively or alternatively) include, for example, a chimeric hinge region. For instance, the chimeric hinge may be partially derived from IgG1, IgG2, or IgG4 molecules (e.g., upper and lower intermediate hinge sequences) and partially derived from IgG3 molecules (e.g., an intermediate hinge sequence). In another example, the Fc region or Fc portion may include a chimeric hinge partially derived from IgG1 molecules and partially derived from IgG4 molecules. In yet another example, the chimeric hinge may include upper and lower hinge domains from IgG4 molecules and an intermediate hinge domain from IgG1 molecules. Such a chimeric hinge can be prepared, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the intermediate hinge domain of the IgG4 hinge region. In some other embodiments, the chimeric hinge may comprise amino acids located at EU positions 233-236 derived from an IgG2 antibody and / or the Ser228Pro mutation, wherein the remaining amino acids of the hinge are derived 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 antibodies according to this disclosure are described in US2005 / 0163783A1.

[0303] In some embodiments, the Fc portion or Fc region comprises or is composed of an amino acid sequence derived from a human immunoglobulin sequence (e.g., the Fc region or Fc portion of a human IgG molecule). However, the polypeptide may contain one or more amino acids from another mammalian species. For example, a primate Fc portion or primate binding site may be included in the target polypeptide. Alternatively, one or more mouse amino acids may be present in the Fc portion or Fc region.

[0304] c. HBC34 and HBC24 antibodies

[0305] In some embodiments, the anti-HBV antibody is HBC34 or its engineered variant, or HBC24 or its engineered variant. 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 spherical SVPs at low stoichiometry. The activity of HBC34, as determined by immunoassay, is 5000 IU / mg. For comparison, the activity of HBIG is ~1 IU / mg.

[0306] As indicated herein, unless otherwise stated, the terms “HBC34 antibody” and “HBC antibody” may include wild-type HBC34 antibody or engineered variants thereof (e.g., HBC34 and HBC34 variants described in Table 3).

[0307] Table 3 shows the amino acid sequences of the CDR, heavy chain variable region (VH), and light chain variable region (VL) of HBC34, as well as its engineered variants (“HBC34v7”, “HBC34v23”, “HBC34v31”, “HBC34v32”, “HBC34v33”, “HBC34v34”, and “HBC34v35”) and “HBC24”. The full-length heavy chain (HC) and light chain (LC) amino acid sequences of exemplary antibodies of this disclosure are also shown. (One or more amino acid residues are indicated in the name).

[0308] Table 3: Sequences of HBC34 and HBC24 antibodies

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324] In some embodiments, the antibody of this disclosure is HBC34, or a non-natural variant of an HBC34 antibody. Examples of non-natural variants of HBC34 include, for example, “HBC34v7”, “HBC34v23”, “HBC34v31”, “HBC34v32”, “HBC34v33”, “HBC34v34”, and “HBC34v35”.

[0325] In some embodiments, the anti-HBV antibody comprises one or more amino acid sequences listed in Table 3. In some embodiments, the antibody or antigen-binding fragment thereof according to this disclosure comprises the CDR sequence and V shown in Table 3. H Sequence, V L The sequence, HC sequence, and / or LC sequence have amino acid sequence identity of 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%. In any currently disclosed embodiment, the antibody or antigen-binding fragment may comprise CDR, V, etc., as shown in Table 3. H V L HC and / or LC sequences.

[0326] In some embodiments, the antibody or antigen-binding fragment of this disclosure comprises: (i) the amino acid sequences of CDRH1, CDRH2, and CDRH3 according to SEQ ID NO:44, 45, or 46, and 47, respectively; and (ii) the amino acid sequences of CDRL1, CDRL2, and CDRL3 according to SEQ ID NO:48, 49, or 50, and 51, or 52, respectively.

[0327] Therefore, in some embodiments, CDRH1, CDRH2, and CDRH3 are based on SEQ ID NO:44, 45, and 47, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 are based on SEQ ID NO:44, 46, and 47, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are based on SEQ ID NO:48, 49, and 51, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are based on SEQ ID NO:48, 49, and 52, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are based on SEQ ID NO:48, 50, and 51, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are based on SEQ ID NO:48, 50, and 52, respectively.

[0328] It will be understood that the antibody or antigen-binding fragment of this disclosure may comprise any combination of the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 according to SEQ ID NO:44-52.

[0329] In certain embodiments, the antibody or antigen-binding fragment of this disclosure comprises: the amino acid sequences of CDRH1, CDRH2, and CDRH3 according to SEQ ID NO:44, 45, and 47, respectively; and the amino acid sequences of CDRL1, CDRL2, and CDRL3 according to SEQ ID NO:48, 49, and 51, respectively. In other embodiments, the antibody or antigen-binding fragment of this disclosure comprises: the amino acid sequences of CDRH1, CDRH2, and CDRH3 according to SEQ ID NO:44, 45, and 47, respectively; and the amino acid sequences of CDRL1, CDRL2, and CDRL3 according to SEQ ID NO:48, 49, and 52, respectively.

[0330] In some embodiments, the antibody or antigen-binding fragment of this disclosure includes: (a) a light chain variable domain (V L (a) an amino acid sequence comprising or consisting of an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence shown in any of SEQ ID NO:55-69; and (b) a heavy chain variable domain (V H ), which contains 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.

[0331] In some embodiments, the antibody or antigen-binding fragment of this disclosure comprises:

[0332] (a) Light chain variable structural domain (V) L (a) an amino acid sequence comprising or consisting of an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence shown in any of SEQ ID NO:55-63; and (b) a heavy chain variable domain (V H ), which contains 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.

[0333] In some embodiments, the antibody or antigen-binding fragment of this disclosure comprises:

[0334] (a) Light chain variable structural domain (V) L (a) an amino acid sequence comprising or consisting of an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence shown in any of SEQ ID NO: 55-57 or 64-69; and (b) a heavy chain variable domain (V H ), which contains 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] In some embodiments, the antibody or antigen-binding fragment of this disclosure comprises:

[0336] (i)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:55; and (b) a heavy chain variable domain (V H ), which contains 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;

[0337] (ii)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:55; and (b) a heavy chain variable domain (V H ), which contains 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;

[0338] (iii)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:56; and (b) a heavy chain variable domain (V H ), which contains 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] (iv)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:56; and (b) a heavy chain variable domain (V H ), which contains 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;

[0340] (v)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:57; and (b) a heavy chain variable domain (V H), which contains 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] (vi)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:57; and (b) a heavy chain variable domain (V H ), which contains 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;

[0342] (vii)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:58; and (b) a heavy chain variable domain (V H ), which contains 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] (viii)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:59; and (b) a heavy chain variable domain (V H ), which contains 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;

[0344] (ix)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:60; and (b) a heavy chain variable domain (V H ), which contains 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;

[0345] (x)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:61; and (b) a heavy chain variable domain (V H), which contains 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;

[0346] (xi)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:62; and (b) a heavy chain variable domain (V H ), which contains 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;

[0347] (xii)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:63; and (b) a heavy chain variable domain (V H ), which contains 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;

[0348] (xiii)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:64; and (b) a heavy chain variable domain (V H ), which contains 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] (xiv)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:65; and (b) a heavy chain variable domain (V H ), which contains 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;

[0350] (xv)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:66; and (b) a heavy chain variable domain (V H), which contains 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;

[0351] (xvi)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:67; and (b) a heavy chain variable domain (V H ), which contains 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;

[0352] (xvii)(a) Light chain variable structural domain (V) L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:68; and (b) a heavy chain variable domain (V H ), which contains 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

[0353] (xviii)(a) Light chain variable structural domain (V L (a) an amino acid sequence that is at least 90%, at least 95%, or 100% identical to or constitutes the amino acid sequence shown in SEQ ID NO:69; and (b) a heavy chain variable domain (V H ), which contains 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.

[0354] In some embodiments, the antibody or antigen-binding fragment of this disclosure comprises:

[0355] (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 shown in 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 shown in any one of SEQ ID NO:70-72 and 97; or

[0356] (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 shown 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 shown in any one of SEQ ID NO:70-72 and 97; or

[0357] (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 shown in any of SEQ ID NO: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 shown in any of SEQ ID NO:70-72, 97, and 98; or

[0358] In a particular embodiment, the antibody or antigen-binding fragment of this 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.

[0359] In a particular embodiment, the antibody or antigen-binding fragment of this 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.

[0360] In other embodiments, the antibody or antigen-binding fragment of this 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:72.

[0361] In other embodiments, the antibody or antigen-binding fragment of this 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:97.

[0362] In other embodiments, the antibody or antigen-binding fragment of this 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:70.

[0363] In other embodiments, the antibody or antigen-binding fragment of this 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:71.

[0364] In yet another embodiment, the antibody or antigen-binding fragment of this 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.

[0365] In other embodiments, the antibody or antigen-binding fragment of this 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:97.

[0366] In some embodiments, the antibody or antigen-binding fragment of this disclosure comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, having amino acid sequences according to SEQ ID NO:77-82, respectively. In some embodiments, the antibody or antigen-binding fragment of this disclosure comprises (a) a light chain variable domain (V) according to SEQ ID NO:76. L (a) amino acid sequence; and (b) the heavy chain variable domain (V) according to SEQ ID NO:75. H ) amino acid sequence.

[0367] In some embodiments, the antibody or antigen-binding fragment of this disclosure includes (a) a light chain variable domain (V L (b) The amino acid sequence shown in SEQ ID NO:76 has at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:76, and the heavy chain variable domain (V H It has at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:75.

[0368] d. Pharmaceutical composition

[0369] In some embodiments, the combination therapy antibody or its antigen-binding fragment is provided as a pharmaceutical composition comprising an anti-HBV antibody and optionally a pharmaceutically acceptable carrier. In some embodiments, the composition may comprise an anti-HBV antibody, wherein the antibody may constitute 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 weight. In such compositions, the antibody may be in a purified form.

[0370] Pharmaceutical compositions for anti-HBV antibodies may contain antimicrobial agents, especially if packaged in multiple doses. They may contain detergents, such as Tween (polysorbate), e.g., Tween 80. When present, detergents are typically present at low levels (e.g., less than 0.01%). The composition may also contain sodium salts (e.g., sodium chloride) to improve tolerability. For example, in some embodiments, the pharmaceutical composition contains NaCl at a concentration of 10 ± 2 mg / ml.

[0371] In addition, the pharmaceutical composition may contain sugar alcohols (e.g., mannitol) or disaccharides (e.g., sucrose or trehalose), for example, at about 15-30 mg / ml (e.g., 25 mg / ml), particularly if it is lyophilized, or if it contains materials reconstituted from lyophilized material. Prior to lyophilization, the pH of the composition used for lyophilization may be adjusted to between 5 and 8, or between 5.5 and 7, or about 6.1.

[0372] The antibody compositions disclosed herein may also contain one or more immunomodulators. In some embodiments, one or more immunomodulators include adjuvants.

[0373] A method for preparing a pharmaceutical composition of anti-HBV antibody may include the steps of: (i) preparing an antibody; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

[0374] IV. Treatment methods using combination therapy

[0375] In some embodiments, this disclosure provides methods for treating HBV infection or hepatitis B virus-related diseases.

[0376] As used herein, “object” is an animal, such as a mammal, including any mammal that can be infected with HBV, such as primates (e.g., humans, non-human primates, e.g., monkeys or chimpanzees), or an animal considered an acceptable clinical model of HBV infection, an HBV-AAV mouse model (see, e.g., 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 object has hepatitis B virus (HBV) infection. In some other embodiments, the object has both hepatitis B virus (HBV) infection and hepatitis D virus (HDV) infection. In some other embodiments, the object is a human, such as a person with HBV infection, particularly chronic hepatitis B virus (CHBV) infection.

[0377] As used herein, the terms “treating” or “treatment” refer to the following favorable or desired outcomes, including but not limited to: reduction 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 (generally considered to be within the normal range of approximately 10 to 34 U / L), absence or low levels of anti-HBV antibodies; liver injury; 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; fatigue; taste and smell disturbances (aversion to food and cigarettes); or right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; somnolence; sleep pattern disturbances; confusion; coma; ascites; gastrointestinal bleeding; coagulation disorders; jaundice; hepatomegaly (mildly enlarged, soft liver); splenomegaly; palmar erythema; spider angiomas; muscle wasting; spider hemangiomas; vasculitis; varicose vein bleeding; peripheral edema; gynecomastia. Developmental disorders; testicular atrophy; abdominal collateral veins (peritoneal varicose veins); ALT levels higher than AST levels; elevated gamma-glutamyl transferase (GGT) (normal range is generally considered to be about 8 to 65 U / L) and elevated alkaline phosphatase (ALP) levels (normal range is generally considered to be about 44 to 147 IU / L, and not exceeding 3 times the ULN); slightly low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened erythrocyte survival time; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); 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; presence of tissue nonspecific antibodies, such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%); presence of tissue specific antibodies, such as anti-thyroid antibodies (10-20%); elevated rheumatoid factor (RF) levels; decreased platelet and white blood cell counts; degenerative and regenerative hepatocyte changes in the lobules, accompanied by inflammation; and major centrilobular necrosis, whether or not it can be detected. This reduces the likelihood of developing 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 compared to a group with the same risk factors but not receiving the treatments described herein."Treatment" can also refer to extending survival compared to the expected survival without treatment.

[0378] As used herein, the terms “preventing” or “prevention” refer to the prevention of the development of a disease, condition, or illness, or the reduction of the development of signs or symptoms associated with such a disease, condition, or illness (e.g., by a clinically relevant amount), or the delay in the manifestation of delayed signs or symptoms (e.g., by days, weeks, months, or years). Prevention may require the administration of more than one dose.

[0379] In some implementations, treatment of HBV infection results in a “functional cure” for hepatitis B. As used herein, a functional cure is understood as the clearance of circulating HBsAg and may be accompanied by a transition to a state where HBsAg antibodies are detectable using clinically relevant assays. For example, detectable antibodies may include signals above 10 mIU / ml, such as by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay. A functional cure does not require the clearance of all replicating forms of HBV (e.g., cccDNA from the liver). Approximately 0.2–1% of patients with chronic infection will spontaneously undergo anti-HB seroconversion each year. However, even after anti-HB seroconversion, low levels of HBV are frequently observed persisting for decades, indicating a functional cure rather than a complete cure. Without being constrained by a specific mechanism, the immune system may be able to control HBV under conditions where a functional cure has been achieved. A functional cure allows for the cessation of any treatment for HBV infection. However, it should be understood that a “functional cure” for HBV infection may not be sufficient to prevent or treat disease or condition caused by HBV infection, such as liver fibrosis, HCC, or cirrhosis. In some specific implementations, "functional cure" may refer to a sustained decrease in serum HBsAg, such as <1 IU / mL, at the start or end of a treatment regimen, for at least 3 months, at least 6 months, or at least one year.

[0380] As used herein, the term "hepatitis B virus-related disease" or "HBV-related illness" refers to a disease or condition caused by or associated with HBV infection or replication. The term "HBV-related illness" includes diseases, conditions, or illnesses that would benefit from a reduction in HBV gene expression or replication. Non-limiting examples of HBV-related illness 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.

[0381] In some implementations, 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, relying on the mandatory auxiliary function provided by hepatitis B virus (HBV) for transmission; in fact, HDV requires an associated or pre-existing HBV infection to become infectious and reproductive, particularly the viral envelope containing hepatitis B surface antigen. HDV can cause severe acute and chronic liver disease 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 (excluding the Mediterranean).

[0382] HDV transmission can occur through co-infection with HBV (co-infection) or superimposed on chronic hepatitis B or a hepatitis B carrier state (reinfection). Double infection and co-infection with HDV generally lead to more severe complications compared to HBV infection alone. These complications include a greater likelihood of liver failure in acute infection and rapid progression to cirrhosis, and an increased chance of liver cancer in chronic infection. Hepatitis D, combined with HBV, has the highest mortality rate among all hepatitis infections, at 20%.

[0383] In some implementations, HBV-related disease is acute hepatitis B. Acute hepatitis B involves liver inflammation lasting less than six months. Typical symptoms of acute hepatitis B are fatigue, loss of appetite, nausea, and vomiting. Very high transaminase levels (>1000 U / L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B can rapidly progress to acute liver failure, characterized by impaired hepatic synthesis. This is typically defined as a prothrombin time (PT) of 16 seconds or an international normalized ratio (INR) of 1.5 in the absence of prior liver disease. Acute hepatitis B can evolve into chronic hepatitis B.

[0384] In some implementations, HBV-related disease is chronic hepatitis. Chronic hepatitis B (CHB) includes liver inflammation lasting more than six months. Individuals with CHB are HBsAg positive and have high viremia (≥10). 4 HBV-DNA copies / ml of blood or hypoviremia (<10 HBV-DNA copies / ml of blood) or low viremia 3(1 HBV-DNA copy / ml blood). In some embodiments, the subject has been infected with HBV for at least 5 years. In some embodiments, the subject has been infected with HBV for at least 10 years. In some embodiments, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease may be immune-tolerant or have inactive chronic infection without any evidence of active disease and are asymptomatic. Patients with chronic active hepatitis, especially those in a replicating state, may experience symptoms similar to acute hepatitis. Subjects with chronic hepatitis B disease may have active chronic infection with necrotizing inflammatory liver disease, increased hepatocyte turnover without detectable necrotizing inflammation, or inactive chronic infection without any signs of active disease and are also asymptomatic. The persistence of HBV infection in CHB subjects is a result of cccHBV DNA. In some embodiments, subjects with CHB are HBeAg positive. In some other embodiments, subjects with CHB are HBeAg negative. Subjects with CHB have serum HBV DNA levels below 10. 5 Furthermore, transaminases (e.g., ALT, AST, and gamma-glutamyl transferase) are persistently elevated. Individuals with CHB may have a liver biopsy score (e.g., necrotizing inflammation score) of less than 4.

[0385] In some implementations, HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B exhibit symptoms of acute hepatitis, along with other symptoms such as confusion or coma (due to the liver's inability to detoxify chemicals) and bruising or bleeding (due to a lack of clotting factors).

[0386] Subjects with HBV infection (e.g., CHB) may develop liver fibrosis. Therefore, in some embodiments, HBV-related disease is liver fibrosis. The histological definition of liver fibrosis (or cirrhosis) is a diffuse liver process characterized by fibrosis (excessive fibrous connective tissue) and the transformation of normal liver structure into structurally abnormal nodules.

[0387] Subjects with HBV infection (e.g., CHB) may develop end-stage liver disease. Therefore, in some embodiments, HBV-related disease is end-stage liver disease. For example, liver fibrosis may progress to a degree that the body cannot compensate for, such as decreased liver function leading to liver fibrosis (i.e., decompensated liver), and result in, for example, psychiatric and neurological symptoms and liver failure.

[0388] Individuals with HBV infection (e.g., CHB) may develop hepatocellular carcinoma (HCC), also known as malignant liver cancer. Therefore, in some implementations, HBV-related disease is HCC. HCC typically develops in individuals with CHB and can be fibrous lamellar, pseudoglandular (adenoids), pleomorphic (giant cells), or clear cell.

[0389] "HDV-related conditions" or "Hepatitis D virus-related conditions" are diseases or conditions associated with the expression of HDV. Exemplary HDV-related conditions 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.

[0390] As used herein, "therapeutic effective dose" refers to an amount, including RNAi agents or anti-HBV antibodies, sufficient to effectively treat an individual with HBV infection and / or HBV-related disease (e.g., by reducing or maintaining existing disease or one or more symptoms of the disease). "Therapeutic effective dose" may vary depending on the RNAi agent and / or anti-HBV antibody, the route of administration, the disease and its severity, and the patient's medical history, age, weight, family history, genetic makeup, stage of the HBV gene-mediated pathological process, type of prior or concomitant treatment (if any), and other individual characteristics. A therapeutic effective dose may require more than one dose.

[0391] "Therapeutic effective amount" also includes the amount of RNAi agent or anti-HBV antibody that produces some of the desired effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutic agents (e.g., RNAi agents, anti-HBV antibodies) used in the methods of this disclosure can be administered in sufficient amounts to produce a reasonable benefit / risk ratio applicable to such treatment.

[0392] As used herein, the term "sample" includes a collection of similar fluids, cells, or tissues separated from an object, as well as fluids, cells, or tissues present within the object. Examples of biological fluids include blood, serum and serous fluids, plasma, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized regions. For example, a sample may originate from a specific organ, a portion of an organ, or fluids or cells within those organs. In some embodiments, the sample may be derived from the liver (e.g., the entire liver or portions thereof, or certain cell types within the liver, such as, for example, hepatocytes). In some embodiments, "sample derived from an object" refers to blood, plasma, or serum obtained from a blood sample drawn from the object. In other embodiments, "sample derived from an object" refers to liver tissue (or a subcomponent thereof) or blood tissue (or a subcomponent thereof, such as serum) derived from the object.

[0393] Some embodiments of this disclosure provide methods for treating chronic HBV infection or HBV-related disease in subjects in need, comprising: (i) administering an agent that reduces HBV antigen load to the subject; and (ii) administering an anti-HBV antibody to the subject. In some embodiments, the agent that reduces HBV antigen load is administered prior to the anti-HBV antibody. In some embodiments, the agent that reduces HBV antigen load is administered prior to the anti-HBV antibody such that the viral load is reduced when the anti-HBV antibody is administered. In some embodiments, the therapeutically effective amount of the anti-HBV antibody in a 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.

[0394] In some embodiments, this disclosure provides a method for treating chronic HBV infection or HBV-related disease in a subject in need, comprising: administering an agent that reduces HBV antigen load 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 agent that reduces HBV antigen load, wherein a reduction in HBsAg indicates a decrease in the expression of at least one HBV gene.

[0395] In some embodiments, this disclosure provides a medicament for reducing HBV antigen load in treating a subject with chronic HBV infection or HBV-related disease, wherein an anti-HBV antibody is subsequently administered to the subject. In some other embodiments, this disclosure provides an anti-HBV antibody for treating a subject with chronic HBV infection or HBV-related disease, and the subject has previously been administered a medicament for reducing HBV antigen load. In other embodiments, the expression of at least one HBV gene is reduced after administration of the medicament for reducing 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.

[0396] In some embodiments, this disclosure provides the use of agents that reduce HBV antigen load and / or anti-HBV antibodies in the preparation of agents for treating chronic HBV infection or HBV-related diseases.

[0397] Some embodiments of this disclosure provide methods for treating chronic HBV infection or HBV-related disease in subjects in need, comprising: (i) administering an HBV gene expression inhibitor to the subject; and (ii) administering an anti-HBV antibody to the subject. In some embodiments, the HBV gene expression inhibitor is administered prior to the administration of the anti-HBV antibody. In some embodiments, administering the HBV gene expression inhibitor prior to the anti-HBV antibody results in a reduction in viral load when the anti-HBV antibody is administered. In some embodiments, the therapeutically effective amount of the anti-HBV antibody in a combination therapy is lower than the therapeutically effective amount of the anti-HBV antibody delivered when the subject has not yet been administered an HBV gene expression inhibitor (e.g., when the anti-HBV antibody is administered alone as a monotherapy).

[0398] In some embodiments, the 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 the expression of said at least one HBV gene is reduced. In a particular embodiment, the at least one HBV gene is the HBV X gene and / or HBsAg.

[0399] In some embodiments, this disclosure provides a method for treating chronic HBV infection or HBV-related disease in a subject in need, comprising: administering an HBV gene expression inhibitor to the subject; 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 decrease in HBsAg indicates a reduction in the expression of at least one HBV gene.

[0400] In some embodiments, this disclosure provides an HBV gene expression inhibitor for treating chronic HBV infection or HBV-related disease in subjects, wherein the subjects are subsequently administered an anti-HBV antibody. In some other embodiments, this disclosure provides the use of an anti-HBV antibody for treating chronic HBV infection or HBV-related disease in subjects, wherein the subjects have previously been administered a gene expression inhibitor. In other embodiments, at least one HBV gene expression decreases after administration of the HBV gene expression inhibitor, and the anti-HBV antibody is administered to the subject when the expression of at least one HBV gene decreases.

[0401] In some embodiments, this disclosure provides the use of HBV gene expression inhibitors and / or anti-HBV antibodies in the preparation of medicaments for treating chronic HBV infection or HBV-related diseases.

[0402] In any of the above methods, compositions, or uses in their preparation, the methods and compositions can be used to treat chronic HBV infection.

[0403] In some embodiments, the HBV gene expression inhibitor is administered as a single, two, three, four, or five doses. In certain specific embodiments, at least one dose of the HBV gene expression inhibitor is administered prior to the administration of the anti-HBV antibody.

[0404] In some implementations, the HBV gene expression inhibitor is administered as a single, two, three, four, five, six, seven, or eight dose. One or more doses may be administered, for example, twice daily, once daily, every two days, every three days, twice weekly, once weekly, every other week, every four weeks, or once monthly.

[0405] In some implementations, administration of anti-HBV antibodies includes administration twice a week, once a week, every other week, every two weeks, or once a month.

[0406] In some embodiments, administering anti-HBV antibodies includes administering at least two doses of a therapeutically effective amount of anti-HBV antibodies. In some other embodiments, at least two doses are administered twice a week, once a week, every other week, every two weeks, or once a month.

[0407] In some embodiments, anti-HBV antibody administration is initiated at least one week after administration of the HBV gene expression inhibitor. In some embodiments, anti-HBV antibody administration is initiated two weeks after administration of the HBV gene expression inhibitor. In some embodiments, anti-HBV antibody administration is initiated eight weeks after administration of the HBV gene expression inhibitor.

[0408] In some implementations, the anti-HBV antibody and the HBV gene expression inhibitor are administered subcutaneously, respectively.

[0409] In specific embodiments of the above methods, compositions, or uses in preparation, the anti-HBV antibody can recognize HBV genotypes A, B, C, D, E, F, G, H, I, and J.

[0410] In certain embodiments of the methods, compositions, or uses described above, the anti-HBV antibody may be a human antibody; a monoclonal antibody; or a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating an immune effector (e.g., a second specificity for stimulating cytotoxic or vaccination effects). In some other embodiments of the methods, compositions, or uses described above disclosed herein, the anti-HBV antibody is a monoclonal antibody.

[0411] In certain embodiments of the methods, compositions, or uses described above, the anti-HBV antibody may be HBC34 or a non-natural variant of HBC34 as disclosed herein. For example, in some embodiments, the anti-HBV antibody comprises a CDR having the following amino acid sequences: (i) according to SEQ ID NO: 44, 45, 47-49 and 51; or (ii) according to SEQ ID NO: 44, 45, 47-49 and 52. In some embodiments, the anti-HBV antibody comprises a CDR having the amino acid sequences according to SEQ ID NO: 44, 45, 47, 48, 49 and 51. In some embodiments, the anti-HBV antibody comprises a CDR having the amino acid sequences according to SEQ ID NO: 44, 45, 47, 48, 49 and 52. In some embodiments, the anti-HBV antibody comprises: (1)(a) a light chain variable domain (V L (a) having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:55-63; and (b) a heavy chain variable domain (V H ), which has at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:53; or (2)(a) a light chain variable domain (V L (a) having at least 90%, at least 95%, or 100% identity with the amino acid sequences shown in any one of SEQ ID NO: 55-57 and 64-69; and (b) a heavy chain variable domain (V H The amino acid sequence of SEQ ID NO:54 is at least 90%, at least 95%, or 100% identical to that shown.

[0412] In some embodiments, the anti-HBV antibody comprises: (1)(a) a light chain variable domain (V L (a) having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 55-69; and (b) a heavy chain variable domain (V H ), which has at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:53; or (2)(a) a light chain variable domain (V L (a) having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO:55-69; and (b) a heavy chain variable domain (V H The amino acid sequence of SEQ ID NO:54 is at least 90%, at least 95%, or 100% identical to that shown.

[0413] In some embodiments, the anti-HBV antibody comprises: (a) a light chain variable domain (V) according to SEQ ID NO:59 L (a) sequence; and (b) heavy chain variable domain (V) according to SEQ ID NO:53 H )sequence.

[0414] In some embodiments, the anti-HBV antibody comprises: (a) a light chain variable domain (V) according to SEQ ID NO:58 L (a) sequence; and (b) heavy chain variable domain (V) according to SEQ ID NO:53 H )sequence.

[0415] In a particular embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:73, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:70-72 and 97.

[0416] In a particular embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:74, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:70-72 and 97.

[0417] In a particular embodiment of the method, composition, or use in preparation, the anti-HBV antibody comprises: (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:83-95, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:70-72, 97, and 98.

[0418] In a particular 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.

[0419] In a particular 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.

[0420] In a particular 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.

[0421] 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 NO: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) of an amino acid sequence according to SEQ ID NO:76. L (a) and (b) the heavy chain variable domain (V) according to the amino acid sequence of SEQ ID NO:75. H ).

[0422] In certain embodiments of the above methods, compositions, or uses in preparation, the anti-HBV antibody comprises (a) a light chain variable domain (V L (a) has at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:76, and (b) heavy chain variable domain (V H The amino acid sequence of SEQ ID NO:75 is at least 90%, at least 95%, or 100% identical to that shown.

[0423] In some implementations, the therapeutically effective dose of the anti-HBV antibody is lower than the therapeutically effective dose of the anti-HBV antibody delivered when the subject is not administered an HBV gene expression inhibitor. For example, combination therapy may reduce the effective dose of the anti-HBV antibody compared to administration of the anti-HBV antibody alone.

[0424] In some implementations, the anti-HBV antibody is administered in at least two separate doses. In certain implementations, at least two doses are administered twice a week, once a week, every other week, every two weeks, or once a month.

[0425] In some embodiments, the subject is a human, and the anti-HBV antibody is administered at a therapeutically effective amount; wherein the therapeutically effective amount is from about 3 mg / kg to about 30 mg / kg.

[0426] In certain embodiments of the methods, compositions, or uses described above, the inhibitor is an RNAi agent that inhibits the 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 decrease in protein levels as measured by ELISA.

[0427] In some 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 from nucleotides 1579-1597 of SEQ ID NO:1 by no more than 3 nucleotides. In some 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.

[0428] In a particular embodiment of the above methods, compositions, or for use in preparation, at least one strand of the RNAi agent may contain a 3' overhang of at least one nucleotide or at least two nucleotides.

[0429] In a specific embodiment of the above method, composition, or use in preparation, the double-stranded region of the RNAi agent may 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.

[0430] In a particular embodiment of the above methods, compositions, or uses in preparation, each strand of the RNAi agent may be 15-30 nucleotides or 19-30 nucleotides.

[0431] In certain embodiments of the methods, compositions, or uses described above, the RNAi agent is siRNA. In certain embodiments, the siRNA inhibits the expression of HBV transcripts encoding HBsAg, HBcAg, and HBx proteins or HBV DNA polymerase protein. In some embodiments, the siRNA binds to at least 15 adjacent nucleotides encoded by the following targets: P gene, nucleotides 2309-3182 and 1-1625 of NC_003977.2; 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 C gene, nucleotides 1816-2454 of NC_003977.2.

[0432] In certain embodiments of the methods, compositions, or uses described above, the RNAi agent is siRNA, and the antisense strand of said siRNA comprises at least 15 or 19 adjacent nucleotides of the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:4). In some embodiments, the antisense strand of the siRNA comprises the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:4). In some embodiments, the antisense strand is composed of the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:4). In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO:3). In some embodiments, the sense strand of the siRNA is composed of the nucleotide sequence 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO:3).

[0433] In certain embodiments of the methods, compositions, or uses described above, the RNAi agent is siRNA, and the antisense strand of the siRNA comprises at least 15 or 19 adjacent nucleotides of the nucleotide sequence 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107). In some embodiments, the antisense strand of the siRNA comprises the nucleotide sequence 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107). In some embodiments, the antisense strand consists of the nucleotide sequence 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107). In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 106). In some embodiments, the sense strand of the siRNA consists of the nucleotide sequence 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 106).

[0434] In certain embodiments of the methods, compositions, or uses described above, the RNAi agent is 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, divalent, or trivalent linker. In some embodiments, the GalNAc derivative attached via a linker is or comprises the following:

[0435]

[0436] In a particular embodiment, the siRNA is conjugated to the ligand as shown in the figure below (i.e., the GalNAc derivative attached via the adapter is):

[0437]

[0438] Where X is O or S.

[0439] In a specific embodiment of the above methods, compositions, or uses in preparation, the RNAi agent is siRNA, wherein at least one nucleotide of the siRNA is a modified nucleotide, including deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, and 2'-O-allyl nucleotides. The siRNA may contain nucleotides modified with α-, 2'-C-alkyl, 2'-hydroxy, 2'-methoxyethyl, 2'-O-alkyl, morpholino, phosphatase, nucleotides containing non-natural bases, tetrahydropyran, 1,5-anhydrohexyl alcohol, cyclohexenyl, phosphate-thioester, methylphosphonate, 5'-phosphate, adenosine diol, or nucleotides containing 5'-phosphate analogs. In some embodiments, the siRNA contains phosphate backbone modification, 2'-ribose modification, 5'-triphosphate modification, or GalNAc conjugation modification. In some embodiments, the phosphate backbone modification contains a phosphate-thioester bond. In some embodiments, the 2'-ribose modification contains fluorine or -O-methyl substitution.

[0440] In specific embodiments of the above methods, compositions, or uses in preparation, the RNAi agent is a siRNA having a sense strand containing 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO:5) and an antisense strand containing 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:6).

[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] s is a thiophosphate linkage; and

[0444] L96 is N-[tris(GalNAc-alkyl)-amidodecyl)]-4-hydroxyprolyl.

[0445] In specific embodiments of the above methods, compositions, or uses in preparation, the RNAi agent is a siRNA having a sense strand containing 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO:7) and an antisense strand containing 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:8).

[0446] 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;

[0447] Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively;

[0448] (Agn) is adenosine diol nucleic acid (GNA);

[0449] s is a thiophosphate linkage; and

[0450] L96 is N-[tris(GalNAc-alkyl)-amidodecyl)]-4-hydroxyprolyl.

[0451] In specific 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).

[0452] 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;

[0453] Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively;

[0454] s is a thiophosphate linkage; and

[0455] L96 is N-[tris(GalNAc-alkyl)-amidodecyl)]-4-hydroxyprolyl.

[0456] In a particular embodiment of the above methods, compositions, or uses in preparation, the subject is a human being, and the RNAi or siRNA is administered to the subject at 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.

[0457] In some embodiments of the methods, compositions, or uses disclosed herein, siRNA is administered to the subject twice daily, once daily, every two days, every three days, twice weekly, once weekly, every other week, every four weeks, or once monthly. In some embodiments, the siRNA is administered to the subject every four weeks.

[0458] In some embodiments, the method includes administering two HBV gene expression inhibitors and an anti-HBV antibody. The two HBV gene expression inhibitors may be two siRNAs, such as two siRNAs targeting different HBV genes. The two different HBV genes may be, for example, HBsAg and HBV X. The two HBV gene expression inhibitors may be administered simultaneously. In some 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 a siRNA with a sense strand comprising at least 15 adjacent nucleotides from nucleotides 2850 to 3182 of SEQ ID NO:1. In some 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 a siRNA with a sense strand comprising at least 15 adjacent nucleotides from nucleotides 2850 to 3182 of SEQ ID NO:1. In some embodiments, two siRNAs, each directed to the HBV gene, are administered, wherein 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 some 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.

[0459] In some embodiments, anti-HBV antibodies and HBV gene expression inhibitors exhibit synergistic therapeutic effects. The term "synergistic effect" is used to describe the combined effect of two or more active agents that 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 the therapeutic effect observed with a combination of two or more therapies, where the therapeutic effect (e.g., measured by any of a plurality of parameters) is greater than the sum of the individual therapeutic effects observed with each of the respective individual therapies.

[0460] In some embodiments, an RNAi agent targeting HBV mRNA is administered to a subject with HBV infection and / or HBV-related disease, resulting in a reduction of 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%, or 47% in the 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). 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.

[0461] In some embodiments, an RNAi agent targeting HBV mRNA is administered to a subject with HBV infection and / or HBV-related disease, inhibiting HBV gene expression 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%, and 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%, or 99%, or approximately 100% (i.e., below the measured detection level).

[0462] In some embodiments, the combination therapy according to this disclosure comprises administration of 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 a nucleotide or nucleoside 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), clavidine, ritonavir, dipyridoxine, lobucavir, famciclovir, N-acetylcysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-α, ganciclovir, basiloxane (ANA-380 / LB-80380), and tenofovir exaliades (TLX / CMX157). In some embodiments, the nucleoside (acid) analogue is entecavir (ETV). Nucleoside (acid) analogues are commercially available from many sources and are used in the methods provided herein according to their label instructions (e.g., typically administered orally at a specific dose) or as determined by those skilled in the art for treating HBV.

[0463] Anti-HBV antibodies or HBV gene expression inhibitors may be present in the same pharmaceutical composition as the third active ingredient, or anti-HBV antibodies, HBV gene expression inhibitors, and the third active ingredient may be present in three different pharmaceutical compositions. These different pharmaceutical compositions may be administered in combination / simultaneously, at different times, or at different locations (e.g., different parts of the body).

[0464] V. Kits for HBV combination therapy

[0465] This document provides kits containing components of HBV therapy. In some embodiments, the kit contains one or more anti-HBV antibodies, one or more HBV gene expression inhibitors, and optionally a third component of HBV combination therapy (e.g., a nucleoside (acid) analog). The kit may additionally include instructions for preparing and / or administering the components of HBV combination therapy.

[0466] This application also provides the following implementation schemes

[0467] Implementation Scheme 1. An HBV gene expression inhibitor for treating chronic HBV infection in a subject, wherein an anti-HBV antibody is subsequently administered to the subject.

[0468] Implementation Scheme 2. An agent for treating chronic HBV infection in a subject by reducing HBV antigen load, wherein an anti-HBV antibody is subsequently administered to the subject.

[0469] Implementation Scheme 3. A composition for treating chronic HBV infection in a subject, wherein the composition comprises an anti-HBV antibody, and the subject has previously been administered a gene expression inhibitor.

[0470] Implementation Scheme 4. A composition for treating chronic HBV infection in a subject, wherein the composition comprises an anti-HBV antibody, and the subject has previously been administered an agent that reduces HBV antigen load.

[0471] Implementation Scheme 5. The composition according to Implementation Scheme 2 or Implementation Scheme 3, wherein the expression of at least one HBV gene is reduced after administration of the HBV gene expression inhibitor or the agent for reducing 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.

[0472] Implementation Plan 6. Use of HBV gene expression inhibitors and anti-HBV antibodies in the preparation of drugs for the treatment of chronic HBV infection.

[0473] Implementation Plan 7. Use of agents that reduce HBV antigen load and anti-HBV antibodies in the preparation of medicaments for the treatment of chronic HBV infection.

[0474] Implementation Plan 8. A method for treating chronic HBV infection in individuals in need, comprising:

[0475] Administering an agent that reduces HBV antigen load to the subject; and

[0476] Anti-HBV antibody was administered to the subject.

[0477] Implementation Scheme 9. A method for treating chronic HBV infection in individuals in need, comprising:

[0478] Administering an HBV gene expression inhibitor to the subject; and

[0479] Anti-HBV antibody was administered to the subject.

[0480] Implementation Scheme 10. The method according to Implementation Scheme 8 or Implementation Scheme 9, wherein the expression of at least one HBV gene is reduced after administration of an agent that reduces HBV antigen load or an HBV gene expression inhibitor, and the anti-HBV antibody is administered when the expression of said at least one HBV gene is reduced.

[0481] Implementation Scheme 11. The method according to any one of Implementation Schemes 8-10, further comprising measuring the amount of HBsAg present in a blood sample from the subject before and after administration of the agent reducing HBV antigen load or the HBV expression inhibitor, wherein a reduction in HBsAg indicates a decrease in the expression of the at least one HBV gene.

[0482] Implementation Scheme 12. The method, composition, or use according to any of the preceding embodiments, wherein the therapeutically effective amount of the anti-HBV antibody is lower than the therapeutically effective amount of the anti-HBV antibody delivered when the subject is not administered the HBV gene expression inhibitor or the agent that reduces HBV antigen load.

[0483] Implementation Scheme 13. The method, composition, or use according to any of the preceding embodiments, wherein administering the anti-HBV antibody comprises administering at least two doses of a therapeutically effective amount of the anti-HBV antibody.

[0484] Implementation Scheme 14. The method, composition, or use according to Implementation Scheme 13, wherein the at least two agents are applied twice a week, once a week, every other week, every two weeks, or once a month.

[0485] Implementation Scheme 15. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody is administered at least one week after administration of the HBV gene expression inhibitor or the agent reducing HBV antigen load. Implementation Scheme 16. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody is administered 8 weeks after administration of the HBV gene expression inhibitor or the agent reducing HBV antigen load.

[0486] Implementation Scheme 17. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody is administered subcutaneously.

[0487] Implementation Scheme 18. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody recognizes HBV genotypes A, B, C, D, E, F, G, H, I, and J.

[0488] Implementation Scheme 19. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody is a human antibody.

[0489] Implementation Scheme 20. The method, composition, or use according to any of the preceding embodiments, wherein the antibody is HBC34 or a non-natural variant of HBC34.

[0490] Implementation Scheme 21. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0491] (i) Based on the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 44, 45 or 46, and 47, respectively; and

[0492] (ii) Based on the amino acid sequences of CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO: 48, 49 or 50, and 52 or 51, respectively.

[0493] Implementation Scheme 22. The method, composition, or use according to Implementation Scheme 21, wherein the anti-HBV antibody comprises: (i) amino acid sequences of CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NO: 44, 45, and 47, respectively; and

[0494] (ii) amino acid sequences of CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NO: 48, 49, and 52, respectively. Embodiment 23. The method, composition, or use according to Embodiment 21, wherein the anti-HBV antibody comprises: (i) amino acid sequences of CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NO: 44, 45, and 47, respectively; and

[0495] (ii) Based on the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 48, 49, and 51, respectively. Embodiment 24. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0496] (a) Light chain variable structural domain (V) L (a) having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 55-69; and (b) a heavy chain variable domain (V H ), which has at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 53; or

[0497] (a) Light chain variable structural domain (V) L (a) having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 55-69; and (b) a heavy chain variable domain (V H It has at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 54.

[0498] Implementation Scheme 25. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0499] (a) According to the light chain variable domain (V) of SEQ ID NO: 59 L (a) amino acid sequence; and (b) the heavy chain variable domain (V) according to SEQ ID NO: 53. H ) amino acid sequence.

[0500] Implementation Scheme 26. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0501] (a) The light chain variable domain (V) according to SEQ ID NO: 56 or 58 L (a) amino acid sequence; and (b) the heavy chain variable domain (V) according to SEQ ID NO: 53. H ) amino acid sequence.

[0502] Implementation Scheme 27. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0503] (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 73, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 70-72 and 97; or

[0504] (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 74, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 70-72 and 97; or

[0505] (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 83-95, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 70-72, 97, and 98.

[0506] Implementation Scheme 28. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0507] (a) the light chain amino acid sequence shown in SEQ ID NO: 73, and (b) the heavy chain amino acid sequence shown in SEQ ID NO: 70; or

[0508] (a) the light chain amino acid sequence shown in SEQ ID NO: 73, and (b) the heavy chain amino acid sequence shown in SEQ ID NO: 71.

[0509] Implementation Scheme 29. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0510] (a) the light chain amino acid sequence shown in SEQ ID NO: 74, and (b) the heavy chain amino acid sequence shown in SEQ ID NO: 70.

[0511] Implementation Scheme 30. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0512] (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 73, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 70-72 and 97; or

[0513] (a) a light chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 74, and (b) a heavy chain having at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 70-72 and 97.

[0514] Implementation Scheme 31. The method, composition, or use according to any one of Implementation Schemes 1-19, wherein the anti-HBV antibody comprises:

[0515] (a) A CDR having the amino acid sequence according to SEQ ID NO: 77-82; or

[0516] (b)(i) According to the light chain variable structural domain (V) of SEQ ID NO: 76 L (i) amino acid sequence; and (ii) the heavy chain variable domain (V) according to SEQ ID NO: 75 H ) amino acid sequence.

[0517] Implementation Scheme 32. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody is a monoclonal antibody.

[0518] Implementation Scheme 33. The method, composition, or use according to any of the preceding embodiments, wherein the anti-HBV antibody is a bispecific antibody having a first specificity against HBsAg and a second specificity against an immune effector.

[0519] Implementation Scheme 34. The method, composition, or use according to Implementation Scheme 33, wherein the second specific stimulus cytotoxicity or vaccine effect is described.

[0520] Implementation Scheme 35. The method, composition, or use according to any of the preceding embodiments, wherein the subject is a human, and a therapeutically effective amount of the anti-HBV antibody is administered; wherein the therapeutically effective amount is from about 3 mg / kg to about 30 mg / kg. Implementation Scheme 36. The method, composition, or use according to any of the preceding embodiments, wherein the HBV gene expression inhibitor or the agent for reducing HBV antigen load is an RNAi agent that inhibits HBV transcript expression.

[0521] Implementation Scheme 37. The method, composition, or use according to Implementation Scheme 36, wherein the inhibition of HBV transcript expression is measured by rtPCR.

[0522] Implementation Scheme 38. The method, composition, or use according to Implementation Scheme 36, wherein the inhibition of HBV transcript expression is measured by a reduction in protein levels, said reduction in protein levels being measured by enzyme-linked immunosorbent assay (ELISA).

[0523] Implementation Scheme 39. The method, composition, or use according to any one of Implementation Schemes 36-38, wherein 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 from nucleotides 1579-1597 of SEQ ID NO: 1 by no more than 3 nucleotides.

[0524] Implementation Scheme 40. The method, composition, or use according to any one of Implementation Schemes 36-39, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotides 1579-1597 of SEQ ID NO: 1.

[0525] Implementation Scheme 41. The method, composition, or use according to any one of Implementation Schemes 36-40, wherein at least one strand of the RNAi agent comprises a 3' overhang of at least one nucleotide.

[0526] Implementation Scheme 42. The method, composition, or use according to any one of Implementation Schemes 36-40, wherein at least one strand of the RNAi agent comprises a 3' overhang of at least two nucleotides.

[0527] Implementation Scheme 43. The method, composition, or use according to any one of Implementation Schemes 36-42, wherein the length of the double-stranded region of the RNAi agent is 15-30 nucleotide pairs.

[0528] Implementation Scheme 44. The method, composition, or use according to any one of Implementation Schemes 36-42, wherein the length of the double-stranded region of the RNAi agent is 17-23 nucleotide pairs.

[0529] Implementation Scheme 45. The method, composition, or use according to any one of Implementation Schemes 36-42, wherein the length of the double-stranded region of the RNAi agent is 17-25 nucleotide pairs.

[0530] Implementation Scheme 46. The method, composition, or use according to any one of Implementation Schemes 36-42, wherein the length of the double-stranded region of the RNAi agent is 23-27 nucleotide pairs.

[0531] Implementation Scheme 47. The method, composition, or use according to any one of Implementation Schemes 36-42, wherein the length of the double-stranded region of the RNAi agent is 19-21 nucleotide pairs.

[0532] Implementation Scheme 48. The method, composition, or use according to any one of Implementation Schemes 36-42, wherein the length of the double-stranded region of the RNAi agent is 21-23 nucleotide pairs.

[0533] Implementation Scheme 49. The method, composition, or use according to any one of Implementation Schemes 36-48, wherein each strand of the RNAi agent has 15-30 nucleotides.

[0534] Implementation Scheme 50. The method, composition, or use according to any one of Implementation Schemes 36-48, wherein each strand of the RNAi agent has 19-30 nucleotides.

[0535] Implementation Scheme 51. The method, composition, or use according to any one of Implementation Schemes 36-50, wherein the RNAi agent is siRNA.

[0536] Implementation Scheme 52. The method, composition, or use according to Implementation Scheme 51, wherein the siRNA inhibits the expression of HBV transcripts, the HBV transcripts encoding HBsAg protein, HBcAg protein, and HBx protein or HBV DNA polymerase protein.

[0537] Implementation Scheme 53. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the siRNA binds to at least 15 consecutive nucleotides encoded by the following: P gene, nucleotides 2309-3182 and 1-1625 of NC_003977.2; 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 C gene, nucleotides 1816-2454 of NC_003977.2.

[0538] Implementation Scheme 54. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of said siRNA comprises at least 15 consecutive nucleotides of said nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0539] Implementation Scheme 55. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of said siRNA comprises at least 19 consecutive nucleotides of said nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0540] Implementation Scheme 56. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of said siRNA comprises the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0541] Implementation Scheme 57. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of the siRNA consists of the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0542] Implementation Scheme 58. The method, composition, or use according to any one of Implementation Schemes 54-57, wherein the positive strand of the siRNA comprises the nucleotide sequence 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

[0543] Implementation Scheme 59. The method, composition, or use according to any one of Implementation Schemes 54-57, wherein the sense strand of the siRNA comprises the nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

[0544] Implementation Scheme 60. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of said siRNA comprises at least 15 consecutive nucleotides of said nucleotide sequence 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107).

[0545] Implementation Scheme 61. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of said siRNA comprises at least 19 consecutive nucleotides of said nucleotide sequence 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107).

[0546] Implementation Scheme 62. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of said siRNA comprises the nucleotide sequence 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107).

[0547] Implementation Scheme 63. The method, composition, or use according to Implementation Scheme 51 or Implementation Scheme 52, wherein the antisense strand of the siRNA consists of the nucleotide sequence of 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 107).

[0548] Implementation Scheme 64. The method, composition, or use according to any one of Implementation Schemes 54-57, wherein the positive strand of the siRNA comprises the nucleotide sequence 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 106).

[0549] Implementation Scheme 65. The method, composition, or use according to any one of Implementation Schemes 54-57, wherein the positive strand of the siRNA consists of the nucleotide sequence 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 106).

[0550] Implementation Scheme 66. The method, composition, or use according to any one of Implementation Schemes 51-65, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached to the 3'-terminus.

[0551] Implementation Scheme 67. The method, composition, or use according to Implementation Scheme 66, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branching connector.

[0552] Implementation Scheme 68. The method, composition, or use according to Implementation Scheme 66 or 67, wherein the ligand is

[0553] Implementation Scheme 69. The method, composition, or use according to Implementation Scheme 68, wherein the siRNA is conjugated to the ligand as shown in the following structure:

[0554]

[0555] Where X is O or S.

[0556] Implementation Scheme 70. The method, composition, or use according to any one of Implementation Schemes 51-69, wherein at least one nucleotide of said siRNA is a modified nucleotide, including deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleosyl nucleotides, etc. Nucleotides, nucleotides modified with 2'-C-allyl, nucleotides modified with 2'-hydroxyl, nucleotides modified with 2'-methoxyethyl, nucleotides modified with 2'-O-alkyl, morpholinonucleotides, aminophosphates, non-natural bases containing nucleotides, nucleotides modified with tetrahydropyran, nucleotides modified with 1,5-dehydrohexyl alcohol, nucleotides modified with cyclohexenyl, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, adenosine glycol nucleic acids, or nucleotides containing 5'-phosphate mimics.

[0557] Implementation Scheme 71. The method, composition, or use according to any one of Implementation Schemes 51-70, wherein the siRNA comprises a phosphate backbone modification, a 2' ribose modification, a 5' triphosphate modification, or a GalNAc conjugation modification.

[0558] Implementation Scheme 72. The method, composition, or use according to Implementation Scheme 71, wherein the phosphate backbone modification comprises a thiophosphate bond.

[0559] Implementation Scheme 73. The method, composition, or use according to Implementation Scheme 71 or Implementation Scheme 72, wherein the 2' ribose modification comprises fluorine or -O-methyl substitution.

[0560] Implementation Scheme 74. The method, composition, or use according to any one of Implementation Schemes 51-59 and 66-73, 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),

[0561] 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;

[0562] Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively;

[0563] s is a thiophosphate linker; and

[0564] L96 is N-[tris(GalNAc-alkyl)-amide-decanoyl)]-4-hydroxyprolyl.

[0565] Embodiment 75. The method, composition, or use according to any one of Embodiments 51-59 and 66-73, wherein 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),

[0566] 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;

[0567] Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively;

[0568] (Agn) is adenosine-glycol nucleic acid (GNA);

[0569] s is a thiophosphate linker; and

[0570] L96 is N-[tris(GalNAc-alkyl)-amide-decanoyl)]-4-hydroxyprolyl.

[0571] Embodiment 76. The method, composition, or use according to any one of Embodiments 51-53 and 60-73, 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).

[0572] 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;

[0573] Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively;

[0574] s is a thiophosphate linker; and

[0575] L96 is N-[tris(GalNAc-alkyl)-amide-decanoyl)]-4-hydroxyprolyl.

[0576] Implementation Scheme 77. The method, composition, or use according to any one of Implementation Schemes 36-76, wherein the subject is a human being, and a therapeutically effective amount of RNAi agent or siRNA is administered to the subject; and wherein the effective amount of said RNAi agent or siRNA is from about 1 mg / kg to about 8 mg / kg.

[0577] Implementation Scheme 78. The method, composition, or use according to any one of Implementation Schemes 36-77, wherein the RNAi agent or siRNA is administered to the subject twice daily, once daily, once every two days, once every three days, twice weekly, once weekly, once every other week, once every four weeks, or once monthly.

[0578] Implementation Scheme 79. The method, composition, or use according to any one of Implementation Schemes 36-77, wherein the RNAi agent or siRNA is administered to the subject every four weeks.

[0579] Implementation Scheme 80. The method, composition, or use according to any one of Implementation Schemes 51-79, wherein two siRNAs, each targeting the 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 consecutive nucleotides of nucleotides 2850-3182 of SEQ ID NO: 1.

[0580] Implementation Scheme 81. The method, composition, or use according to any one of Implementation Schemes 51-79, wherein two siRNAs targeting HBV genes are administered, wherein the two siRNAs comprise: an siRNA targeting the HBV X gene and an siRNA targeting the HBV S gene.

[0581] Implementation Scheme 82. The method, composition, or use according to any one of Implementation Schemes 51-79, wherein two siRNAs, each targeting the 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 has an antisense strand comprising SEQ ID NO: 107 or SEQ ID NO: 109.

[0582] Implementation Scheme 83. The method, composition, or use according to Implementation Scheme 82, wherein the first siRNA has a positive strand comprising SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; and the second siRNA has a positive strand comprising SEQ ID NO: 106 or SEQ ID NO: 108.

[0583] Implementation Scheme 84. The method, composition, or use according to any one of Implementation Schemes 80-83, wherein the two siRNAs are administered simultaneously.

[0584] Implementation Scheme 85. The method, composition, or use according to any of the preceding embodiments further includes administering a nucleoside (acid) analog to the subject, or wherein a nucleoside (acid) analog is further administered to the subject.

[0585] Implementation Scheme 86. The method, composition, or use according to Implementation Scheme 85, wherein the nucleoside (acid) analogue is tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavidine, ritonavir, dipivoxil, adefovir dipivoxil, lobucavir, famciclovir, N-acetylcysteine ​​(NAC), PC1323, theradigm-HBV, thymosin α, and ganciclovir, basil (ANA-380 / LB-80380), or tenofovir exaliades (TLX / CMX157).

[0586] Implementation Scheme 87. A reagent kit comprising:

[0587] A pharmaceutical composition comprising an RNAi agent targeting mRNA encoded by the HBV gene and a pharmaceutically acceptable excipient; and

[0588] A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient.

[0589] Implementation Scheme 88. The kit according to Implementation Scheme 69, wherein the RNAi agent comprises siRNA as described in any one of Embodiments 51-76.

[0590] Implementation Scheme 89. The kit according to Implementation Scheme 87 or Implementation Scheme 88, wherein the anti-HBV antibody comprises an antibody as described in any one of Embodiments 18-34.

[0591] Example

[0592] Example 1

[0593] Combination therapy of antibodies and HBV-targeting siRNA reduces biomarkers of HBV infection in AAV-HBV mice.

[0594] To determine whether siRNA-antibody combination therapy might be effective in treating HBV infection, AAV / HBV-infected C57BL / 6 mice were administered one of the following fourteen different treatments: (1) HBV-specific siRNA (HBV02, with the antisense strand of SEQ ID NO: 8); (2)-(5) one of four doses of anti-HBV antibody (HBC34 antibody HBC34v7, 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).

[0595] Table 4: Treatment Levels and Doses

[0596]

[0597] HBV02 is a chemically synthesized double-stranded oligonucleotide covalently linked to a ligand containing three GalNAc residues. All nucleotides are modified with 2'-OMe or 2'-F, and one nucleic acid in the antisense strand is replaced with (S)-1-(2,3-dihydroxypropyl)adenosine (Agn). The nucleotides of the sense and antisense strands are linked by 3'-5' phosphodiester linkages or 3'-5' thiophosphate linkages to form the sugar-phosphate backbone of the oligonucleotide.

[0598] HBC34 is a highly neutralizing monoclonal antibody against HBV surface antigens (PreS1, PreS2, and S). The HBC34 antibody used in this experiment was a fully murine-derived HBC34v7, except for the Fab fragment portion that binds to the HBV surface antigens. Human HBC34v7 has the V-shaped structure shown in SEQ ID NO:53. H Sequence and V as shown in SEQ ID NO:56 L Sequence. The mouse chimeric form of the HBC34v7 sequence in the HBC34-v7-mu-IgG2a antibody used in this experiment has heavy and light chain amino acid sequences as shown in SEQ ID NO:99 and 100, respectively.

[0599] The siRNA targeting the human thyroxine transporter gene was used as a control siRNA because it was not expected to cause a reduction in HBV infection markers in serum.

[0600] The control monoclonal antibody (mAb) used in this embodiment is a respiratory syncytial virus-specific antibody, which is not expected to cause a reduction in HBV markers of infection in the serum.

[0601] Mice (C57BL / 6 strain) were inoculated with the following amount of rAAV8-1.3HBV strain ayw, type D: via tail vein injection, 1.0 x 10^6 mg / m² per mouse. 11 One viral genome was collected in a 200 μl volume. Treatment with the test compound was initiated four weeks after viral inoculation.

[0602] Dosing regimen such as Figure 1 As shown. 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 weeks three and four. A portion of the mice were sacrificed in week four, and another portion in week six.

[0603] Twice weekly, viral load, HBsAg, and free HBBC34 antibody were measured from serum samples. Serum HBeAg, serum alanine transferase (ALT), liver HBcAg, liver HBsAg, total HBV DNA in the liver (by qPCR), and serum anti-HBV antibodies were also measured. Hepatic lymphocytes, spleen cells, and lymph nodes (portal vein / peritoneal and groin) were analyzed to determine HBV-specific IFNg. + CD4 + Cells and IFNg + CD8 + The proportion of cells.

[0604] The mean HBsAg values ​​in the treatment group are shown in Table 5.

[0605]

[0606] Figure 2A and 2B The viral load, as measured by HBV DNA copy number, is shown. Figure 3A and 3B Serum HBsAg levels were displayed. Figure 2A and 3A The results show viral load and HBsAg levels after administration of HBV02 siRNA or HBC34 antibody (15 mg / kg) alone. Compared to the saline control, HBV02 siRNA reduced serum HBV DNA and HBsAg by approximately 0.5-log. 10 and 1-log 10 Compared to the saline control, the HBC34 antibody alone had no effect on HBV DNA and reduced serum HBsAg by <1-log. 10 . Figure 2B and 3B The results showed that, compared with the saline control, treatment with HBV02 siRNA and HBC34 antibody (15 mg / kg) reduced viral load and HBsAg levels by approximately 3-log. 10 Compared to either molecule alone, the combined use of HBV02 siRNA and HBC34 antibody resulted in significantly stronger reductions in serum HBV DNA and HBsAg, with the combined effect exceeding the sum of the effects of single therapies. The combination therapy also reduced viral load and HBsAg levels more effectively than entecavir alone. The effect of the HBV02 siRNA and HBC34 antibody combination was observed regardless of whether entecavir was administered. These results suggest that HBV02 siRNA and HBC34 antibody have the potential for a synergistic effect in reducing viral load and HBsAg, independent of entecavir treatment.

[0607] Figure 4 The study depicted free HBC34 antibody levels measured between 14 and 42 days after the start of the study on day 1 (day 1 = administration of siRNA to the selected treatment group). Compared to HBC34 treatment alone, regardless of whether entecavir was included in the treatment, combination therapy with HBV02 siRNA and HBC34 antibody resulted in higher initial free antibody levels, which were maintained for more than 28 days. Figure 4 The results shown, along with viral load and serum HBsAg levels, indicate that treatment efficacy depends on the amount of free circulating HBBC34 antibody, and that lower doses of antibody may become effective as HBsAg load decreases. For example, combination therapy may allow for effective treatment via lower doses of antibody, lower antibody dosage, and / or less invasive routes of administration (e.g., subcutaneous rather than intravenous), based at least in part on reducing HBsAg load prior to antibody therapy.

[0608] In summary, this study demonstrates that administering siRNA targeting HBV followed by HBV antibody treatment effectively reduces serum HBV DNA and HBsAg. Furthermore, the components appear to interact synergistically, thus this combination therapy is more effective than using each component alone, and also greater than the expected effect if the effects were merely additive. Finally, the results indicate that siRNA administration reduces serum HBsAg, thereby enhancing the effectiveness of the antibody treatment.

[0609] Example 2

[0610] Combination therapy of one of two anti-HBV antibodies and HBV-targeting siRNA

[0611] To determine whether the siRNA-antibody combination therapy using siRNA and anti-HBV antibody HBC24 was effective in treating HBV infection, C57BL / 6 mice infected with AAV / HBV 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 murine 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).

[0612] Table 6: Treatment levels and dosages in Example 2

[0613]

[0614] The HBC24 and HBC34 antibodies used in this experiment were completely murine-derived, except for the Fab fragment portion that binds to the HBV surface antigen. Human HBC24 has the V-shaped structure shown in SEQ ID NO:75. H The amino acid sequence and V as shown in SEQ ID NO:76 L Amino acid sequences. The murine HBC24 sequence of the antibody used in this experiment has a heavy chain and a light chain, comprising the amino acid sequences listed in SEQ ID NO:103 and 104, respectively. The HBC34 antibody is a murine HBC34v35 variant, HBC34-v35-mu-IgG2a. 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 murine HBC34v35 sequence of the HBC34-v35-mu-IgG2a antibody used in this experiment has a heavy chain and a light chain, comprising the amino acid sequences listed in SEQ ID NO:101 and 102, respectively.

[0615] siRNA targeting the human transthyretin gene is not expected to lead to a reduction in serum HBV infection markers.

[0616] Control monoclonal antibodies (mAbs) are specific antibodies against respiratory syncytial virus and are not expected to cause a reduction in HBV infection markers in serum.

[0617] This study investigated the treatment of immunocompromised HBV mice (WuXi immunocompromised HBV mouse) by WuXi AppTec. This mouse model was created by transducing hepatocytes containing the HBV genome into immunocompetent mice. Using this model, HBV protein production was controlled by an endogenous HBV promoter, and the mice produced HBV-specific cellular and humoral T-cell responses. However, no HBV infection occurred, no cccDNA was produced, replication was transient, and the immune response was hampered by vector-driven interference.

[0618] Mice (C57BL / 6 strain) were inoculated with the following amount of rAAV8-1.3HBV strain ayw, type D: via tail vein injection, 1.0 x 10^6 mg / m² per mouse. 11 One viral genome, in a 200 μl volume.

[0619] Each treatment group consisted of five mice. At the start of the study, HBV-specific siRNA was administered subcutaneously once, followed by intraperitoneal administration of anti-HBV antibodies twice weekly during weeks two and three. Mice were sacrificed at week six.

[0620] Serum samples were collected periodically throughout the study to measure viral load, HBsAg, and free HBBC34 antibodies. Serum HBeAg, serum alanine transferase (ALT), liver HBcAg, liver HBsAg, total HBV DNA in the liver (by qPCR), and serum anti-HBV antibodies were also measured. Hepatic lymphocytes, spleen cells, and lymph nodes (portal vein / peritoneal and groin) were analyzed to determine HBV-specific IFNg. + CD4 + Cells and IFNg + CD8 + The proportion of cells.

[0621] Experimental results are as follows Figure 5A and 5B (serum HBV DNA concentration) Figure 6A and 6B (serum HBsAg concentration) and Figure 7A and 7B(Serium HBeAg concentration) is shown. Mice treated with HBV02 and one of the anti-HBV antibodies had lower serum HBV DNA, HBsAg, and HBeAg concentrations compared to mice treated with siRNA alone or as a control. This effect was observed for both HBC34 and HBC24 antibodies. Furthermore, higher doses of HBV02 were more effective, and when higher doses of HBV02 were used, a reduction in HBsAg was achieved at lower antibody doses. These results provide further evidence that combination therapy with HBV02 and an HBV-targeting monoclonal antibody is more effective at reducing HBsAg and HBeAg than HBV02 monotherapy. These results also suggest that higher doses of siRNA prior to antibody administration can provide a similar reduction in HBsAg at lower antibody doses.

[0622] Example 3

[0623] Serum clearance of HBsAg and inhibition of viral entry in mouse models

[0624] 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) used uPA / SCID mice to generate mice with ≥70% mouse liver proliferating human hepatocytes (Ohshita H and Tateno C, Methods MolBiol. 1506:91-100, (2017)). Unlike the AAV-HBV model, cccDNA was established and intrahepatic transmission of HBV is possible.

[0625] Primary human hepatocytes were transplanted into SCID mice, whose hepatocytes had been previously enzymatically destroyed. The mice lacked T cells and B cells. This model can be used to study HBV infection, including entry, transmission, cccDNA regulation, intrinsic hepatocyte 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 adaptive immune responses. Mice were inoculated via tail vein, with each mouse receiving 1.0 x 10^6 mg of the drug. 7 The viral genome is HBV genotype C. Treatment begins eight weeks post-infection.

[0626] HBV-infected mice (n=4, each treatment group) were administered one of seven different treatments: (1) PBS alone; (2–4) anti-HBV antibody (fully murine 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, with the antisense strand of SEQ ID NO:8; see description in Example 1) administered subcutaneously once at the start of the study, and fully murine HBC34v35 administered intraperitoneally twice weekly at one of three doses during weeks 2 and 3 (see Table 7). Mice were sacrificed at week 6. Study design also see Figure 8 .

[0627] Table 7: Treatment Levels and Doses

[0628]

[0629] The HBC34 antibody HBC34v35 used in this experiment was completely murine-derived, except for the Fab fragment portion 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 murine form of the HBC34-v35-mu-IgG2a antibody used in this experiment, HBC34v35, has a heavy chain and a light chain, containing the amino acid sequences listed in SEQ ID NO:101 and 102, respectively.

[0630] Serum samples were collected periodically throughout the study to measure viral load, HBsAg, and free HBBC34 antibodies. Serum HBeAg, serum alanine transferase (ALT), liver HBcAg, liver HBsAg, total HBV DNA in the liver (by qPCR), and serum anti-HBV antibodies were also measured. Hepatic lymphocytes, spleen cells, and lymph nodes (portal vein / peritoneal and groin) were analyzed to determine HBV-specific IFNg. + CD4 + Cells and IFNg + CD8 + The proportion of cells.

[0631] Compared to administering the same dose of antibody, the combination of siRNA and anti-HBV antibody administration reduced serum HBV DNA concentration. Figure 9 ) and serum HBsAg concentration ( Figure 10 Regarding serum HBeAg concentration ( Figure 11 ) and serum HBcrAg concentration ( Figure 12Similar trends were observed. Furthermore, in this model system, the antibody also acted as an inhibitor of viral entry into hepatocytes; when 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 The value is also relatively low.

[0632] This study provides experimental support in a real infection model that when HBV02 siRNA and HBC34 antibody are used in combination, the reduction in HBV DNA and HBsAg levels is greater compared to HBC34 monotherapy.

[0633] Example 4

[0634] Clinical evaluation of siRNA antibody combination therapy for chronic HBV infection

[0635] A phase 2 clinical trial of siRNA-antibody combination therapy was conducted to evaluate its efficacy in human patients with chronic HBV infection. Table 8 shows the treatment regimen of this study. The study may include additional cohorts to test the effect of additional treatments on the combination therapy (e.g., nine cohorts if two additional treatments are tested). Each group / cohort includes fifteen patients.

[0636] Table 8: Treatment levels and dosages in clinical trials

[0637]

[0638] Figure 13The treatment regimen designed for a phase 2 study is shown. The study consisted of 24 weeks of treatment and 24 weeks of follow-up. All patients were free of cirrhosis and had suppressed NUC (treated with nucleoside (acid) analogs) at study entry. Throughout the study, all patient groups could receive NUC treatment (e.g., daily oral tenofovir or entecavir). The study began with all cohorts receiving an eight-week induction therapy with HBV02 siRNA. For example, the dose of HBV02 could be two 400 mg doses administered subcutaneously every four weeks. However, an appropriate dose could be determined through a monotherapy trial prior to the phase 2 trial. After eight weeks of the study, all cohorts continued treatment with HBV02; cohorts 2, 4, and 6 began treatment with a low dose of HBC34 antibody (HBC34v35); cohorts 3, 5, and 7 began treatment with a higher dose of HBC34 antibody; and cohort 1 did not r...

Claims

1. An HBV gene expression inhibitor for treating chronic HBV infection in a subject, wherein an anti-HBV antibody is subsequently administered to the subject.

2. An agent for treating chronic HBV infection in a subject by reducing HBV antigen load, wherein an anti-HBV antibody is subsequently administered to the subject.

3. A composition for treating chronic HBV infection in a subject, wherein the composition comprises an anti-HBV antibody, and the subject has previously been administered a gene expression inhibitor.

4. A composition for treating chronic HBV infection in a subject, wherein the composition comprises an anti-HBV antibody, and the subject has previously been administered an agent that reduces HBV antigen load.

5. The composition according to claim 2 or claim 3, wherein the expression of at least one HBV gene is reduced after administration of the HBV gene expression inhibitor or the agent for reducing 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.

6. Use of HBV gene expression inhibitors and anti-HBV antibodies in the preparation of drugs for the treatment of chronic HBV infection.

7. Use of agents that reduce HBV antigen load and anti-HBV antibodies in the preparation of drugs for the treatment of chronic HBV infection.

8. A method for treating chronic HBV infection in individuals in need, comprising: The agent that reduces HBV antigen load was administered to the subject; and Anti-HBV antibody was administered to the subject.

9. A method for treating chronic HBV infection in individuals in need, comprising: Administer an HBV gene expression inhibitor to the subject; and Anti-HBV antibody was administered to the subject.

10. A reagent kit comprising: A pharmaceutical composition comprising an RNAi agent that targets mRNA encoded by the HBV gene and a pharmaceutically acceptable excipient; and A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient.

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