Compositions and methods for treating hepatitis delta virus (HDV) infection and related diseases

Abstract

The present disclosure provides methods of treating hepatitis D virus (HDV) infection and / or HDV-related diseases using combination therapy, as well as related kits and compositions for use in such methods. Components of these combination therapies may include one or more of: an anti-HBV antibody; siRNA that is targeted to HBV mRNA; and a nucleoside (acid) reverse transcriptase inhibitor (NRTI).

Description

[0001] References to Electronic Sequence Listings

[0002] The contents of the electronic sequence listing (444WO_SeqListing.xml; size: 70.5KB; creation date: May 5, 2023) are incorporated herein by reference in their entirety. Background Art

[0003] Hepatitis D, also known as "hepatitis D", is a viral infection caused by the hepatitis D virus (HDV). HDV is a defective RNA satellite virus that does not encode its own envelope protein and is dependent on the expression of the hepatitis B virus (HBV) surface antigen (HBsAg) to complete its life cycle and produce infectious HDV virions. Therefore, HDV must be co-infected or superinfected with HBV.

[0004] Approximately 300 million people worldwide live with chronic HBV infection (Polaris Observatory Collaborators, Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study, Lancet Gastroenterol Hepatol. 2018 Jun; 3(6):383-403) and are at risk for serious sequelae, including cirrhosis, liver failure, and hepatocellular carcinoma (HCC). The global prevalence of HDV was estimated in three recent meta-analyses, with results ranging from 12 million (Stockdale AJ et al., The global prevalence of hepatitis D virus infection: Systematic review and meta-analysis, J Hepatol. 2020 Sep; 73(3):523-532) to 60 to 72 million (Chen HY et al., Prevalence and burden of hepatitis D virus infection in the global population: a systematic review and meta-analysis, Gut 2019 Mar; 68(3):512-521; Miao Z et al., Estimating the Global Prevalence, Disease Progression, and Clinical Outcome of Hepatitis Delta Virus Infection, J Infect Dis. 2020 Apr 27; 221(10):1677-1687). HDV infection is the most aggressive form of viral hepatitis, as rapid progression to liver failure, cirrhosis, HCC, and death has been observed in patients with chronic HBV / HDV co-infection (Lee AU and Lee C, Hepatitis D Review: Challenges for the Resource-Poor Setting, Viruses. 2021 Sep 23, 13(10):1912; Stockdale et al., 2020, supra).Among people with chronic HDV infection, 85% to 95% may develop cirrhosis and liver failure within 10 years of infection, with some developing these complications as early as 1 to 2 years after infection (-15%) (Kamili S et al., Delta hepatitis: Toward improved diagnostics, Hepatology, Dec 2017, 66(6):1716-1718; NIH, National Institute of Diabetes and Digestive and Kidney Diseases, United States 2017, Department of Health and Human Services, accessed April 10, 2020; Rizzetto M, Hepatitis D Virus: Introduction and Epidemiology, Cold Spring Harb Perspect Med. Jul 1, 2015, 5(7), a021576; WHO, Hepatitis B, https: / / www.who.int / news-room / factsheets / detail / hepatitis-b, published in July 2021, accessed on April 10, 2022). It is worth noting that approximately 50% of patients with HDV infection have cirrhosis at the time of diagnosis (Fattovich G et al., Influence of hepatitis delta virus infection on progression to cirrhosis in chronic hepatitis type B, J Infect Dis. 1987 May, 155(5):931-5).

[0005] Hepatitis B virus (HBV) is a DNA virus that infects, replicates and persists in human hepatocytes (Protzer U et al., Living in the liver: hepatic infections, Nature Reviews Immunology 2012, 12: 201-213). The small viral genome (3.2 kb) consists of partially double-stranded relaxed circular DNA (rcDNA) with four open reading frames encoding the following seven proteins: HBcAg (HBV core antigen, viral capsid protein), HBeAg (hepatitis B e antigen), HBV Pol / RT (polymerase, reverse transcriptase), PreSl / PreS2 / HBsAg (large, medium and small surface envelope glycoproteins), and HBx (HBV× antigen, transcriptional regulator required for infection) (Seeger C et al., Molecular biology of hepatitis B virus infection, Virology 2015, 479-480: 672-686; Tong S et al., Overview of viral replication and genetic variability, Journal of Hepatology, 2016, 64(1): S4-S16).

[0006] In hepatocytes, rcDNA in the form of HBV nucleic acid introduced by infected virions is converted into covalently closed circular DNA (cccDNA), which persists in the host cell nucleus as an additional chromatin structure (Allweiss L et al., The Role of cccDNA in HBV Maintenance, Viruses 2017, 9: 156). CccDNA serves as a transcription template for all viral transcripts (Lucifora J et al., Attacking hepatitis B virus cccDNA—The holygrail to hepatitis B cure, Journal of Hepatology 2016, 64 (1): S41-S48). Pregenomic RNA (pgRNA) transcripts are reverse transcribed into new rcDNA for new virions, which are secreted without causing cytotoxicity. In addition to secreting infectious virions, infected hepatocytes also secrete a large number of genome-free subviral particles, which may exceed 10,000 times the number of secreted virions (Seeger et al., 2015, supra). The virus can also randomly integrate into the host genome, a mechanism that contributes to hepatocyte transformation (Levrero M et al., Mechanisms of HBV-induced hepatocellular carcinoma, Journal of Hepatology 2016, 64(l): S84-S101). HBV persists in hepatocytes in the form of cccDNA and integrated DNA (intDNA).

[0007] Hepatitis B infection is characterized by serological viral markers and antibodies. In acute lytic infection, the virus is cleared by the following mechanisms: effective innate and adaptive immune responses, including cytotoxic T cells that cause death of infected hepatocytes; and induction of B cells to produce neutralizing antibodies that prevent viral spread (Bertoletti A, Adaptive immunity in HBV infection, Journal of Hepatology 2016, 64 (1): S71-S83; Maini MK et al., The role of innate immunity in the immunopathology and treatment of HBV infection, Journal of Hepatology 2016, 64 (1): S60-S70; Li Y et al., Genome-wide association study identifies 8p21.3 associated with persistent hepatitis B virus infection among Chinese, Nature Communications 2016, 7: 11664). In contrast, chronic infection is associated with T-cell and B-cell dysfunction and is mediated by multiple regulatory mechanisms, including viral epitope presentation on hepatocytes and subviral particle secretion (Bertoletti et al., 2016, supra; Maini et al., 2016, supra; Burton AR et al., Dysfunctional surface antigen specific memory B cells accumulate in chronic hepatitis B infection, EASL International Liver Congress, Paris, France 2018). Therefore, the continuous expression and secretion of viral proteins due to the persistence of cccDNA in hepatocytes is considered a key step in the host's inability to clear the infection.

[0008] Treatment options for HDV infection are limited to pegylated interferon alpha (PEG-IFNa) and brevirin. PEG-IFNa induces sustained virological response (SVR (serum HDV clearance rate maintained for 6 months after stopping treatment)) in only about 25% to 30% of individuals treated for 48 weeks. Late relapse is observed in about 50% of these patients, reducing long-term efficacy to about 15% (Abbas Z et al., Interferon alpha for chronic hepatitis D. Cochrane Database Syst Rev. December 7, 2011, 2011 (12): CD006002; Heidrich B et al., Late HDV RNA relapse after peginterferon alpha-based therapy of chronic hepatitis delta, Hepatology 2014; 60: 87-97). Current guidelines from the American Association for the Study of Liver Diseases (AASLD), the Asia-Pacific Association for the Study of the Liver (APASL), and the European Association for the Study of the Liver (EASL) recommend administration of PEG-IFNa for at least 48 weeks to patients with chronic HDV infection (European Association for the Study of the Liver (EASL), EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017 August, 67(2):370-398; Sarin SK et al., Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update, Hepatol Int. 2016 January, 10(1):1-98; Terrault NA et al., Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance, Hepatology 2018 April, 67(4):1560-1599).In addition to the low SVR rate, the major adverse effects of PEG-IFNa therapy have been well described, and PEG-IFNa is contraindicated for patients with autoimmune diseases, severe psychiatric syndromes, and Child-Pugh-Turcotte (CPT)-B or CPT-C cirrhosis (Rizzetto M, Hepatitis D Virus: Introduction and Epidemiology, Cold Spring Harb Perspect Med. July 1, 2015, 5(7): a021576; Sleijfer S et al., Side effects of interferon alpha therapy, Pharm World Sci. December 2005, 27(6): 423-31). Reduced efficacy of PEG-IFNa has been observed in the treatment of cirrhotic patients with chronic HDV, which further limits the usefulness of this therapy (Gunsar F et al., Two-year interferon therapy with or without ribavirin in chronic delta hepatitis, Antivir Ther. 2005, 10(6): 721-6). The limitations of using PEG-IFNa in cirrhotic HDV patients are particularly evident, as 50% of HDV-infected patients have cirrhosis at the time of diagnosis (Fattovich et al., 1987, supra). These limitations and low efficacy of existing agents highlight the unmet needs of patients with chronic HDV infection at all stages of the disease. Blevirtide (BLV), an entry inhibitor targeting the sodium taurocholate cotransporting polypeptide (NTCP) receptor, is conditionally approved in the European Union for chronic HDV therapy. Interim results from the Phase 3 MYR301 study showed that after 24 weeks of administration of 2 mg daily subcutaneous (SC) doses of BLV, 36.7% of participants achieved a combined virological and biochemical endpoint. Nucleoside reverse transcriptase inhibitor (NRTI) therapy can block HBV replication but does not directly affect HBsAg production, intrahepatic HDV replication, or HDV viremia. Summary of the invention

[0009] In some aspects, the present disclosure provides a method for treating hepatitis delta virus (HDV) infection or HDV-related disease in a subject in need thereof, comprising administering to the subject: (a) an anti-HBV antibody; and (b) an siRNA targeting HBV mRNA. In some embodiments, the subject has cirrhosis, for example, a liver biopsy METAVIR grade of F4 within 12 months prior to treatment, or a liver elastography grade of F4. Kilopascals (kPa); creatine clearance (CLcr) calculated by the Cockcroft-Gault formula before treatment ≥ 60 mL / min; Child-Pugh-Turcotte (CPT) score of 5 or higher before treatment.

[0010] In some aspects, compositions for use in therapy, compositions for the manufacture of a medicament, and kits are provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The study protocol for Cohort 1 of the clinical study in Example 1 is depicted.

[0012] Figure 2 The study protocol for Cohorts 2 to 4 of the clinical study in Example 1 is depicted.

[0013] FIG. 3A to FIG. 3D Depicted is a timeline of activity during the induction phase for Cohort 1a and Cohort 1b of the clinical study in Example 1.

[0014] FIG. 4A to FIG. 4C Depicted is the activity schedule for Cohort 1a and Cohort 1b of the clinical study in Example 1 during the maintenance phase.

[0015] FIG. 5A to FIG. 5D Depicted is the activity timeline of Cohorts 2 to 4 of the clinical study in Example 1 during the induction phase.

[0016] FIG. 6A to FIG. 6C Depicted is the activity schedule for Cohorts 2 to 4 during the maintenance phase for participants transitioning to the maintenance phase at Week 24 in the clinical study of Example 1.

[0017] 7A to 7C Depicted is the activity schedule for Cohorts 2 to 4 during the maintenance phase for participants transitioning to the maintenance phase at Week 48 in the clinical study of Example 1.

[0018] FIG. 8A to FIG. 8C The follow-up period activity schedule for all cohorts of the clinical study in Example 1 is depicted.

[0019] Fig. 9 The in vitro HBV neutralization activity of AB01 is shown.

[0020] Fig.10 The in vitro HDV neutralization activity of AB01 is shown.

[0021] Fig.11 The in vitro HBV antiviral activity of SIRNA01 is shown.

[0022] FIG. 12A to FIG. 12B The study design for Cohorts 1 to 4 of the clinical study in Example 3 is depicted.

[0023] FIG. 13A to FIG. 13D Depicted is a timeline of activity during the induction phase for Cohort 1a and Cohort 1b of the clinical study in Example 3.

[0024] FIG. 14A to FIG. 14D Depicted is the activity schedule for Cohort 1a and Cohort 1b of the clinical study in Example 3 during the maintenance phase.

[0025] FIG. 15A to FIG. 15D Depicted is a timeline of activity for Cohorts 2a, 2b1, 2b2, and 2c of the clinical study in Example 3 during the first 48 weeks of the treatment period.

[0026] FIG. 16A to FIG. 16D Depicted is the timeline of activity for Cohorts 2a, 2b1, 2b2, and 2c of the clinical study in Example 3 during Week 49 to Week 96 of the treatment period.

[0027] FIG. 17A to FIG. 17E Depicted is a timeline of activity for Cohort 3 of the clinical study in Example 3 during the first 48 weeks of the treatment period.

[0028] 18A to 18D Depicted is a timeline of activity for Cohort 3 of the clinical study in Example 3 during Week 49 to Week 96 of the treatment period.

[0029] 19A to 19D Depicted is a timeline of activities during the delayed treatment period of Cohort 4 of the clinical study in Example 3.

[0030] FIG. 20A to FIG. 20B The follow-up period activity schedule for all cohorts of the clinical study in Example 3 is depicted.

[0031] FIG. 21A to FIG. 21G The event timeline for the pharmacokinetic analysis of the clinical study in Example 3 is depicted.

[0032] FIG. 22A to FIG. 22B A timeline of optional study activities for the clinical study in Example 3 is depicted.

[0033] Fig.23 Shown are the changes in HDV RNA from baseline over the first 8 weeks for six subjects in Cohort 1b of the clinical study in Example 3. DETAILED DESCRIPTION

[0034] The present disclosure provides methods and compositions for treating hepatitis delta virus (HDV) infection or HDV-related diseases, wherein one or more of anti-HBV antibodies and anti-HBV siRNAs are administered to a subject, as well as related kits.

[0035] I. Glossary

[0036] The following sections provide detailed descriptions of combination therapies for treating HDV infection or HDV-related diseases and kits related to these combination therapies. Before describing the present disclosure in more detail, it may be helpful to understand the present disclosure to provide definitions of certain terms that will be used herein. Additional definitions are set forth throughout the present disclosure.

[0037] In the description of the present invention, unless otherwise specified, the term "about" means +20% of the indicated range, value or structure.

[0038] The term "comprising" (and similar terms such as "including" and "consisting of") means the presence of the specified features, integers, steps, or components mentioned in the claims, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups of these. The term "consisting essentially of limits the scope of a claim to the specified materials or steps, and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0039] It should be understood that the terms "a" and "an" as used herein refer to "one or more" / "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of these alternatives, and can be used synonymously with "and / or". As used herein, the terms "including" and "having" are used synonymously, and these terms and variations thereof are intended to be interpreted as non-restrictive or open-ended.

[0040] The word "substantially" does not exclude "completely"; for example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definitions provided herein.

[0041] The term "disease" as used herein is intended to be generally synonymous with the terms "disorder" and "condition" (as in "medical condition"), and to be used interchangeably, as all of these terms reflect an abnormal condition in the human or animal body or one of its components that impairs the normal functioning of the body, usually manifests itself by distinctive signs and symptoms, and results in a shortened life span or reduced quality of life in a human or animal.

[0042] As used herein, the terms "peptide", "polypeptide" and "protein" and variant forms of these terms refer to molecules comprising at least two amino acids linked to each other by normal peptide bonds or by modified peptide bonds (such as, in the case of isosteric peptides), particularly peptides, oligopeptides, polypeptides or proteins, including fusion proteins. For example, a peptide, polypeptide or protein can be composed of amino acids selected from the 20 kinds of amino acids defined by the genetic code, which are linked to each other by normal peptide bonds ("classical" polypeptides). A peptide, polypeptide or protein can be composed of L-amino acids and / or D-amino acids. In particular, the terms "peptide", "polypeptide" and "protein" also include "peptide mimetics", which are defined as peptide analogs containing non-peptide structural elements, which can simulate or antagonize the biological effects of natural parent peptides. Peptide mimetics lack classic peptide features, such as enzymatically scissile peptide bonds. In particular, a peptide, polypeptide or protein may also include other amino acids different from these amino acids in addition to the 20 kinds of amino acids defined by the genetic code, or may be composed of amino acids different from the 20 kinds of amino acids defined by the genetic code. In particular, peptides, polypeptides or proteins in the context of the present disclosure may also be composed of amino acids modified by natural processes (such as post-translational maturation processes) or by chemical processes (well known to those skilled in the art). Such modifications are fully described in detail in the literature. These modifications may occur at any position in the polypeptide: in the peptide backbone, in the amino acid chain, or even at the carboxyl or amino terminus. In particular, the peptide or polypeptide may form a branched structure after ubiquitination, or may be a ring with or without a branched structure. This type of modification may be the result of a natural or synthetic post-translational process well known to those skilled in the art. In the context of the present disclosure, the terms "peptide", "polypeptide" or "protein" also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositols, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation (including pegylation), hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, seneloylation, sulfation, addition of amino acids (such as arginylation) or ubiquitination.Such modifications are well described in the literature (Proteins Structure and Molecular Properties, 2nd Edition, T. C. Creighton, New York (1993); Post-translational Covalent Modifications of Proteins, ed. B. C. Johnson, Academic Press, New York (1983); Seifter et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 1990, 182: 626-46; and Rattan et al., Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci 1992, 663: 48-62). Thus, the terms "peptide", "polypeptide" and "protein" include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like.

[0043] 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) (poly)peptides, i.e., one or more chains of amino acid monomers linked by peptide bonds as explained above. In specific embodiments, a protein according to the present disclosure comprises 1, 2, 3 or 4 polypeptides.

[0044] As used herein, the term "recombinant" (e.g., recombinant antibodies, recombinant proteins, recombinant nucleic acids, etc.) refers to any molecule (antibody, protein, nucleic acid, siRNA, etc.) that is prepared, expressed, produced, or isolated by recombinant means and that does not exist naturally. As used herein, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably and are intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded nucleic acid molecules or double-stranded nucleic acid molecules. In a specific embodiment, the nucleic acid molecule is a double-stranded RNA.

[0045] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be identical in DNA content, due to intentional or unintentional mutations. Included are variant progeny screened for the same function or biological activity as in the originally transformed cell. Where a different designation is intended, it will be clear from the context.

[0046] As used herein, the term "sequence variant" refers to any sequence having one or more changes compared to a reference sequence, wherein the reference sequence is any sequence listed in the sequence table, i.e., SEQ ID NO: 1 to SEQ ID NO: 61. Therefore, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. For sequence variants in the context of nucleotide sequences, the reference sequence is also a nucleotide sequence, and for sequence variants in the context of amino acid sequences, the reference sequence is also an amino acid sequence. As used herein, "sequence variant" is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the reference sequence. Unless otherwise indicated, sequence identity is typically calculated over the full length of the reference sequence (i.e., the sequence listed in the present application). Percent identity as referred to herein can be determined, for example, using BLAST, using the default parameters specified by NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; Gap open penalty = 1, Gap extension penalty = 1]. In the context of nucleic acid (nucleotide) sequences, "sequence variants" have altered sequences in which one or more nucleotides in a reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of a reference nucleotide sequence. Nucleotides are represented 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 may or may not result in a change in the corresponding reference amino acid sequence, i.e., produce an amino acid "sequence variant". In certain embodiments, a nucleotide sequence variant is a variant that does not produce an amino acid sequence variant (i.e., a silent mutation). However, nucleotide sequence variants that cause "non-silent" mutations are also within the scope, particularly 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 a reference amino acid sequence. In the context of amino acid sequences, a "sequence variant" has an altered sequence in which one or more amino acids are deleted, substituted or inserted compared to a reference amino acid sequence. As a result of these changes, 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 a reference amino acid sequence. For example, a variant sequence having no more than 10 changes (i.e., any combination of deletions, insertions or substitutions) for every 100 amino acids of the reference sequence is "at least 90% identical" to the reference sequence.

[0047] Although non-conservative amino acid substitutions may exist, in certain embodiments, the substitution is a conservative amino acid substitution, in which the substituted amino acid has similar structural properties or chemical properties to the corresponding amino acid in the reference sequence. For example, a conservative amino acid substitution involves a substitution of an aliphatic or hydrophobic amino acid (e.g., alanine, valine, leucine, and isoleucine) by another amino acid of the same type; a hydroxyl-containing amino acid (e.g., serine and threonine) by another amino acid of the same type; an acidic residue (e.g., glutamic acid or aspartic acid) by another residue of the same type; an amide-containing residue (e.g., asparagine and glutamine) by another residue of the same type; an aromatic residue (e.g., phenylalanine and tyrosine) by another residue of the same type; a basic residue (e.g., lysine, arginine, and histidine) by another residue of the same type; and a small amino acid (e.g., alanine, serine, threonine, methionine, and glycine) by another amino acid of the same type.

[0048] Amino acid sequence insertions include amino terminal fusions and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusing the N-terminus or C-terminus of an amino acid sequence to a reporter molecule or enzyme.

[0049] Unless otherwise specified, the changes in the sequence variants do not eliminate the function of the corresponding reference sequence, for example, in this case, the function of the sequence of the anti-HBV antibody or siRNA is sufficient to neutralize HBV infection or reduce HBV protein expression, respectively. Guidance in determining which nucleotides and amino acid residues can be substituted, inserted or deleted, respectively, without eliminating such function can be found by using computer programs well known in the art.

[0050] As used herein, the nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide or protein refers to the source of the nucleic acid, peptide, polypeptide or protein. In some embodiments, the nucleic acid sequence or amino acid sequence derived from a specific sequence has an amino acid sequence substantially identical to the sequence or a portion of the sequence from which it is derived, wherein "substantially identical" includes sequence variants as defined above. In certain embodiments, the nucleic acid sequence or amino acid sequence derived from a specific peptide or protein is derived from a corresponding domain in a specific peptide or protein. Thus, "corresponding" refers in particular to the same function. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another "transmembrane domain" (of another protein). Therefore, the "corresponding" parts of peptides, proteins and nucleic acids are identifiable by those of ordinary skill in the art. Similarly, sequences "derived from" other sequences can usually be identified by those of ordinary skill in the art as having their source in the sequence.

[0051] In some embodiments, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein can be identical with the starting 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 can also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), and in particular, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein can be the functional sequence variant as described above of the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). For example, in a peptide / protein, one or more amino acid residues can be replaced by other amino acid residues, or one or more amino acid residues can be inserted or deleted.

[0052] As used herein, the term "mutation" refers to changes in a nucleic acid sequence and / or an amino acid sequence compared to a reference sequence (e.g., a corresponding genomic sequence). Mutations, for example, can be (naturally occurring) somatic mutations, spontaneous mutations, induced mutations (e.g., induced by enzymes, chemicals, or radiation), or mutations obtained by site-directed mutagenesis (a molecular biological method for making specific and intentional changes in nucleic acid sequences and / or amino acid sequences), for example, compared to genomic sequences. Therefore, the term "mutation" should be understood to also include, for example, physically making mutations in nucleic acid sequences or amino acid sequences. 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. In order to achieve mutations in an amino acid sequence, mutations can be introduced into the nucleotide sequence encoding the amino acid sequence so as to express (recombinantly) mutated polypeptides. Mutations can be achieved, for example, by changing (e.g., by site-directed mutagenesis) the codons of a nucleic acid molecule encoding one amino acid to produce codons encoding a different amino acid, or by synthesizing sequence variants, for example, by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and then designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide variant without mutating one or more nucleotides of the nucleic acid molecule.

[0053] As used herein, the term "coding sequence" is intended to refer to a polynucleotide molecule that encodes the amino acid sequence of a protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon.

[0054] As used herein, the term "expression" refers to any step involved in the production of a polypeptide including transcription, post-transcriptional modification, translation, post-translational modification, secretion, and the like.

[0055] As used herein, the term "vaccine" is generally understood to provide at least one antigen or immunogen preventive or therapeutic material, including a viral vector vaccine containing a nucleic acid encoding the antigen or immunogen. Antigens or immunogens can be derived from any material suitable for vaccination. For example, antigens or immunogens can be derived from pathogens, such as bacteria or viral particles, or tumors or cancerous tissues. Antigens or immunogens stimulate the body's adaptive immune system to provide an adaptive immune response. In particular, "antigen" or "immunogen" generally refers to a substance that can be recognized by the immune system (e.g., an adaptive immune system) and can, for example, trigger an antigen-specific immune response by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or can include a peptide or protein that can be presented to a T cell by MHC.

[0056] As used herein, "hepatitis B virus", which can be used interchangeably with the term "HBV", refers to a well-known non-cytopathic hepadnavirus belonging to the Hepadnaviridae family. The HBV genome is a partially double-stranded circular DNA with four overlapping reading frames (which may be referred to as "genes", "open reading frames" or "transcripts" herein): C, X, P, S. The core protein is encoded by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the pre-core (pre-C) protein. DNA polymerase is encoded by gene P. Gene S is a gene encoding a surface antigen (HBsAg). The HBsAg gene is a long open reading frame containing three in-frame "start" (ATG) codons, producing three polypeptides of different sizes, called large, medium and small S antigens, pre-S1+pre-S2+S, pre-S2+S, or S. In addition to decorating the HBV envelope, surface antigens are also part of subviral particles that are produced in excess compared to virion particles and play a role in immune tolerance and sequestration of anti-HBsAg antibodies, thereby allowing infectious particles to evade immune detection. The protein encoded by gene X plays a role in transcriptional transactivation and replication and has been associated with the development of hepatocellular carcinoma.

[0057] Nine genotypes of HBV have been identified, named A to I, and another genotype J has been proposed, each with a unique geographical distribution (Velkov S et al., The Global Hepatitis B Virus Genotype Distribution Approximated from Available Genotyping Data, Genes 2018, 9 (10): 495). The term "HBV" includes any genotype (A to J) of HBV. The complete coding sequence of the reference sequence of the HBV genome can be found in, for example, GenBank accession numbers GI: 21326584 and GI: 3582357. The amino acid sequences of C, X, P, S proteins can be found, for example, with the following NCBI accession numbers: YP_009173857.1 (C protein), YP_009173867.1 and BAA32912.1 (X protein), YP 009173866.1 and BAA32913.1 (P protein), and YP 009173869.1, YP_009173870.1, YP_009173871.1 and BAA32914.1 (S protein). Additional examples of HBV messenger RNA (mRNA) sequences are available using public databases (e.g., GenBank, UniProt, and OMIM). The international repository of hepatitis B virus strain data can be obtained at http: / / www.hpa-bioinformatics.org.uk / HepSEQ / main.php. As used herein, the term "HBV" also refers to naturally occurring DNA sequence variations of the HBV genome, ie, genotypes AJ and variants thereof.

[0058] In some embodiments, the present disclosure provides a combined therapy for treating HBV, including anti-HBV siRNA. siRNA mediates the targeted cutting of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, thereby achieving gene expression inhibition. This process is often referred to as "RNA interference" (RNAi). Not wishing to be bound by a particular theory, the long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is decomposed into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15: 485). Dicer is a ribonuclease III-like enzyme that processes dsRNA into 19-23 base pair siRNAs with characteristic double base 3' overhangs (Bernstein et al., Nature 2001, 409: 363). Then siRNA is incorporated into RISC, where one or more helicases unwind the siRNA duplex so that the complementary antisense strand can guide target recognition (Nykanen et al., Cell 2001 107: 309). After binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir et al., Genes Dev. 2001, 15:188).

[0059] The terms "silencing", "inhibiting the expression of", "downregulating the expression of", "preventing the expression of", etc., insofar as they refer to HBV genes, refer herein to at least a partial reduction in HBV gene expression, as evidenced by a reduction in the amount of HBV mRNA that can be isolated from or detected in a first cell or a first group of cells, in which the HBV gene is transcribed, and the first cell or the first group of cells has been treated with an inhibitor of HBV gene expression, such that the expression of the HBV gene is inhibited compared to a second cell or a second group of cells (control cells) that is substantially identical to the first cell or the first group of cells but has not been so treated. The degree of inhibition can be measured, for example, by subtracting the difference between the degree of mRNA expression in the control cells and the degree of mRNA expression in the treated cells. Alternatively, the degree of inhibition can be given by a reduction in a parameter that is functionally related to HBV gene expression, such as the amount of protein encoded by the HBV gene, or the number of cells that display a certain phenotype (e.g., an HBV infection phenotype). In principle, HBV gene silencing can be determined in any cell expressing an HBV gene, such as an HBV infected cell or a cell engineered to express an HBV gene, by any appropriate assay.

[0060] The level of HBV RNA expressed by a cell or a cell population or the level of circulating HBV RNA can be determined using any method known in the art for evaluating mRNA expression, such as the rtPCR method provided in Example 2 of International Application Publication No. WO 2016 / 077321A1 and U.S. Patent Application Publication No. US2017 / 0349900A1, which is incorporated herein by reference. In some embodiments, the expression level of the HBV gene in the sample (e.g., total HBV RNA, HBV transcript (e.g., HBV3.5kb transcript)) is determined by detecting a transcribed polynucleotide or a portion thereof (e.g., RNA of the HBV gene). RNA can be extracted from cells using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit Or PAXgene (PreAnalytix, Switzerland). Typical assay formats using RNA hybridization include nuclear conjugation assay, RT-PCR, ribonuclease protection assay (Melton DA et al., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nuc. Acids Res. 1984, 12: 7035-56), Northern blotting, in situ hybridization and microarray analysis. Circulating HBV mRNA can be detected using the methods described in International Application Publication WO 2012 / 177906A1 and U.S. Patent Application Publication US2014 / 0275211A1, which are incorporated herein by reference.

[0061] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HBV gene, including mRNA as an RNA processing product of the primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-guided cleavage at or near the portion. For example, the target sequence is typically 9 to 36 nucleotides in length, for example, 15 to 30 nucleotides in length, including all subranges therebetween. As non-limiting examples, a target sequence can have 15 to 30 nucleotides, 15 to 26 nucleotides, 15 to 23 nucleotides, 15 to 22 nucleotides, 15 to 21 nucleotides, 15 to 20 nucleotides, 15 to 19 nucleotides, 15 to 18 nucleotides, 15 to 17 nucleotides, 18 to 30 nucleotides, 18 to 26 nucleotides, 18 to 23 nucleotides, 18 to 22 nucleotides, 18 to 21 nucleotides, 18 to 20 nucleotides, 19 to 3 ... In the embodiment of the present invention, the present invention comprises a nucleic acid sequence of at least one nucleotide and a nucleic acid sequence of at least one nucleotide. In the embodiment of the present invention, the nucleic acid sequence of at least one nucleotide and a nucleic acid sequence of at least one nucleotide is at least one nucleotide.

[0062] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0063] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence associated with a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions and form a duplex structure, which is understood by the skilled person. Such conditions may, for example, be stringent conditions, wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12 to 16 hours, followed by washing. Other conditions may be applied, such as physiologically relevant conditions that may be encountered in an organism. Depending on the ultimate application of the hybridizing nucleotides, the skilled person will be able to determine a set of conditions that are most suitable for testing the complementarity of the two sequences.

[0064] As described herein, the complementary sequence in siRNA includes the base pairing of an oligonucleotide or polynucleotide with a first nucleotide sequence and an oligonucleotide or polynucleotide with a second nucleotide sequence in the total length of one or two nucleotide sequences. Such sequences may be referred to herein as "completely 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 completely complementary, or may be formed when hybridizing to a duplex comprising up to 30 base pairs, one or more, but generally no more than 5, 4, 3 or 2 mispaired base pairs, while retaining the ability to hybridize under the conditions most relevant to its final application, for example, inhibiting gene expression via RISC approach. However, when two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs should not be considered as mispairing when determining complementarity. For example, a siRNA comprising an oligonucleotide having a length of 21 nucleotides and another oligonucleotide having a length of 23 nucleotides, wherein a longer oligonucleotide comprises a sequence with 21 nucleotides that is completely complementary to a shorter oligonucleotide, and for purposes described herein, may still be referred to as "completely complementary".

[0065] As used herein, "complementary" sequences may also include non-Watson-Crick base pairs and / or base pairs formed by non-natural modified nucleotides, or may be formed entirely by non-Watson-Crick base pairs and / or base pairs formed by non-natural modified nucleotides, as long as 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 base pairing.

[0066] The terms "complementary," "fully complementary," and "substantially complementary" may be used herein in reference to base matching between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA agent and a target sequence, as will be understood from the context in which these terms are used.

[0067] As used herein, a polynucleotide that is "substantially complementary" to at least a portion of an mRNA refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding an HBV protein). For example, if a polynucleotide is substantially complementary to a non-interrupted portion of an HBV mRNA, then the sequence is complementary to at least a portion of an HBV mRNA.

[0068] As used herein, the term "siRNA" refers to the following RNA interference molecule: it includes an RNA molecule or a molecular complex having a hybrid duplex region, the hybrid duplex region comprising two antiparallel and substantially complementary nucleic acid strands, which will be referred to as having a "sense strand" orientation and an "antisense" orientation relative to the target RNA. The duplex region can have any length that permits the desired target RNA to be specifically degraded by the RISC pathway, but typically its length will be in the range of 9 to 36 base pairs (e.g., 15 to 30 base pairs). In some embodiments, the duplex comprises between 9 and 36 base pairs, and the duplex can have 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 33, 34, 35 or 36 base pairs, and any subranges between these values, including but not limited to: 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs , 18 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 20 to 26 base pairs, 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs, and 21 to 22 base pairs. siRNAs generated in cells by treatment with Dicer and similar enzymes are typically in the range of 19 to 22 base pairs in length.

[0069] One strand of the duplex region of siRNA comprises a sequence that is substantially complementary to a certain region of the target RNA. The two strands forming the duplex structure may be from a single RNA molecule having at least one self-complementary region, or may be formed by two or more separate RNA molecules. When the duplex region is formed by two strands of a single molecule, the molecule may have a duplex region separated by a single strand of nucleotides (referred to herein as a "hairpin loop"), the single strand being between the 3'-end of a strand forming the duplex structure and the 5'-end of the corresponding other strand forming the duplex structure. The hairpin loop may comprise at least one unpaired nucleotide; in some embodiments, the hairpin loop may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. When the two substantially complementary strands of siRNA are composed of separate RNA molecules, those molecules do not need to be covalently linked, but may be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the connecting structure is called a "linker."

[0070] siRNA as described herein can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer, such as commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0071] The term "antisense strand" or "guide strand" refers to a siRNA strand that includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence as defined herein). In the case where the region of complementarity is not completely complementary to the target sequence, mismatches may occur in the internal regions or terminal regions of the molecule. Generally speaking, the most tolerated mismatches are located in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' end and / or the 3' end.

[0072] The term "sense strand" or "passenger strand" as used herein refers to the strand of a siRNA that includes a region that is substantially complementary to a region of the "antisense strand" as that term is defined herein.

[0073] The term "RNA molecule" or "ribonucleic acid molecule" not only encompasses RNA molecules expressed or found in nature, but also encompasses RNA analogs and derivatives comprising one or more ribonucleotides / ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, "ribonucleoside" comprises nucleoside bases and ribose, and "ribonucleotide" is a ribonucleoside with one, two or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleotide" can be considered as equivalent terms used herein. RNA can be modified in core base structure or ribose-phosphate backbone structure, for example, as described in more detail below. However, the siRNA molecule comprising ribonucleoside analogs or derivatives retains the ability to form duplexes. As non-limiting examples, RNA molecules can also include at least one modified ribonucleoside, including but not limited to: 2'-O-methyl modified nucleosides, nucleosides comprising 5' thiophosphate groups, terminal nucleosides connected to cholesterol derivatives or dodecanoic acid bisdecylamide groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluorine modified nucleosides, 2'-amino modified nucleosides, 2'-alkyl modified nucleosides, morpholino nucleosides, aminophosphoroside, or nucleosides comprising non-natural bases, or any combination of these. In another example, RNA molecules can include at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, until the total length of siRNA molecules. For each of this type of multiple modified ribonucleosides in RNA molecules, modification need not be identical. In some embodiments, modified ribonucleosides include deoxyribonucleosides. For example, the siRNA may comprise one or more deoxynucleosides, including, for example, deoxynucleoside overhangs, or one or more deoxynucleosides within the double-stranded portion of the siRNA. However, the term "siRNA" as used herein does not include a complete DNA molecule.

[0074] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the duplex structure of the siRNA. For example, when the 3'-end of one strand of the siRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang. The siRNA may comprise an overhang having at least one nucleotide; alternatively, the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The nucleotide overhang may comprise nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides), or be composed of nucleotide / nucleoside analogs. The overhang may be located on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0075] The term "flat" or "flat-ended" when used herein with respect to siRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the siRNA, i.e., no nucleotide overhangs. One or both ends of the siRNA can be flat. If both ends of the siRNA are flat, the siRNA is referred to as "flat-ended." A "flat-ended" siRNA is an siRNA with both ends being flat (i.e., no nucleotide overhangs at either end of the molecule). Typically, such a molecule will be double-stranded over its entire length.

[0076] In some embodiments, the present disclosure provides a combination therapy for treating HBV, which includes an anti-HBV antibody. In certain embodiments, the anti-HBV antibody or its antigen-binding fragment binds to the antigenic loop region of HBsAg and neutralizes hepatitis B virus infection. In certain embodiments, the anti-HBV antibody or its antigen-binding fragment binds to the antigenic loop region of HBsAg and neutralizes hepatitis D virus infection.

[0077] As used herein, the term "antibody" covers various forms of antibodies, including but not limited to: complete antibodies, antibody fragments, antigen binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered antibodies (variants or mutant antibodies), as long as the characteristic properties of the antibody are retained. In some embodiments, the antibody is a human antibody and / or a monoclonal antibody. In specific embodiments, the antibody is a human monoclonal antibody. In certain specific embodiments, the antibody is a recombinant human monoclonal antibody. As used herein, the terms "antigen binding fragment", "fragment" and "antibody fragment" are used interchangeably to refer to any antibody fragment that retains the antigen binding activity of the antibody in combination therapy. Examples of antibody fragments include but are not limited to single-chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. In addition, the term "antibody" as used herein includes both antibodies and antigen binding fragments thereof.

[0078] As used herein, "neutralizing antibodies" are antibodies that are able to neutralize, i.e., prevent, inhibit, attenuate, hinder or interfere with the ability of a pathogen to initiate and / or maintain an infection in a host. The terms "neutralizing antibody" and "neutralizing antibody" are used interchangeably herein. These antibodies can be used alone or in combination as a prophylactic or therapeutic agent when properly formulated; in conjunction with active vaccination, as a diagnostic tool, or as a manufacturing tool as described herein.

[0079] Human antibodies are well known in the prior art (van Dijk MA and van de Winkel JC, Curr. Opin. Chem. Biol. 2001, 5: 368-74). Human antibodies can also be produced in transgenic animals (e.g., mice) that are able to produce a complete repertoire or collection of human antibodies in the absence of endogenous immunoglobulin production after immunization. Transferring the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen attack (see, e.g., Jakobovits A et al., Proc. Natl. Acad. Sci. USA 1993, 90: 2551-55; Jakobovits A et al., Nature 1993, 362: 255-258; Bruggemann M et al., Year Immunol. 1993, 7: 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom HR and Winter G, Mol.Biol.1992,227:381-88; Marks JD et al., Mol Biol.1991,222:581-97). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p. 77 (1985); Boerner P et al., Immunol.1991,147:86-95). In some embodiments, human monoclonal antibodies are prepared by using improved EBV-B cell immortalization, as described by Traggiai E et al. (Nat Med.2004,10(8):871-5). As used herein, the term "human antibody" also includes antibodies modified (e.g., in the variable region) to generate the properties described herein.

[0080] The antibody of the combined therapy can be any isotype antibody (e.g., IgA, IgG, IgM, i.e., K, γ or β heavy chain), 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 of the combined therapy can have a K or λ light chain. Based on the antigen-independent uptake of IgG into hepatocytes through the FcRN-IgG receptor, IgG-type HBsAg-specific antibodies can also advantageously block the release of HBV and HBsAg from infected cells. Therefore, IgG-type HBsAg-specific antibodies can bind intracellularly, thereby blocking the release of HBV virions and HBsAg.

[0081] As used herein, the term "variable region" (light chain variable region (V L ), heavy chain variable region (V H )) refers to such a portion of an antibody light chain (LC) or heavy chain (HC) (usually near the 105th to 120th amino-terminal amino acids of a mature antibody heavy chain or light chain): it contains the complementarity determining region ("CDR") and the framework region ("FR"), and is directly involved in the binding of the antibody to an antigen. The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR", and are known in the art to refer to non-continuous amino acid sequences within an antibody variable region that confer antigen specificity and / or binding affinity. Generally speaking, there are three CDRs in each variable region of an immunoglobulin binding protein. For example, for an antibody, the VH region and VL region typically contain six CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3). Immunoglobulin sequences can be aligned with numbering schemes (e.g., Kabat, EU, International Immunogenetics Information System (IMGT), and Aho), which allows annotation of equivalent residue positions and comparison of different molecules using the Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (Bioinformatics 2016, 15: 298-300). It should be understood that in certain embodiments, the antibodies or antigen-binding fragments of the present disclosure may include all or part of a heavy chain (HC), a light chain (LC), or both. For example, a full-length intact IgG antibody monomer typically includes a V H 、CH1、CH2、CH3、V L and CL.

[0082] In certain embodiments, the anti-HBV antibody or antigen-binding fragment thereof according to the combined therapy of the present disclosure is a purified antibody, a single-chain antibody, Fab, Fab', F(ab')2, Fv or scFv. Therefore, the antibody of the combined therapy can be a human antibody, a monoclonal antibody, a human monoclonal antibody, a recombinant antibody and / or a purified antibody. The present disclosure also provides fragments of these antibodies, in particular fragments that retain the antigen-binding activity of these antibodies. Such fragments include, but are not limited to, single-chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Although in some places, the present disclosure may explicitly refer to the antigen-binding fragments, antibody fragments, variants and / or derivatives of antibodies, as used herein, the term "antibody" or "antibody for combined therapy" includes all classes of antibodies, that is, antigen-binding fragments, antibody fragments, variants and derivatives of antibodies.

[0083] Fragments of these antibodies 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, fragments of these antibodies can be obtained by cloning and expressing partial sequences of heavy or light chains. The present disclosure also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of antibodies of the present disclosure. For example, the present disclosure includes scFv containing CDRs from antibodies of the present disclosure. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies (e.g., single chain Fv), in which the heavy and light chain variable domains are linked by a peptide linker.

[0084] Antibody fragments disclosed herein can confer monovalent or multivalent interactions and are included in a variety of structures as described above. For example, scFv molecules can be synthesized to produce trivalent "trivalent antibodies" or tetravalent "quadruplexes". These scFv molecules can include Fc region domains that produce divalent miniantibodies. In addition, the sequence of antibody / antibody fragment can be a component of a multispecific molecule, wherein these sequences target epitopes as described herein, and other regions of the multispecific molecule are bound to other targets. Exemplary multispecific molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv and diabodies (Holliger and Hudson, Nature Biotechnology 2005, 9: 1126-36).

[0085] Antibodies according to the present disclosure can be provided in purified form. Typically, the antibody will be present in a composition substantially free of other polypeptides, e.g., wherein less than 90% (by weight), typically less than 60%, more typically less than 50% of the composition is composed of other polypeptides.

[0086] In embodiments, the antibodies and antigen-binding fragments of the present disclosure may have multispecificity (e.g., bispecific, trispecific, tetraspecific, etc.), and may be provided in any multispecific format, as disclosed herein. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are multispecific antibodies, such as bispecific or trispecific antibodies. The form of bispecific antibodies is disclosed, for example, in Spiess et al. (Mol. Immunol. 2015, 67(2):95) and Brinkmann and Kontermann (mAbs 2017, 9(2):182-212), the bispecific formats and preparation methods thereof are incorporated herein by reference, including, for example: bispecific T cell engagers (BiTEs), DARTs, knob-in-hole (KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, TandAbs, triabodies, TriBi minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, intracellular antibodies, CrossMabs, bispecific antibodies ... Heavy-acting Fab (DAF) (two-in-one or four-in-one), DutaMab, DT-IgG, charge pair, Fab arm exchange, SEED body, trifunctional antibody, LUZ-Y assembly, Fcab, Kk 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). A bispecific or multispecific antibody may comprise an HBV and / or HDV-specific binding domain of the present disclosure in combination with another such binding domain of the present disclosure, or with a different binding domain that specifically binds to HBV and / or HDV (e.g., at the same epitope or different epitopes), or with binding domains that specifically bind to different antigens.

[0087] II. siRNA targeting HBV

[0088] In some embodiments, the present disclosure provides a therapeutic method involving the administration of siRNA targeting HBV mRNA, as well as related compositions and kits. Because HDV is a defective satellite virus that relies on HBsAg to complete its life cycle, siRNA that reduces HBsAg production hinders the production of infectious HDV virions. In in vitro experiments, treatment of primary human hepatocytes co-infected with siRNA targeting HBV mRNA (SIRNA01) caused a dose-dependent reduction in secreted infectious HDV virions. In HBV / HDV co-infected patients, HBsAg levels were correlated with HDV RNA levels, indicating that reducing serum HBsAg can lead to a reduction in circulating HDV (Zachou K et al., HIDT-1 Study Group. Quantitative HBsAg and HDV-RNA levels in chronic delta hepatitis, Liver Int., March 2010, 30 (3): 430-7). In addition, in preclinical models, reducing intrahepatic HBsAg with siRNA agents alleviated HDV viremia (Ye X et al., Hepatitis B Virus Therapeutic Agent ARB-1740 Has Inhibitory Effect on Hepatitis Delta Virus in a New Dually-Infected Humanized Mouse Model, ACS Infect Dis. 2019 May 10, 5(5):738-749) and limited the spread of the virus to uninfected hepatocytes.

[0089] In some embodiments described herein, the siRNA targeting HBV mRNA is SIRNA01. SIRNA01 is a synthetic siRNA that is chemically modified and targets HBV RNA, which has a covalently linked triantennary N-acetylgalactosamine (GalNAc) ligand, allowing specific uptake by hepatocytes. SIRNA01 targets the HBV genomic region common to all HBV viral transcripts and has pharmacological activity against HBV genotypes A to J. In preclinical models, SIRNA01 has been shown to inhibit viral replication, translation and secretion of HBsAg, and can provide or contribute to functional cure of chronic HBV infection. SIRNA can have a variety of antiviral effects, including degradation of pgRNA, thereby inhibiting viral replication, and degradation of all viral mRNA transcripts, thereby preventing the expression of viral proteins. This may cause the recovery of a functional immune response to HBV, whether used alone or in combination with other therapies. The ability of SIRNA01 to reduce non-infectious subviral particles containing HBsAg also distinguishes it from currently available treatments.

[0090] SIRNA01 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). More specifically, SIRNA01 targets mRNA encoded by a portion of the HBV genome containing the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO:2), which corresponds to nucleotides 1579 to 1597 of SEQ ID NO:1. Because transcription of the HBV genome produces overlapping RNAs of multiple cistrons, SIRNA01 causes significant inhibition of the expression of most or all HBV transcripts. Exemplary methods for synthesizing SIRNA01 and experimental data demonstrating silencing of HBV gene expression are described in International Application Publication No. WO 2020 / 036862A1, and these methods and data are incorporated herein by reference.

[0091] SIRNA01 has a sense strand comprising 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO:3) and an antisense strand comprising 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:4), wherein the nucleotides include 2'-fluoro (2'F) and 2'-O-methoxy (2'OMe) ribose modifications, phosphorothioate backbone modifications, glycol nucleic acid (GNA) modifications, and conjugation to a triantennary N-acetylgalactosamine (GalNAc) ligand at the 3' end of the sense strand to facilitate delivery to hepatocytes through the asialoglycoprotein receptor (ASGPR). Including the modifications, the sense strand of SIRNA01 comprises 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5) and the antisense strand comprises 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO: 6), wherein the modifications are abbreviated as shown in Table 1.

[0092] Table 1. Abbreviations for nucleotide monomers used in the representation of modified nucleic acid sequences. It should be understood that unless otherwise indicated Otherwise, these monomers, when present in an oligonucleotide, are linked to each other via 5'-3'-phosphodiester bonds.

[0093]

[0094]

[0095] abbreviation Nucleotides dA 2'-deoxyadenosine-3'-phosphorothioate dC 2'-deoxycytidine-3'-phosphate dC 2'-deoxycytidine-3'-phosphorothioate dG 2'-deoxyguanosine-3'-phosphate dG 2'-deoxyguanosine-3'-phosphorothioate dT 2'-Deoxythymidine-3'-phosphate dT 2'-deoxythymidine-3'-phosphorothioate Ud 2'-Deoxyuridine dU 2'-Deoxyuridine-3'-phosphorothioate

[0096] In some embodiments, the siRNA used in the methods, compositions, or kits described herein is SIRNA01. In some embodiments, the siRNA used in the methods, compositions, or kits described herein comprises a sequence variant of SIRNA01. In a specific embodiment, the portion of the HBV transcript targeted by the sequence variant of SIRNA01 overlaps with the portion of the HBV transcript targeted by SIRNA01.

[0097] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein (1) the sense strand comprises SEQ ID NO:3 or SEQ ID NO:5, or a sequence that differs from SEQ ID NO:3 or SEQ ID NO:5 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively; or (2) the antisense strand comprises SEQ ID NO:4 or SEQ ID NO:6, or a sequence that differs from SEQ ID NO:4 or SEQ ID NO:6 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively.

[0098] In some embodiments, a shorter duplex having one of the sequences SEQ ID NO:4 or SEQ ID NO:6 minus only a few nucleotides at one or both ends is used. Therefore, such siRNA is contemplated herein: it has a partial sequence comprising at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one or both of SEQ ID NO:4 and SEQ ID NO:6, and the ability to inhibit HBV gene expression differs from the siRNA comprising the complete sequence by no more than 5%, 10%, 15%, 20%, 25% or 30%. In some embodiments, a siRNA having a blunt end at one or both ends is provided, which is formed by removing nucleotides from one or both ends of siRNAO 1.

[0099] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein (1) the sense strand comprises SEQ ID NO:7, or a sequence that differs from SEQ ID NO:7 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively; or (2) the antisense strand comprises SEQ ID NO:8, or a sequence that differs from SEQ ID NO:8 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively.

[0100] In some embodiments, a shorter duplex having the sequence SEQ ID NO: 8 minus only a few nucleotides at one or both ends is used. Thus, siRNAs are contemplated herein that have a partial sequence comprising at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from SEQ ID NO: 8, and that have an ability to inhibit HBV gene expression that differs by no more than 5%, 10%, 15%, 20%, 25% or 30% from siRNAs comprising the complete sequence. In some embodiments, siRNAs having blunt ends at one or both ends are provided, which are formed by removing nucleotides from one or both ends of SEQ ID NO: 8.

[0101] In some embodiments, siRNA as described herein may contain one or more mismatches with the target sequence. In some embodiments, siRNA as described herein contains no more than 3 mismatches. In some embodiments, if the antisense strand of siRNA contains a mismatch with the target sequence, the mismatch region is not located at the center of the complementarity region. In a specific embodiment, if the antisense strand contains a mismatch with the target sequence, the mismatch is limited to the last 5 nucleotides starting from the 5' end or 3' end of the complementarity region. For example, for a 23 nucleotide siRNA chain complementary to a certain region of the HBV gene, the RNA chain may not contain any mismatches in the 13 nucleotides at the center. Methods described herein or methods known in the art can be used to determine whether the siRNA containing a mismatch with the target sequence is effective in suppressing the expression of the HBV gene.

[0102] In some embodiments, the siRNA used in the methods, compositions and kits described herein comprises two oligonucleotides, wherein one oligonucleotide is described as a sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described herein and known in the art, the complementary sequence of the siRNA can also be included as a self-complementary region of a single nucleic acid molecule, rather than being located on separate oligonucleotides.

[0103] In some embodiments, a single-stranded antisense RNA molecule comprising the antisense strand of a siRNA described herein is used in the methods, compositions, and kits described herein.Antisense RNA molecules can have 15 to 30 nucleotides complementary to the target.

[0104] In some embodiments, a single-stranded antisense RNA molecule comprising an antisense strand of SIRNA01 or a sequence variant thereof is used in the methods, compositions, and kits described herein. The antisense RNA molecule may have 15 to 30 nucleotides complementary to the target. For example, the antisense RNA molecule may have a sequence comprising at least 15, 16, 17, 18, 19, 20, 21 or more consecutive nucleotides from SEQ ID NO: 4 or SEQ ID NO: 6.

[0105] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 5 and the antisense strand comprises SEQ ID NO: 6, and further comprises additional nucleotides, modifications or conjugates as described herein. For example, in some embodiments, the siRNA may comprise additional modifications in addition to those modifications indicated in SEQ ID NO: 5 and 6. Such modifications may be generated using methods 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, which are incorporated herein by reference. Examples of such modifications are described in more detail below.

[0106] In some embodiments, substantially all or all nucleotides of the sense strand and substantially all or all nucleotides of the antisense strand of the siRNA are modified nucleotides. The nucleotides can be modified as described below.

[0107] a. Modified siRNA

[0108] Modifications disclosed herein include, for example: (a) sugar modification (e.g., at the 2' or 4' position) or replacement of sugars; (b) backbone modifications, including modification or replacement of phosphodiester bonds; (c) base modifications, for example, replacement of bases with stabilized bases, destabilized bases, or bases that are base-paired with an extended partner library, removal of bases (absic nucleotides), or conjugation of bases; and (d) terminal modifications, for example, 5' terminal modifications (phosphorylation, conjugation, reverse linkage, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.). Some specific examples of modifications that can be incorporated into the siRNA of the present application are shown in Table 1.

[0109] Modifications include substituted sugar moieties. The siRNAs featured herein can include one of the following at the 2' position: OH, F, O-alkyl, S-alkyl, or N-alkyl, O-alkenyl, S-alkenyl, or N-alkenyl, O-alkynyl, S-alkynyl, or N-alkynyl, or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2) n O] mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2 and O(CH2)nON[(CH2)nCH3)]2, wherein n and m are 1 to about 10. In some other embodiments, the siRNA includes one of the following at the 2' position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of siRNA, or group for improving the pharmacodynamic properties of siRNA, 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 1995, 78: 486-504), i.e., alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2*-O-dimethylaminoethoxyethyl or 2*-DMAEOE), i.e., 2*-O-CH2-O-CH2-N(CH2)2. Other exemplary modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the RNA of the siRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked siRNAs and at the 5' position of the 5' terminal nucleotide. Modifications can also include sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar.

[0110] Representative U.S. patents that teach methods for preparing such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576, , 427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920; each of which is incorporated herein by reference for teachings relating to methods of making such modifications.

[0111] Modified RNA backbones include, for example: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters; methyl and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates; phosphites; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thiocarbonylphosphoramidates; thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5'-linked analogs of these esters, and those with reverse polarity, wherein adjacent nucleoside unit pairs are linked with 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0112] Representative U.S. patents that teach the above-mentioned phosphorus-containing bond preparation methods include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,13 No. 1, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,587,361, No. 5, No. 625,050, No. 6,028,188, No. 6,124,445, No. 6,160,109, No. 6,169,170, No. 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639, No. 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. Pat. No. RE39464; each of these U.S. Pat. Nos. 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. Pat. No. RE39464; each of these U.S. Pat. Nos. 6,683,167,

[0113] RNA with modified main chain is particularly included in those without phosphorus atom in the main chain. For the purpose of this specification, and as sometimes mentioned in the art, modified RNA without phosphorus atom in its internucleoside main chain can also be regarded as oligonucleoside. The modified RNA main chain not including phosphorus atom has a main chain formed by the following items: short chain alkyl or cycloalkyl nucleoside interkey, mixed heteroatoms and alkyl or cycloalkyl nucleoside interkey, or one or more short chain heteroatoms or heterocyclic nucleoside interkey. These main chains include a main chain with morpholino bond (partially formed by the sugar part of nucleoside); siloxane main chain; sulfide, sulfoxide and sulfone main chain; formyl and thioformyl main chain; methyleneformyl and thioformyl main chain; olefin-containing main chain; aminosulfonate main chain; methyleneimino and methylenehydrazine main chain; sulfonate and sulfonamide main chain; amide main chain; and other main chains with mixed N, O, S and CH2 constituent parts.

[0114] Representative U.S. patents that teach the above oligonucleoside preparation methods include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677 , 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439; each of which is incorporated herein by reference for teachings relating to methods of making such modifications.

[0115] In some embodiments, both the sugar and the internucleoside bond (i.e., the main chain) of the nucleotide unit are replaced by novel groups. The base unit is retained to hybridize with an appropriate nucleic acid target compound. One such oligomeric compound (i.e., an RNA mimic that has been shown to have excellent hybridization properties) is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and is directly or indirectly bound to the aza nitrogen atom of the amide portion of the main chain. Representative U.S. patents that teach methods for preparing PNA compounds include, but are not limited to, U.S. Patents 5,539,082, 5,714,331, and 5,719,262; each of these patents is incorporated herein by reference to obtain teachings related to such preparation methods. Further teachings of PNA compounds can be found, for example, in Nielsen et al. (Science 1991, 254: 1497-1500).

[0116] Some embodiments characterized in the technology described herein include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, wherein the heteroatom backbones are particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as methylene (methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-] of U.S. Pat. No. 5,489,677, and the amide backbones of U.S. Pat. No. 5,602,240. In some embodiments, the RNA characterized herein has a morpholino backbone structure of U.S. Pat. No. 5,034,506.

[0117] Modifications of the siRNA disclosed herein may also include modifications or substitutions of nucleobases (commonly referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic nucleobases and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Additional nucleobases include those disclosed in the following documents: U.S. Pat. No. 3,687,808; Modified Nucleosides in Biochemistry, Biotechnology and Medicine (Herdewijn P, ed., Wiley-VCH, 2008); The Concise Encyclopedia Of Polymer Science And Engineering (pp. 858-859, Kroschwitz JL, ed., John Wiley & Sons, 1990); Englisch et al. (Angewandte Chemie, International Edition, 30, 613, 1991); and Sanghvi YS (Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke ST and Lebleu B, ed., CRC Press, 1993). Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds characterized in the technology described herein. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6°C to 1.2°C (Sanghvi YS et al., eds., dsRNA Research and Applications, CRC Press, Boca Raton, pp. 276-278, 1993) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0118] Representative U.S. patents that teach methods for preparing some of the modified nucleobases noted above, as well as other modified nucleobases, include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, and 5,595,191. Nos. 96,091, 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 teachings relating to methods of making such modifications.

[0119] siRNA can also be modified to include one or more diol nucleic acids, such as adenosine-diol nucleic acids (GNA). A description of adenosine-GNA can be found, for example, in Zhang et al. (JACS 2005, 127 (12): 4174-75), which is incorporated herein by reference for providing guidance related to methods for preparing GNA modifications.

[0120] The RNA of siRNA can also be modified to include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety contains an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo structural 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 2005, 33 (1): 439-47; Mook OR et al., Mol Cane Ther 2007, 6 (3): 833-43; Grunweller A et al., Nucleic Acids Research 2003, 31 (12): 3185-93).

[0121] Representative U.S. patents that teach methods for making 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 teachings relating to methods for making such modifications.

[0122] In some embodiments, the siRNA includes modifications involving chemical attachment of one or more ligands, moieties, or conjugates to the RNA that enhance the activity, cellular distribution, or cellular uptake of the siRNA. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA 1989, 86:6553-56), cholic acid (Manoharan et al., Biorg. Med. Chem. Let. 1990, 4:1053-60), thioethers (e.g., beryl-S-tritylthiol) (Manoharan et al., Ann. NY Acad. Sci. 1992, 660:306-9); Manoharan et al., Biorg. Med. Chem. Let. 1993, 3:2765-70), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20:533-38), an aliphatic chain, for example, dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J 1991, 10: 1111-18; Kabanov et al., FEBS Lett. 1990, 259: 327-30; Svinarchuk et al., Biochimie 1993, 75: 49-54), phospholipids (e.g., di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-phosphonic acid triethylammonium (Manoharan et al., Tetrahedron Lett. 1995, 36: 3651-54; Shea et al., Nucl. Acids Res. 1990, 18: 3777-83), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides 1995, 14:969-73) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36:3651-54), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264:229-37), or a stearylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277:923-37).

[0123] In some embodiments, the ligand alters the distribution, targeting, or lifetime of the siRNA into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a cell or organ compartment), tissue, organ, or body region, e.g., compared to a species in which such ligand is not present. In such embodiments, the ligand does not participate in duplex pairing in a duplex nucleic acid.

[0124] The ligand can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipids. The ligand can also be a recombinant molecule or a synthetic molecule, such as a synthetic polymer, for example a synthetic polyamino acid. Examples of polyamino acids include, the polyamino acid is polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, and alpha helical peptides.

[0125] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein (e.g., antibody) that binds to a specified cell type (such as a liver cell). The targeting group can be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipid, cholesterol, a steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) ) lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate esters, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP and AP.

[0126] The ligand can be a protein (e.g., a glycoprotein), or a peptide (e.g., a molecule with a specific affinity for a co-ligand), or an antibody (e.g., an antibody that binds to a specified cell type (such as a hepatocyte). The ligand can also include hormones and hormone receptors. The ligand can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, and multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[0127] The ligand can be a substance, such as a drug, that can increase the uptake of siRNA into a cell, for example, by disrupting the cytoskeleton of the cell, for example, by disrupting the microtubules, microfilaments and / or intermediate filaments of the cell. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine or myoservin.

[0128] In some embodiments, the ligand is a moiety (e.g., a vitamin) that is taken up by a target cell (e.g., a hepatocyte). Exemplary vitamins include vitamin A, vitamin E, and vitamin K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by a target cell (such as a hepatocyte). HSA and low-density lipoprotein (LDL) are also included.

[0129] In some embodiments, the ligand attached to siRNA as described herein acts as a pharmacokinetic (PK) regulator. As used herein, "PK regulator" refers to a pharmacokinetic regulator. PK regulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK regulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides comprising multiple thiophosphate bonds are also known to be combined with serum proteins, so the short oligonucleotides (for example, oligonucleotides with about 5 bases, 10 bases, 15 bases or 20 bases) comprising multiple thiophosphate bonds in the main chain are also suitable for being described as ligands (for example, described as PK regulating ligands) technology herein. In addition, the aptamers in conjunction with serum components (for example, serum proteins) are also suitable for use as PK regulating ligands in embodiments described herein.

[0130] (i) Lipid conjugates. In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. The lipid or lipid-based ligand can (a) increase the degradation resistance of the conjugate, (b) increase targeting or transport into target cells or cell membranes, and / or (c) can be used to regulate binding to serum proteins (e.g., HSA). Such lipids or lipid-based molecules can bind serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used.

[0131] Lipid-based ligands can be used to inhibit (e.g., control) the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

[0132] In some embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand is capable of binding to HSA with sufficient affinity so that the conjugate will be distributed to non-renal tissues. In certain specific embodiments, the HSA-ligand binding is reversible.

[0133] In some embodiments, the lipid-based ligand binds HSA weakly or not at all, such that the conjugate will distribute to the kidney.Other moieties that target renal cells may also be used in place of the lipid-based ligand or in addition.

[0134] (ii) cell penetrating peptides and cell penetrants. On the other hand, the ligand is a cell penetrant, such as a helical cell penetrant. In some embodiments, the cell penetrant is amphoteric. Exemplary cell penetrants are peptides, such as tat peptides or antennapedia peptides. If the cell penetrant is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide bonds, and using D-amino acids. In some embodiments, the helical agent is an alpha-helical agent. In certain specific embodiments, the helical agent has a lipophilic phase and a lipophobic phase.

[0135] "Cell penetrating peptides" are capable of penetrating cells, e.g., microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin PI), disulfide bond-containing peptides (e.g., α-defensins, β-defensins or bovine antimicrobial peptides), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indomethacin).

[0136] The part can be a peptide or a peptide mimetic. Peptide mimics (also referred to as oligopeptide mimics in this article) are molecules that can be folded into a defined three-dimensional structure similar to a natural peptide. The connection of peptide and peptide mimics to siRNA can affect the pharmacokinetic distribution of RNAi, such as by enhancing cell recognition and absorption. The length of the peptide or peptide mimic part can be about 5 to 50 amino acids, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.

[0137] The peptide or peptide mimetic can be, for example, a cell permeable peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide portion can be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). RFGF analogs containing a hydrophobic MTS (e.g., 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 including peptides, oligonucleotides and proteins across the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWK (SEQ ID NO: 12) have been found to be able to act as delivery peptides. The peptide or peptide mimetic can be encoded by a random DNA sequence, such as a peptide identified from a phage display library, or a one-bead-one-cell (OBOC) combinatorial library (Lam et al., Nature 1991, 354: 82-84).

[0138] The cell penetrating peptide may also include a nuclear localization signal (NLS). For example, the cell penetrating peptide may be a bipartite amphipathic peptide such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 1993, 31: 2717-24).

[0139] (iii) Carbohydrate conjugates. In some embodiments, the siRNA oligonucleotides described herein further comprise carbohydrate conjugates. Carbohydrate conjugates may be advantageous for in vivo delivery of nucleic acids, and compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate consisting of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms bonded to each carbon atom; or a compound having a carbohydrate moiety consisting of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms bonded to each carbon atom as part of it. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of C5 and above (in some embodiments, C5-C8 sugars); disaccharides and trisaccharides include sugars having two or three monosaccharide units (in some embodiments, C5-C8 sugars).

[0140] In some embodiments, the carbohydrate conjugate is selected from the group consisting of:

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] Another representative carbohydrate conjugate for use in the embodiments described herein includes

[0148]

[0149] (Formula XXII), wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0150] In some embodiments, the carbohydrate conjugate further comprises another ligand, such as, but not limited to, a PK modulator, an endosomal lytic ligand, or a cell penetrating peptide.

[0151] (iv) Linkers. In some embodiments, the conjugates described herein can be attached to siRNA oligonucleotides using various cleavable or non-cleavable linkers.

[0152] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound. The linker typically comprises a direct bond or an atom such as oxygen or sulfur; a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH; or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl , alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl and alkynylheteroaryl, 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 heterocyclyl; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between 1 and 24 atoms, between 4 and 24 atoms, between 6 and 18 atoms, between 8 and 18 atoms, or between 8 and 16 atoms in length.

[0153] A cleavable linking group is a linking group that is sufficiently stable outside of a cell, but which cleaves after entering a target cell to release the two parts of the linker that are held together. In certain embodiments, the cleavable linking group cleaves faster in a target cell or under a first reference condition (which can, for example, be selected to simulate or represent intracellular conditions) than in the blood of a subject or under a second reference condition (which can, for example, be selected to simulate or represent conditions present in blood or serum), at least 10 times or at least 100 times faster.

[0154] Cleavable linking groups are sensitive to the presence of cleavage agents such as pH, redox potential or degradation molecules. Generally speaking, cleavage agents are more common inside cells than in serum or blood, or their levels or activities are found to be higher. Examples of such degradation agents include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidases or reductases present in cells, or reductants, such as thiols, which can degrade redox cleavable linking groups by reduction; esterases; endosomes or agents that can produce an acidic environment, for example, those that make pH 5 or lower; enzymes, peptidases (which can have substrate specificity) and phosphatases that can hydrolyze or degrade acid cleavable linking groups by acting as universal acids. Cleavable linking groups (such as disulfide bonds) may be sensitive to pH. The pH of human serum is 7.4, while the average pH in cells is slightly lower, in the range of about 7.1 to 7.3. Endosomes have a relatively acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have a cleavable linking group that is cleaved at a specific pH, thereby releasing the cationic lipid from the ligand inside the cell, or into a desired cellular compartment.

[0155] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the targeted cell. For example, a liver targeting ligand can be connected to a cationic lipid by a linker that includes an ester group. Liver cells are rich in esterases, so the linker will be more efficiently cleaved in liver cells than in cell types that are not rich in esterases. Other cell types that are rich in esterases include cells of the lung, kidney cortex, and testis.

[0156] When targeting cell types rich in peptidases, such as liver cells and synoviocytes, linkers containing peptide bonds can be used.

[0157] Generally speaking, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to crack a candidate linking group. It may also be necessary to test the ability of a candidate cleavable linking group to resist cracking in blood or cracking when in contact with other non-target tissues. Therefore, the relative cracking susceptibility between the first condition and the second condition can be determined, wherein the first condition is selected to indicate the cracking in the target cell, and the second condition is selected to indicate the cracking in other tissues or biological fluids (e.g., blood or serum). These evaluations can be carried out in a cell-free system, cell, cell culture, organ or tissue culture, or a complete animal. It may be useful to perform an initial evaluation under cell-free conditions or culture conditions, and then confirm it by further evaluation in a complete animal. In certain embodiments, compared with blood or serum (or under in vitro conditions selected to simulate extracellular conditions), the cracking of available candidate compounds in cells (or under in vitro conditions selected to simulate intracellular conditions) is faster, at least 2 times, at least 4 times, at least 10 times or at least 100 times.

[0158] A class of cleavable linking groups is a redox cleavable linking group that is cleaved when reduced or oxidized. An example of a reduction cleavable linking group is a disulfide linking group (-SS-). In order to determine whether a candidate cleavable linking group is a suitable "reduction cleavable linking group", or whether it is suitable for use with a specific RNAi part and a specific targeting agent, it can be found in the method described herein. For example, a candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art, and these reagents simulate the cleavage rate observed in cells (e.g., target cells). The candidate can also be evaluated under conditions selected to simulate blood or serum conditions. In some embodiments, the candidate compound is cleaved up to 10% in blood. In certain embodiments, compared with blood (or under in vitro conditions selected to simulate extracellular conditions), the degradation of the available candidate compound in cells (or under in vitro conditions selected to simulate intracellular conditions) is faster, at least 2 times, at least 4 times, at least 10 times or at least 100 times. The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0159] Phosphate-based cleavable linking groups are cleaved by factors that degrade or hydrolyze the phosphate group. Examples of factors that cleave the phosphate group in cells are enzymes in the cell, such as phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In certain 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-, -OP(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 specific embodiments, the phosphate linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0160] Acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, the acid cleavable linking group is cleaved in an acidic environment where pH is about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or is cleaved by a reagent (such as an enzyme) that can serve as a general acid. In cells, specific low pH organelles (such as endosomes and lysosomes) can provide a cleavage environment for acid cleavable linking groups. Examples of acid cleavable linking groups include, but are not limited to, esters of hydrazones, esters, and amino acids. Acid cleavable groups can have the general formula -C=N-, C(O)O or -OC(O). In some embodiments, the carbon (alkoxy group) attached to the oxygen in the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or tert-butyl. These candidates can be evaluated using a method similar to the above method.

[0161] The cleavable linking group based on ester is cracked by enzymes (such as esterase and amidase) in cells. The example of the cleavable linking group based on ester includes but is not limited to the ester of alkylene group, alkenylene group and alkynylene group. The ester cleavable linking group has the general formula-C (O) O- or-OC (O)-. These candidates can be evaluated using a method similar to the above method.

[0162] The cleavable linking group based on peptide is cleaved by enzymes (such as peptidases and proteases) in cells. The cleavable linking group based on peptide is formed between amino acids to produce peptide bonds of oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The cleavable group based on peptide does not include an amide group (-C (O) NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. The peptide bond is an amide bond of a special type formed between amino acids to produce peptides and proteins. The cleavable group based on peptide is generally limited to the peptide bond (i.e., amide bond) formed between amino acids to produce peptides and proteins, and does not include the entire amide functional group. The cleavable linking group based on peptide has the general formula -NHCHRAC (O) NHCHRBC (O) -, wherein RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to the above method.

[0163] Representative carbohydrate conjugates with linkers include, but are not limited to:

[0164]

[0165]

[0166]

[0167] wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0168] In certain embodiments of the compositions and methods described herein, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker. For example, in some embodiments, the siRNA is conjugated to a GalNAc ligand as shown in the following schematic:

[0169]

[0170] wherein X is O or S. In some of these embodiments, X is O.

[0171] In some embodiments, the combination therapy comprises an siRNA conjugated to a bivalent or trivalent branched linker selected from the group consisting of structures represented by any one of Formulae (XXXI)-(XXXIV):

[0172]

[0173]

[0174] in,

[0175] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0-20 at each occurrence, and wherein the repeating units may be the same or different;

[0176] p 2A , 2B , 3A , 3B , 4A , 4B , 5A , 5B , 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B and T 5C is, independently at each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;

[0177] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B and Q 5C is independently absent at each occurrence and is alkylene or substituted alkylene, wherein one or more methylene groups may be interrupted by 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);

[0178] R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B and R 5C Each occurrence is independently absent and is NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, or a heterocyclic group,

[0179] L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, independently at each occurrence is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a Is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for inhibiting the expression of target genes together with siRNA, such derivatives as derivatives of formula (XXXV):

[0180]

[0181] Where L 5A , L 5B and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0182] Examples of suitable divalent and trivalent branched linker group conjugated GalNAc derivatives include, but are not limited to, the structures listed above in Formula I, Formula VI, Formula X, Formula IX, and Formula XII.

[0183] Representative U.S. patents that teach methods for preparing RNA conjugates include: U.S. Pat. Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, 5,486,603 、No. 5,512,439、No. 5,578,718、No. 5,608,046、No. 4,587,044、No. 4,605,735、No. 4,667,025、No. 4,762,779、No. 4,789,737、No. 4,824,941、No. 4,835,263、No. 4,876,335、No. 4,904,582、No. 4,958,013、No. 5,082,830、No. 5,112,963、No. 5,214,136、No. 5,082,830、No. 5,1 No. 12,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,8 10, 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 its teachings relating to such methods of preparation.

[0184] In some cases, the RNA of the siRNA can be modified with non-ligand groups. Many non-ligand molecules have been conjugated to siRNA in order to enhance the activity, cellular distribution or cellular uptake of the siRNA, and procedures for performing such conjugations can be found in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo T et al., Biochem. Biophys. Res. Comm. 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci. 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20: 533), aliphatic chains such as dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10: 111; Kabanov et al., FEBS Lett. 1990, 259: 327; Svinarchuk et al., Biochimie 1993, 75: 49), phospholipids (e.g., di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonic acid triethylammonium (Manoharan et al., Tetrahedron Lett. 1995, 36: 3651; Shea et al., Nucl. Acids Res. 1990, 18: 3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides 1995, 14:969) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264:229), or a stearylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277:923).

[0185] Typical conjugation protocols involve synthesizing RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using a suitable coupling or activating reagent. The conjugation reaction can be performed in the solution phase while the RNA is still bound to a solid support or after RNA cleavage. Purification of RNA conjugates by HPLC generally yields pure conjugates.

[0186] b. Pharmaceutical compositions and siRNA delivery

[0187] In some embodiments, a pharmaceutical composition containing siRNA as described herein and a pharmaceutically acceptable carrier or excipient is provided. The pharmaceutical composition containing the siRNA can be used to treat HBV infection. Such pharmaceutical compositions are usually formulated based on the delivery mode. For example, the composition can be formulated for systemic administration achieved via parenteral delivery (e.g., by subcutaneous (SC) delivery).

[0188] A "pharmaceutically acceptable carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more agents (such as nucleic acids) to an animal. Excipients can be liquid or solid, selected with the intended mode of administration in mind so as to provide the desired volume, consistency, etc. when combined with the agent (e.g., nucleic acid) and other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers or excipients include, but are not limited to: binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0189] The siRNA compositions can also be prepared using pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not react adversely with nucleic acids. Suitable pharmaceutically acceptable carriers for non-parenteral delivery formulations include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, gelatin, lactose, straight-chain starch, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0190] Preparations for topical administration of nucleic acid may include sterile aqueous solutions and non-sterile aqueous solutions, non-aqueous solutions in common solvents (such as alcohols), or solutions of nucleic acid in liquid oil bases or solid oil bases. These solutions may also contain buffer, diluent and other suitable additives. Pharmaceutically acceptable organic excipients or inorganic excipients suitable for non-parenteral administration that do not have adverse reactions with nucleic acid may be used.

[0191] In some embodiments, the pharmaceutical compositions and pharmaceutical formulations described herein can be administered topically (e.g., by a transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by a nebulizer), intratracheal, intranasal, epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subdermal administration (e.g., via an implant device); or intracranial administration (e.g., by intraparenchymal, intrathecal, or intraventricular administration).

[0192] In some embodiments, the pharmaceutical composition includes a sterile solution of siRNA (eg, SIRNA01) formulated in water for subcutaneous injection. In some embodiments, the pharmaceutical composition includes a sterile solution of SIRNA01 formulated in water for subcutaneous injection, with a free acid concentration of 200 mg / mL.

[0193] In some embodiments, the pharmaceutical composition containing siRNA described herein is applied with a dosage sufficient to inhibit HBV gene expression. In some embodiments, the dosage of siRNA is in the range of 0.001 mg to 200.0 mg / kg recipient body weight per day, or in the range of 1 mg to 50 mg / kg body weight per day. For example, siRNA can be applied with 0.01mg / kg, 0.05mg / kg, 0.5mg / kg, 1mg / kg, 1.5mg / kg, 2mg / kg, 3mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg or 50mg / kg per single dose. The pharmaceutical composition can be applied once a day, or can be applied as two, three or more sub-doses at appropriate intervals throughout the day, or even using continuous infusion or delivery realized by controlled release formulations. In this case, in order to achieve a total daily dose, the siRNA contained in each sub-dose must be correspondingly smaller. The dosage unit can also be compounded for delivery over a few days, for example, using a conventional sustained release formulation that provides sustained release of the siRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents at specific sites, such as can be used with agents of the technology described herein. In such embodiments, the dosage unit comprises a corresponding multiple of the daily dose.

[0194] In some embodiments, a pharmaceutical composition comprising a siRNA targeting an HBV mRNA described herein (eg, SIRNA01) contains the siRNA at a dose of 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg, or 15 mg / kg.

[0195] In some embodiments, the pharmaceutical composition comprising siRNA described herein (e.g., SIRNA01) contains a dosage of 20mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg or 900mg siRNA. In some embodiments, the pharmaceutical composition comprising siRNA described herein (e.g., SIRNA01) contains a dosage of 20mg to 900mg siRNA. In some embodiments, the pharmaceutical composition comprising siRNA described herein (e.g., SIRNA01) contains a dosage of 100mg to 300mg siRNA.

[0196] In some embodiments, a pharmaceutical composition comprising a siRNA described herein (eg, SIRNA01) contains a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg of siRNA.

[0197] In some embodiments, a pharmaceutical composition comprising an siRNA described herein (eg, SIRNA01) contains a dose of 200 mg of the siRNA.

[0198] III. Anti-HBV Antibodies

[0199] The present disclosure also provides anti-HBV antibodies for use in combined therapies for treating HDV or HDV-related diseases. Antibodies that bind to the antigenic loop of HBsAg on the surface of HDV virions can act as entry inhibitors by blocking the interaction between HBsAg and its receptor NTCP. Inhibiting viral entry prevents a new round of HDV infection in the liver and ultimately reduces HDV viremia. Antibodies can also promote HDV clearance by conditioning virions. Antibodies can also have indirect antiviral activity against HDV by stimulating an immune response to HBsAg, where HBsAg will be present in co-infected hepatocytes, thereby producing new HDV virions.

[0200] a. Antibodies that bind to HBV proteins

[0201] In some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy binds to the antigenic loop region of HBsAg. The envelope of the hepatitis B virus contains three "HBV envelope proteins" (also known as "HBsAg", "hepatitis B surface antigen"): S protein ("small", also known as S-HBsAg), M protein ("medium", also known as M-HBsAg) and L protein ("large", also known as L-HBsAg). S-HBsAg, M-HBsAg and L-HBsAg share the same C-terminus (also known as the "S domain", containing 226 amino acids), which corresponds to the S protein (S-HBsAg) and is involved in viral assembly and infectivity. S-HBsAg, M-HBsAg and L-HBsAg are synthesized in the endoplasmic reticulum (ER) and secreted in the form of particles through the Golgi apparatus after assembly. The S domain contains four predicted transmembrane (TM) domains, whereby both the N-terminus and C-terminus of the S domain are exposed to the lumen. Transmembrane domains TM1 and TM2 are both necessary for the integration of co-translational proteins into the ER membrane, and transmembrane domains TM3 and TM4 are located at the C-terminal third of the S domain. The "antigenic loop region" of HBsAg is located between the predicted TM3 transmembrane domain and the TM4 transmembrane domain of the HBsAg S domain, whereby the antigenic loop region comprises amino acids 101 to 172 of the S domain (Salisse J and Sureau C, Journal of Virology 2009, 83: 9321-8). An important determinant of infectivity is present in the antigenic loop region of the HBV envelope protein. In particular, the residues between the 119th and 125th positions of HBsAg contain the CXXC motif, which has been shown to be the most important sequence required for HBV infectivity (Jaoude GA and Sureau C, Journal of Virology 2005, 79: 10460-6).

[0202] As used herein, the S domain of HBsAg refers to the amino acid sequence shown in SEQ ID NO: 13 (shown below), or a natural or artificial sequence variant thereof.

[0203] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTA QGTSMYPSCCCTKPSD GNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVOWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI (SEQ ID NO: 13; amino acids 101 to 172 are underlined)

[0204] For example, the expression "amino acids 101 to 172 of the S domain" refers to amino acid residues 101 to 172 of the polypeptide according to SEQ ID NO: 13. However, it will be understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions, for example, HBsAg of different genotypes or different HBsAg mutants as described herein) may naturally exist in the amino acid sequence of the HBsAg S domain, or be artificially introduced into the amino acid sequence of the HBsAg S domain without affecting its biological properties. Therefore, the term "HBsAg S domain" includes all such polypeptides, for example, including polypeptides according to SEQ ID NO: 13 and natural or artificial mutants thereof. In addition, when describing a sequence fragment of the HBsAg S domain herein (e.g., amino acids 101 to 172 or amino acids 120 to 130 of the HBsAg S domain), not only the corresponding sequence fragments of SEQ ID NO: 13 are included, but also the corresponding sequence fragments of natural or artificial mutants thereof. For example, the expression "amino acid residues from positions 101 to 172 of the HBsAg S domain" includes corresponding fragments of amino acid residues from positions 101 to 172 of SEQ ID NO: 13 and mutants (natural or artificial mutants) thereof.

[0205] As used herein, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at the same position of the sequence when the sequence is optimally aligned (i.e., the sequence is aligned to obtain the highest identity percentage). The M protein (M-HBsAg) corresponds to the S protein extended by an N-terminal domain comprising 55 amino acids and called "pre-S2". The L protein (L-HBsAg) corresponds to the M protein extended by an N-terminal domain comprising 108 amino acids and called "pre-S1" (genotype D). The pre-S1 domain and pre-S2 domain of the L protein can be present on the inner surface of the virus particle (on the cytoplasmic side of the ER), thereby playing a key role in virus assembly, or on the outer surface (on the luminal side of the ER), which can be used to interact with target cells and is necessary for viral infectivity. In addition, the HBV surface protein (HBsAg) is not only incorporated into the virion envelope, but also spontaneously buds from the ER-Golgi intermediate compartment membrane to form an empty "subviral particle" (SVP), which is released from the cell by secretion.

[0206] Because all three HBV envelope proteins S-HBsAg, M-HBsAg and L-HBsAg contain the S domain, all three HBV envelope proteins S-HBsAg, M-HBsAg and L-HBsAg also contain an "antigenic loop region." Therefore, an antibody or antigen-binding fragment thereof that binds to the antigenic loop region of HBsAg binds to all three HBV envelope proteins: S-HBsAg, M-HBsAg and L-HBsAg.

[0207] In addition, in some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy neutralizes hepatitis B virus infection. In other words, the antibody or antigen-binding fragment thereof can reduce the viral infectivity of hepatitis B virus. In some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy neutralizes hepatitis D virus infection. In other words, the antibody or antigen-binding fragment thereof can reduce the viral infectivity of hepatitis D virus (see below for further explanation of hepatitis D virus as an obligate satellite of hepatitis B virus).

[0208] To study and quantify viral infectivity (or "neutralization") in the laboratory, those skilled in the art are aware of various standard "neutralization assays". For neutralization assays, animal viruses are typically propagated in cells and / or cell lines. In the context of the present disclosure, for neutralization assays, cultured cells can be incubated with a fixed amount of HBV in the presence (or absence) of the antibody to be tested. As a readout, the level of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant can be used, and / or HBcAg staining can be assessed. In one embodiment of the HBV neutralization assay, cultured cells (e.g., HepaRG cells, particularly differentiated HepaRG cells) are incubated with a fixed amount of HBV in the presence or absence of the antibody to be tested, for example, at 37°C for 16 hours. The incubation can be carried out in a culture medium (e.g., supplemented with 4% PEG 8000). After incubation, the cells can be washed and further cultured. To measure viral infectivity, the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the culture supernatant, for example from day 7 to day 11 after infection, can be determined by enzyme-linked immunosorbent assay (ELISA). In addition, HBeAg staining can be assessed by immunofluorescence assay.

[0209] In some embodiments, antibodies and antigen-binding fragments have high and medium efficacy. The concentration of the disclosed antibodies required for 50% neutralization of hepatitis B virus (HBV) is, for example, about 10pg / ml or less. In certain embodiments, the concentration of the disclosed antibodies required for 50% neutralization of HBV is about 5pg / ml, about 1pg / ml, or about 750ng / ml. In certain embodiments, the concentration of the disclosed antibodies required for 50% neutralization of HBV is 500ng / ml or less, for example, 450ng / ml, 400ng / ml, 350ng / ml, 300ng / ml, 250ng / ml, 200ng / ml, 175ng / ml, 150ng / ml, 125ng / ml, 100ng / ml, 90ng / ml, 80ng / ml, 70ng / ml, 60ng / ml, or about 50ng / ml or less. This means that only a low concentration of antibodies is required for 50% neutralization of HBV. Specificity and potency can be measured using standard assays known to those of skill in the art.

[0210] In some embodiments, anti-HBV antibodies as components of combination therapy can be used to prevent and / or treat hepatitis B or hepatitis B-related diseases.

[0211] In some embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure promote the removal of HBsAg and HBV. In particular, antibodies or antigen-binding fragments thereof according to the present disclosure can promote the removal of both HBV and hepatitis B virus subviral particles (SVP). The removal of HBsAg or subviral particles can be assessed by measuring, for example, the level of HBsAg in a blood sample (e.g., from a hepatitis B patient). Similarly, the removal of HBV can be assessed by measuring, for example, the level of HBV in a blood sample (e.g., from a hepatitis B patient).

[0212] In the serum of patients infected with HBV, in addition to infectious particles (HBV), there is usually an excess (usually 1,000 times to 100,000 times) of empty subviral particles (SVP) composed only of HBV envelope protein (HBsAg), which are in the form of relatively small spheres and filaments of variable length. Subviral particles have been shown to strongly enhance the intracellular viral replication and gene expression of HBV (Bruns M et al., J Virol 1998, 72 (2): 1462-8). This is also important in the infectivity of serum containing HBV, because infectivity depends not only on the number of viruses, but also on the number of SVP (Bruns et al., 1998, supra). In addition, excessive subviral particles can act as bait by absorbing neutralizing antibodies, thus delaying the removal of infection. Generally, the realization of hepatitis B surface antigen (HBsAg) loss is therefore considered to be the ideal endpoint of treatment and the closest result of curing chronic hepatitis B (CHB). Therefore, in some embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure that promote HBsAg clearance, in particular clearance of subviral particles of hepatitis B virus and HBV, enable improved treatment of hepatitis B, particularly in the case of chronic hepatitis B. Therefore, antibodies or antigen-binding fragments thereof according to the present disclosure can potently neutralize HBV because less antibody is absorbed by SVP as bait. In addition, in certain embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure promote clearance of subviral particles of hepatitis B virus and reduce the infectivity of HBV in serum.

[0213] According to the genome sequence, HBV is differentiated into many genotypes. So far, eight well-known genotypes (A to H) of the HBV genome have been defined. In addition, two new genotypes I and J (Sunbul M, World J Gastroenterol 2014, 20 (18): 5427-34) have been identified. It is known that genotype affects disease progression, and differences in response to antiviral therapy between genotypes have been determined. For example, genotype A has a tendency to chronicity, while genotype c often undergoes viral mutations. Chronicity and mutation frequency are both common in genotype D. In addition, there are differences in the distribution of HBV genotypes worldwide (Sunbul, 2014, supra). In certain embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure are combined with at least 6, at least 8, or all 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. Examples of different genotypes of HBsAg include the following: GenBank Accession No. J02203 (HBV-D, ayw3), GenBank Accession No. FJ899792.1 (HBV-D, adw2), GenBank Accession No. AM282986 (HBV-A), GenBank Accession No. D23678 (HBV-B1 Japan), GenBank Accession No. AB1 1 7758 (HBV-C1 Cambodia), GenBank Accession No. AB205192 (HBV-E Ghana), GenBank Accession No. X69798 (HBV-F4 Brazil), GenBank Accession No. AF 160501 (HBV-G United States), GenBank Accession No. AY090454 (HBV-H Nicaragua), GenBank Accession No. AF241409 (HBV-I Vietnam) and GenBank Accession No. AB486012 (HBV-J Borneo). The amino acid sequences of the antigenic loop region of the S domain of HBsAg with different genotypes are shown in Table 2 (SEQ ID NOs: 14-42).

[0214] Table 2. Antigenic loop sequences from various HBV genotypes.

[0215]

[0216]

[0217] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the following HBsAg mutants having mutations in the antigenic loop region: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121 S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141 E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R and HBsAg N146A. These mutants are naturally occurring mutants of the S domain based on HBsAg genotype D (SEQ ID NO: 43) (GenBank Accession No. FJ899792) (wherein the mutated amino acid residues are indicated by name).

[0218] MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSD GNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVOWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI (SEQ ID NO: 43) (antigenic loop region, ie, amino acids 101 to 172, are underlined).

[0219] In a specific embodiment, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to at least 12, at least 15 or all 18 of the following infectious HBsAg mutants having mutations in the antigenic loop region: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121 S, HBsAg R122D, HBsAg R122I, HBsAgT123N, HBsAg Q129H, HBsAg Q129L, HBsAg M1 33H, HBsAg M133L, HBsAg M1 33T, HBsAgK141 E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R and HBsAg N146A.

[0220] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to an epitope comprising at least one, at least two, at least three, or at least four amino acids of 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 to 133 of the S domain of HBsAg, amino acids 120 to 133 of the S domain of HBsAg, or amino acids 120 to 130 of the S domain of HBsAg. It is noteworthy that the positions of these amino acids (e.g., positions 115 to 133, positions 120 to 133, positions 120 to 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.

[0221] In a specific embodiment, the antibody or antigen-binding fragment thereof 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 a position selected from amino acids 115 to 133, amino acids 120 to 133, or amino acids 120 to 130 of the S domain of HBsAg.

[0222] As used herein in the context of epitopes, the term "formed by..." means that the epitope to which the antibody or antigen-binding fragment thereof of the present disclosure binds may be a linear epitope (continuous) or a conformational epitope (discontinuous). A linear epitope or a sequential epitope is an epitope recognized by an antibody through its linear amino acid sequence or primary structure. In contrast, a conformational epitope has a specific three-dimensional shape and protein structure. Therefore, if the epitope is a linear epitope and comprises more than one amino acid located at the position of amino acids 115 to 133 or amino acids 120 to 133 selected from the S domain of HBsAg, the amino acids contained in the epitope may be located at adjacent positions of the primary structure (i.e., continuous amino acids in the amino acid sequence). In contrast, in the case of a conformational epitope (3D structure), the amino acid sequence generally forms a 3D structure as an epitope, and therefore, the amino acids forming the epitope (or the amino acids "comprising" the epitope) may or may not be located at adjacent positions of the primary structure (i.e., may or may not be continuous amino acids in the amino acid sequence). In certain embodiments, the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed only by amino acids selected from amino acids 115 to 133, amino acids 120 to 133, or amino acids 120 to 130 of the S domain of HBsAg. In specific embodiments, (additional) amino acids located outside of positions 115 to 133, 120 to 133, or 120 to 130 are not required to form the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind.

[0223] In certain embodiments, the epitope in the antigenic loop region of HBsAg to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed by two or more amino acids located at positions selected from amino acids 115 to 133, amino acids 120 to 133, or amino acids 120 to 130 of the S domain of HBsAg. In certain embodiments, the epitope in the antigenic loop region of HBsAg to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed by three or more amino acids located at positions selected from amino acids 115 to 133, amino acids 120 to 133, and amino acids 120 to 130 of the S domain of HBsAg. In some embodiments, the epitope in the antigenic loop region of HBsAg to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed by four or more amino acids located at positions selected from amino acids 115 to 133, amino acids 120 to 133, or amino acids 120 to 130 of the S domain of HBsAg. Therefore, the antibody or antigen-binding fragment thereof according to the present disclosure may bind to at least one, at least two, at least three or at least four amino acids selected from the following amino acids in the antigenic loop region of HBsAg: amino acids 115 to 133 of the S domain of HBsAg, amino acids 120 to 133 of the S domain of HBsAg, or amino acids 120 to 130 of the S domain of HBsAg. In a specific embodiment, the antibody or antigen-binding fragment thereof according to the present disclosure binds to an epitope comprising at least two, at least three or at least four amino acids in the antigenic loop region of HBsAg, wherein the at least two, at least three or at least four amino acids are selected from amino acids 120 to 133, or amino acids 120 to 130 of the S domain of HBsAg, and wherein the at least two, at least three or at least four amino acids are located at adjacent positions (i.e., consecutive amino acids in the amino acid sequence / primary structure).

[0224] In certain embodiments, the epitope to which the antibody or antigen-binding fragment thereof according to the present disclosure binds is a conformational epitope. Therefore, the antibody or antigen-binding fragment thereof according to the present disclosure may bind to an epitope comprising at least two, at least three or at least four amino acids of the HBsAg antigenic loop region, wherein the at least two, at least three or at least four amino acids are selected from amino acids 120 to 133, or amino acids 120 to 130 of the HBsAg S domain, and wherein the at least two, at least three or at least four amino acids are located in adjacent positions (of the primary structure).

[0225] In certain specific embodiments, the antibodies of the present disclosure are bispecific antibodies having a first specificity for HBsAg and a second specificity for stimulating immune effector cells (e.g., by targeting T cell surface proteins, such as the extracellular portion of the CD3 protein). The second specificity can cause, for example, a cytotoxic effect or a vaccine effect.

[0226] b. Fc part

[0227] In some embodiments, the binding protein (e.g., an antibody or an antigen-binding fragment thereof) comprises an Fc portion. In certain embodiments, the Fc portion may be derived from humans, e.g., from human IgG1, IgG2, IgG3 and / or IgG4. In specific embodiments, the antibody or antigen-binding fragment may comprise an Fc portion derived from human IgG1.

[0228] As used herein, the term "Fc portion" refers to a sequence comprising or derived from a portion of an immunoglobulin heavy chain, which portion starts at the hinge region upstream of the papain cleavage site (e.g., the 216th residue in native IgG, taking the first residue of the heavy chain constant region as the 114th residue) and ends at the C-terminal of the immunoglobulin heavy chain. Therefore, the Fc portion can be a complete Fc portion or a portion thereof (e.g., domain). In certain embodiments, the complete Fc portion comprises a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU numbering system amino acids 216 to 446). Additional lysine residues (K) are sometimes present at the extreme C-terminal of the Fc portion, but are usually cut from mature antibodies. The amino acid positions in the Fc portion have been numbered according to the EU numbering system of Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 1983 and 1987). The amino acid positions of the Fc portion may also be numbered according to the IMGT numbering system (including the unique numbering of the C domain and the exon numbering) and the Kabat numbering system.

[0229] In some embodiments, the Fc portion comprises at least one of the following: a hinge (e.g., an upper hinge region, a middle hinge region, and / or a 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. An amino acid sequence of an exemplary Fc portion of a human IgG1 isotype is provided in SEQ ID NO: 60. Relative to a naturally occurring Fc portion, the Fc portion may also comprise one or more amino acid insertions, deletions, or substitutions. For example, a hinge domain, a CH2 domain, or a CH3 domain, or at least one of a portion of these domains may be deleted. For example, the Fc portion may comprise or consist of (i) a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof.

[0230] The Fc portion of the present disclosure can be modified so that it differs from the intact Fc portion of a naturally occurring immunoglobulin molecule in amino acid sequence while retaining (or enhancing) at least one desired function conferred by the naturally occurring Fc portion. These 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. Various portions of naturally occurring Fc portions that are involved in such functions have been described in the art.

[0231] For example, to activate the complement cascade, when an immunoglobulin molecule is attached to an antigen target, the C1q protein complex can bind to at least two IgG1 molecules or one IgM molecule (Ward ES and Ghetie, V, Ther. Immunol. 1995, 277-94). The heavy chain region containing amino acid residues 318 to 337 is involved in complement fixation (Burton DR, Mol. Immunol. 1985, 22: 161-206). Duncan AR and Winter G. (Nature 1988, 332: 738-40) reported using site-directed mutagenesis that Glu318, Lys320 and Lys322 form a binding site for C1q. The role of the Glu318 residue, the Lys320 residue and the Lys 322 residue in binding to C1q was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement-mediated lysis.

[0232] For example, FcR binding can be mediated by the interaction of the Fc portion (of an antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on cells, including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes, as well as the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated with the corresponding antibody via antibody-dependent cell-mediated cytotoxicity (ADCC; Van de Winkel JG and Anderson CL, J. Leukoc. Biol. 1991, 49: 511-24). FCRs are defined according to their specificity for immunoglobulin classes; the Fc receptors for IgG antibodies are called FcyRs, the Fc receptors for IgE antibodies are called FcsRs, the Fc receptors for IgA antibodies are called FcaRs, and so on, and the neonatal Fc receptor is called FcRn. Fc receptor binding is described, for example, in Ravetch JV and Kinet JP, Annu. Rev. Immunol. 1991, 9:457-92; Capel PJ et al., Immunomethods 1994, 4:25-34; de Haas M et al., J Lab. Clin. Med. 1995, 126:330-41; and Gessner JE et al., Ann. Hematol. 1998, 76:231-48.

[0233] Cross-linking of the Fc domain of a natural IgG antibody (FcyR) to a receptor triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory mediators, and immune complex clearance and regulation of antibody production. Contemplated herein are Fc portions (e.g., FcyR) that provide receptor cross-linking. In humans, three classes of FcyRs have been characterized: (i) FcyRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complexed IgG with moderate to low affinity, is widely expressed, especially on leukocytes, is considered a central player in antibody-mediated immunity, and can be divided into FcyRIIA, FcyRIIB and FcyRIIC, 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) FcyRIII (CD16), which binds IgG with moderate to low affinity and has been found in two forms: FcyRIIIA, which has been found on NK cells, macrophages, eosinophils and some monocytes and T cells, and is thought to mediate ADCC; and FcyRIIIB, which is highly expressed on neutrophils.

[0234] FcγRIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils), and appears to be able to activate the killing process. FcγRIIB appears to play a role in the inhibition process and is found on B cells, macrophages, mast cells and eosinophils. It has been confirmed that 75% of all FcγRIIB are found in the liver (Ganesan LP et al., Journal of Immunology 2012, 189: 4981-8). FcγRIIB is expressed in large quantities on the liver sinusoidal endothelium (called LSEC) and in Kupffer cells in the liver, and LSEC is the main site for clearing small immune complexes (Ganesan et al., 2012, supra).

[0235] In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein include an Fc portion for binding to FcγRIIb, particularly an Fc region, such as an IgG type antibody. In addition, it is possible to engineer the Fc portion to enhance FcγRIIB binding by introducing mutations S267E and L328F, as described by Chu SY et al. (Molecular Immunology 2008, 45: 3926-33). Thus, the clearance rate of immune complexes can be enhanced (Chu S et al., Am J Respir Crit, American Thoracic Society International Conference Abstracts 2014). In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include an engineered Fc portion with mutations S267E and L328F, particularly as described by Chu SY et al. (2008, supra).

[0236] On B cells, FcyRIIB appears to act to prevent further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcyRIIB is thought to inhibit phagocytosis mediated by FcyRIIA. On eosinophils and mast cells, the b-form may help prevent these cells from activating by binding of IgE to its independent receptors.

[0237] Regarding FcyRI binding, modifications in native IgG of at least one of E233-G236, P238, D265, N297, A327, and P329 reduced binding to FcyRI. IgG2 residues at positions 233 to 236 substituted with the corresponding positions of IgG1 and IgG4 reduced binding of IgG1 and IgG4 to FcyRI by a factor of 10 3 times, and eliminated the response of human monocytes to antibody-sensitized erythrocytes (Armour KL et al., Eur. J. Immunol. 1999, 29: 2613-2624).

[0238] With respect to FcyRII binding, reduced binding to FcyRIIA was found, for example, for IgG mutations of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

[0239] With respect to FcyRIII binding, reduced binding to FcyRIIIA was found, for example, for mutations in at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376.

[0240] Methods for mapping the Fc receptor binding site on human IgG1, the mutation sites mentioned above, and methods for measuring binding to FcyRI and FcyRIIA are described in Shields RL et al. (J. Biol. Chem. 2001, 276: 6591-6604).

[0241] With regard to binding to FcγRII, two regions of native IgG Fc appear to be involved in the interaction between FcγRII and IgG, namely: (i) the lower hinge site of IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, specifically the loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example, in the P331 region (Wines BD et al., J. Immunol. 2000, 164: 5313-8). In addition, FcγRI appears to bind to the same site on IgG Fc, while FcRn and protein A bind to different sites on IgG Fc, which appear to be located at the CH2-CH3 interface (Wines BD et al., 2000, supra).

[0242] Mutations that increase the binding affinity of the Fc portion of the present disclosure to (i.e., one or more) Fy receptors (e.g., compared to a reference Fc portion or antibody that does not contain these mutations) are also contemplated. See, e.g., Delillo and Ravetch, Cell 2015, 161(5):1035-45, and Ahmed et al., J. Struc. Biol. 2016, 194(1):78, Fc mutations and techniques thereof are incorporated herein by reference. In any of the embodiments disclosed herein, the binding protein may comprise an Fc portion comprising a mutation selected from the group consisting of G236A, S239D, A330L, and I332E, or a combination of these mutations; for example, S239D / I332E, S239D / A330L / I332E, G236A / S239D / I332E, G236A / A330L / I332E, and G236A / S239D / A330L / I332E.

[0243] In certain embodiments, the Fc part may include at least a portion of the Fc part that participates in binding to FcRn, or is composed of at least a portion. In certain embodiments, the Fc part includes one or more amino acid modifications, which improve the binding affinity to FcRn, and in some embodiments, thereby extending the in vivo half-life of the molecule comprising the Fc part (for example, compared with the reference Fc part or antibody that does not include these modifications). In certain embodiments, the Fc part includes or is derived from IgG Fc, and the half-life extension mutation includes any one or more of the following: M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V, T307A, and E380A (EU numbering). In certain embodiments, the half-life extension mutation includes M428L / N434S. In certain embodiments, the half-life extension mutation includes M252Y / S254T / T256E. In certain embodiments, the half-life extension mutation includes T250Q / M428L. In certain embodiments, the half-life extension mutation includes P257VQ31 II. In certain embodiments, the half-life extension mutation includes P257I / N434H. In certain embodiments, the half-life extension mutation includes D376V / N434H. In certain embodiments, the half-life extension mutation includes T307A / E380A / N434A.

[0244] In a specific embodiment, the binding protein comprises an Fc portion comprising the following substitution mutations: M428L / N434S and G236A / A330L / I332E. In certain embodiments, the antibody or antigen-binding fragment comprises an Fc portion comprising the following substitution mutations: M428L / N434S and G236A / S239D / A330L / I332E.

[0245] In specific embodiments, the binding protein comprises an Fc portion comprising the following substitution mutations: G236A / A330L / I332E. In certain embodiments, the antibody or antigen-binding fragment comprises an Fc portion comprising the following substitution mutations: G236A / S239D / A330L / I332E.

[0246] Alternatively or in addition, the Fc portion of the binding protein of the present disclosure may include at least a portion known in the art to be required for binding to protein A; and / or the Fc portion of the antibody of the present disclosure includes at least a portion of the Fc molecule known in the art to be required for binding to protein G. In some embodiments, the effect of retention includes clearing HBsAg and HBVg. Therefore, in certain embodiments, the Fc portion includes at least a portion known in the art to be required for binding to FcyR. As outlined above, the Fc portion may therefore comprise at least: (i) the lower hinge site of native IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering), and (ii) an adjacent region of the CH2 domain of native IgG Fc, specifically the loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example, in the P331 region, for example, a region comprising at least 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids located around P331 in the upper CH2 domain of native IgG Fc (e.g., between amino acids 320 and 340 (EU numbering) of native IgG Fc).

[0247] In some embodiments, the binding protein according to the present disclosure comprises an Fc region. As used herein, the term "Fc region" refers to a portion formed by two or more Fc portions of an antibody heavy chain in an immunoglobulin. For example, the Fc region can be a monomer or a "single-chain" Fc region (i.e., an scFc region). A single-chain Fc region is composed of multiple Fc portions connected within a single polypeptide chain (e.g., encoded with a single continuous nucleic acid sequence). Exemplary scFc regions are disclosed in WO 2008 / 143954 A2 and are incorporated herein by reference. The Fc region can be or may include a dimer Fc region. "Dimer Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two independent immunoglobulin heavy chains. The dimeric Fc region can be a homodimer of two identical Fc parts (e.g., the Fc regions of naturally occurring immunoglobulins), or a heterodimer of two non-identical Fc parts (e.g., one Fc monomer of the dimeric 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 monomer, or one Fc monomer may be truncated compared to the other Fc monomer).

[0248] The Fc part disclosed in the present invention may include Fc sequences or Fc regions of the same or different species and / or subclasses. For example, the Fc part may be derived from an immunoglobulin (e.g., human immunoglobulin) of the IgG1, IgG2, IgG3 or IgG4 subclass, or any combination of these subclass immunoglobulins. In certain embodiments, these Fc parts of the Fc region belong to the same class and subclass. However, the Fc region (or one or more Fc parts of the Fc region) may also be chimeric, whereby the chimeric Fc region may include Fc parts derived from different immunoglobulin classes and / or subclasses. For example, at least two of the Fc parts of a dimer Fc region or a single-chain Fc region may be from different immunoglobulin classes and / or subclasses. In certain embodiments, a dimer Fc region may include sequences from two or more different isotypes or subclasses; for example, a SEED body ("chain exchange engineered domain") (see Davis et al., Protein Eng. Des. Sei. 2010, 23 (4): 195).

[0249] In addition or alternatively, the chimeric Fc region may comprise one or more chimeric Fc portions. For example, a chimeric Fc region or portion may comprise one or more portions derived from a first subclass (e.g., IgG1, IgG2, or IgG3 subclass) immunoglobulin, while the remainder of the Fc region or portion belongs to a different subclass. For example, the Fc region or portion of an Fc polypeptide may comprise a CH2 and / or CH3 domain derived from a first subclass (e.g., IgG1, IgG2, or IgG4 subclass) immunoglobulin, and a hinge region derived from a second subclass (e.g., IgG3 subclass) immunoglobulin. For example, the Fc region or portion may comprise a hinge and / or CH2 domain derived from a first subclass (e.g., IgG4 subclass) immunoglobulin, and a CH3 domain derived from a second subclass (e.g., IgG1, IgG2, or IgG3 subclass) immunoglobulin. For example, a chimeric Fc region may include an Fc portion (e.g., a complete Fc portion) from a first subclass (e.g., IgG4 subclass) immunoglobulin, and an Fc portion from a second subclass (e.g., IgG1, IgG2, or IgG3 subclass) immunoglobulin. For example, an Fc region or portion may include a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, an Fc region or portion may include a CH1 domain and a CH2 domain from an IgG4 molecule, and a CH3 domain from an IgG1 molecule. For example, an Fc region or portion may include a portion of a CH2 domain from a specific subclass of an antibody, for example, EU positions 292-340 of a CH2 domain. For example, an Fc region or portion may include amino acids 292 to 340 in CH2 derived from an IgG4 portion and the remainder of CH2 derived from an IgG1 portion (alternatively, positions 292 to 340 of CH2 may be derived from an IgG1 portion and the remainder of CH2 may be derived from an IgG4 portion).

[0250] In addition, the Fc region or part can (in addition or alternatively) for example comprise a chimeric hinge region. For example, a chimeric hinge can be derived in part from, for example, an IgG1, IgG2 or IgG4 molecule (e.g., upper and lower middle hinge sequences), and in part from an IgG3 molecule (e.g., middle hinge sequences). In another example, the Fc region or Fc part can comprise a chimeric hinge that is derived in part from an IgG1 molecule and in part from an IgG4 molecule. In another example, a chimeric hinge can comprise upper and lower hinge domains from an IgG4 molecule, and middle hinge domains from an IgG1 molecule. This chimeric hinge can be prepared, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 of the middle hinge domain in the IgG4 hinge region. In some other embodiments, the chimeric hinge may comprise amino acids from an IgG2 antibody and / or a Ser228Pro mutation at EU positions 233 to 236, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g., a chimeric hinge of sequence ESKYGPPCPPCPAPPVAGP (SEQ ID NO: 61)). Additional chimeric hinges that can be used in the Fc portion of an antibody according to the present disclosure are described in US 2005 / 0163783A1.

[0251] In some embodiments, the Fc portion or Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., an Fc region or Fc portion derived from a human IgG molecule). However, the polypeptide may comprise one or more amino acids from another mammalian species. For example, a primate Fc portion or primate binding site may be included in the subject polypeptide. Alternatively, one or more mouse amino acids may be present in the Fc portion or Fc region.

[0252] c.HBC34 antibody

[0253] In certain embodiments, the anti-HBV antibody is HBC34 or an engineered variant thereof. HBC34 is a human antibody against HBsAg with high neutralizing activity. HBC34 binds to the HBsAg antigen ring with high affinity (in the pM range), recognizes all 10 HBV genotypes and 18 mutants, and binds to the spherical SVP at a low stoichiometric ratio. The activity of HBC34 measured diagnostically by immunoassay is 5000 IU / mg. In comparison, the activity of HBIG is about 1 IU / mg.

[0254] As referred to herein, the terms "HBC34 antibody" and "HBC antibody" may include wild-type HBC34 antibody or engineered variants thereof (eg, HBC34 and HBC34 variants described in Table 3), unless otherwise specified.

[0255] Table 3 shows the amino acid sequences of the CDRs, heavy chain variable regions (VH) and light chain variable regions (VL) of HBC34 and its engineered variants. Also shown are the full-length heavy chain (HC) amino acid sequences and light chain (LC) amino acid sequences of exemplary antibodies of the present disclosure.

[0256] Table 3. Sequence of the HBC34 antibody.

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] In certain embodiments, the anti-HBV antibody comprises one or more amino acid sequences as listed in Table 3. In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure comprises an amino acid sequence that is 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% identical to the CDR sequence, VH sequence, VL sequence, HC sequence and / or LC sequence as shown in Table 3. In any of the embodiments disclosed herein, the antibody or antigen-binding fragment may comprise the CDR, VH sequence, VL sequence, HC sequence and / or LC sequence as shown in Table 3. H 、V L , HC and / or LC sequences. Exemplary methods for synthesizing antibodies having the sequences shown in Table 3 and experimental data demonstrating the binding and neutralization achieved by AB01 are described in International Application Publication No. WO 2020 / 132091 A2, which are incorporated herein by reference.

[0263] In some embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise: (i) CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NO:44, SEQ ID NO:45 or 46, and SEQ ID NO:47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NO:48, SEQ ID NO:49 or 50, and SEQ ID NO:51 or 52, respectively.

[0264] Thus, in some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 44, 45, and 47, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 44, 46, and 47, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are according to SEQ ID NOs: 48, 49, and 52, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are according to SEQ ID NOs: 48, 50, and 52, respectively.

[0265] It will be understood that the antibodies or antigen-binding fragments of the present disclosure may comprise any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 44-50 and 52.

[0266] In a specific embodiment, the antibodies or antigen-binding fragments of the present disclosure comprise: CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

[0267] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise: (a) a light chain variable domain (VL) comprising or consisting of an amino acid sequence that is at least 90%, at least 95% or 100% identical to an amino acid sequence according to SEQ ID NO:55; and (b) a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence that is at least 90%, at least 95% or 100% identical to an amino acid sequence according to SEQ ID NO:53.

[0268] In a specific embodiment, the antibody or antigen-binding fragment of the present disclosure comprises: (a) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:59, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:57.

[0269] d. Pharmaceutical Compositions

[0270] In some embodiments, the antibody or antigen-binding fragment thereof of the combination therapy is provided as a pharmaceutical composition, which includes an anti-HBV antibody and an optional pharmaceutically acceptable carrier. In some embodiments, the composition may include an anti-HBV antibody, wherein the antibody may constitute at least 50% by weight of the total protein in the composition (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more). In such a composition, the antibody may be in a purified form.

[0271] Pharmaceutical compositions of anti-HBV antibodies may include antimicrobial agents, particularly if packaged in multidose form. These antimicrobial agents may include detergents, for example, Tweens (polysorbates), such as Tween 80. When present, the detergent is typically present at low levels (e.g., less than 0.01%). The composition may also include a sodium salt (e.g., sodium chloride) for adjusting tonicity. For example, in some embodiments, the pharmaceutical composition comprises NaCl at a concentration of 10 ± 2 mg / ml.

[0272] In addition, the pharmaceutical composition can include, for example, about 15 mg / ml to 30 mg / ml (e.g., 25 mg / ml) of a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose), particularly when the pharmaceutical composition is to be lyophilized or when the pharmaceutical composition includes material that has been reconstituted from a lyophilized material. Before the composition for lyophilization is lyophilized, the pH of the composition can be adjusted to between 5 and 8, or between 5.5 and 7, or about 6.1.

[0273] The antibody compositions of the present disclosure may also include one or more immunomodulators. In some embodiments, one or more of the immunomodulators include an adjuvant.

[0274] The method for preparing a pharmaceutical composition of an anti-HBV antibody may include the following steps: (i) preparing the antibody; and (ii) admixing the purified antibody with one or more pharmaceutically acceptable carriers.

[0275] In some embodiments, a pharmaceutical composition comprising an anti-HBV antibody described herein (e.g., AB01) contains a dose of 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg of the antibody. In some embodiments, a pharmaceutical composition comprising an anti-HBV antibody described herein (e.g., AB01) contains a dose of 100 mg to 300 mg of the antibody. In some embodiments, a pharmaceutical composition comprising an anti-HBV antibody described herein (e.g., AB01) contains a dose of 100 mg to 200 mg of the antibody.

[0276] In some embodiments, a pharmaceutical composition comprising an anti-HBV antibody described herein (eg, AB01) contains a dose of 200 mg of the antibody.

[0277] IV. Treatment Methods Using Combination Therapy

[0278] In some embodiments, the present disclosure provides methods for treating HDV infection or HDV-related disease in a subject.

[0279] In some embodiments, a method for treating HDV infection or HDV-related disease in a subject is provided, wherein the method comprises administering to the subject an siRNA and an antibody as described herein. In some embodiments, SIRNA01 and ABO1 are administered to the subject.

[0280] In some embodiments, a method for treating HDV infection or HDV-related disease in a subject is provided, wherein the method comprises administering to the subject an siRNA and an antibody as described herein, and further administering to the subject a nucleoside / nucleotide reverse transcriptase inhibitor. As used herein, "nucleoside / nucleotide reverse transcriptase inhibitor" or "nucleoside (acid) reverse transcriptase inhibitor" (NRTI) refers to an inhibitor of DNA replication, which is structurally similar to nucleotides or nucleosides, specifically inhibits the replication of HBV cccDNA by inhibiting the action of HBV polymerase, and does not significantly inhibit the replication of host (e.g., human) DNA. Such inhibitors include tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil fumarate (TD), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, dipivoxil, lobcavir, famciclovir, N-acetylcysteine ​​(NAC), PC1323, Theradigm-HBV, thymosin alpha, ganciclovir, besifovir (ANA-380 / LB-80380) and tenofovir-exaliade (TLX / CMX157). In some embodiments, the NRTI is tenofovir. In some embodiments, the NRTI is tenofovir disoproxil fumarate (TDF). In some embodiments, the NRTI is dipivoxil (TD). In some embodiments, the NRTI is entecavir (ETV). In some embodiments, the NRTI is lamivudine. In some embodiments, the NRTI is adefovir or adefovir dipivoxil.

[0281] As used herein, "subject" is an animal, such as a mammal, including any mammal that can be infected by HBV, such as a primate (such as a human, a non-human primate, such as a monkey or a chimpanzee), or an animal that is considered to be an acceptable clinical model of HBV infection, including HBV-AAV mouse models (see, for example, Yang et al., Cell and Mol Immunol 2014, 11: 71) or HBV 1,3xfs transgenic mouse models (Guidotti et al., J. Virol. 1995, 69: 6158). In some embodiments, the subject suffers from hepatitis B virus (HBV) infection. In some other embodiments, the subject suffers from both hepatitis B virus (HBV) infection and hepatitis D virus (HDV) infection. In some other embodiments, the subject is a human, such as a human with HBV infection, especially chronic hepatitis B virus (CHBV) infection.

[0282] As used herein, the term "treat" refers to a beneficial or desired result, including but not limited to: alleviation or improvement of one or more signs or symptoms associated with undesirable HBV gene expression or HBV replication, such as the presence of serum or liver HBV cccDNA, the presence of serum HBV DNA, presence of serum or liver HBV antigen (e.g., HBsAg or HBeAg), elevated ALT, elevated AST (normal range is generally considered to be about 10 U / L to 34 U / L), absence of anti-HBV antibodies or low levels of anti-HBV antibodies; liver damage; cirrhosis; hepatitis D; acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness syndrome; anorexia; nausea; vomiting; low-grade fever; myalgia; fatigability; disturbances in taste acuity and smell (aversion to food and cigarettes); or pain in the right upper quadrant and epigastric region (intermittent, mild to moderate); hepatic encephalopathy; lethargy; disturbed sleep pattern; confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (mild enlargement, soft liver); splenomegaly; palmar erythema; spider angiomas; muscular atrophy; spider angiomas; vasculitis; varicose bleeding; peripheral edema gynecomastia; testicular atrophy; abdominal collateral veins (periumbilical varicose veins); ALT levels higher than AST levels; elevated levels of gamma-glutamyl transpeptidase (GGT) (normal range is generally considered to be about 8 U / L to 65 U / L) and alkaline phosphatase (ALP) (normal range is generally considered to be about 44 IU / L to 147 IU / L (international units per liter), not more than 3 times the ULN); slightly low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; elevated erythrocyte sedimentation rate (ESR); shortened red blood cell survival; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum or liver HBsAg, HBeAg, or hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); presence of hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), or HBV DNA; elevated bilirubin levels; hyperglobulinemia; the presence of tissue nonspecific antibodies, such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10% to 20%); the presence of tissue specific antibodies, such as antibodies to the thyroid gland (10% to 20%); elevated rheumatoid factor (RF) levels; low platelet and white blood cell counts; lobular and degenerative and regenerative hepatocyte changes and accompanying inflammation; and significant centrilobular necrosis, whether detectable or undetectable. A reduced likelihood of developing, for example, liver fibrosis occurs, for example, when an individual with one or more risk factors for liver fibrosis (e.g., chronic hepatitis B infection) fails to develop liver fibrosis, or develops less severe liver fibrosis relative to a population with the same risk factors and not receiving treatment as described herein."Treatment" can also mean prolonging survival as compared to expected survival without treatment.

[0283] As used herein, the term "prevent" refers to the failure to develop a disease, disorder, or condition, or a reduction in the development of signs or symptoms associated with such a disease, disorder, or condition (e.g., a reduction by a clinically relevant amount), or a delay in the appearance of signs or symptoms (e.g., a delay of days, weeks, months, or years). Prevention may require administration of more than one dose.

[0284] Doses are usually expressed relative to body weight. Thus, a dose expressed in [g, mg or other unit] / kg (or g, mg, etc.) usually means [g, mg or other unit] "per kg (or g, mg, etc.) body weight", even if the term "body weight" is not explicitly mentioned.

[0285] In some embodiments, treatment of HBV infection causes "functional cure" of hepatitis B. As used herein, functional cure is understood to be the removal of circulating HBsAg, and may be accompanied by conversion to a state in which HBsAg antibodies become detectable using clinically relevant assays. For example, detectable antibodies may include a signal higher than 10mIU / ml, as measured by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay. Functional cure does not require the removal of all replicated forms of HBV (e.g., cccDNA from the liver). Anti-HBs seroconversion occurs spontaneously in about 0.2% to 1% of chronically infected patients each year. However, even after anti-HBs seroconversion, HBV is often observed to persist at low levels for decades, indicating that functional cure rather than complete cure has occurred. Without being constrained by a specific mechanism, the immune system may be able to keep HBV under control under conditions where functional cure has been achieved. Functional cure permits termination of any treatment for HBV infection. However, it should be understood that "functional cure" of HBV infection may not be sufficient to prevent or treat diseases or conditions caused by HBV infection, such as liver fibrosis, HCC or cirrhosis. In some embodiments, "functional cure" may refer to a sustained reduction in serum HBsAg (such as <1 IU / mL) for at least 3 months, at least 6 months or at least 1 year after starting a treatment regimen or completing a treatment regimen.

[0286] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" is a disease or condition caused by or associated with HBV infection or replication. The term "HBV-related disease" includes diseases, disorders or conditions that would benefit from reduced HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example: hepatitis D virus infection, hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0287] In some embodiments, the HBV-related disease is chronic hepatitis B. Chronic hepatitis B is defined by one of the following criteria: (1) at least two positive serum HBsAg, HBV DNA, or HBeAg tests 6 months apart (any combination of these tests performed 6 months apart is acceptable); or (2) negative immunoglobulin M (IgM) antibodies to HBV core antigen (IgM anti-HBc) and a positive result for one of the following tests: HBsAg, HBeAg, or HBV DNA. Chronic HBV typically includes liver inflammation that persists for more than six months. Subjects with chronic HBV are HBsAg positive and have high viremia (>10 4 HBV-DNA copies / ml blood) or low viremia (<10 3 HBV-DNA copies / ml blood). In certain embodiments, the subject has been infected with HBV for at least five years. In certain embodiments, the subject has been infected with HBV for at least ten years. In certain embodiments, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease may be immune tolerant, or there may be inactive chronic infection, no evidence of any active disease, and they are also asymptomatic. Patients with chronic active hepatitis may have symptoms similar to acute hepatitis, especially during the replicating state. Subjects with chronic hepatitis B disease may have active chronic infection with necrotic inflammatory liver disease, increased liver cell turnover in the absence of detectable necrotic inflammation, or there is inactive chronic infection without any evidence of active disease, and they are also asymptomatic. The continued presence of HBV infection in chronic HBV subjects is the result of cccHBV DNA. In some embodiments, subjects with chronic HBV are HBeAg positive. In some other embodiments, subjects with chronic HBV are HBeAg negative. Subjects with chronic HBV have serum HBV DNA levels below 105 and persistent elevations in transaminases (e.g., ALT, AST, and gamma-glutamyl transferase). Subjects with chronic HBV may have a liver biopsy score (e.g., necroinflammatory score) of less than 4. In some embodiments, the ALT ULN value is 34 HJ / mL for females and 43 IU / mL for males.

[0288] In some embodiments, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or hepatitis D virus (HDV) is a human pathogen. However, the virus is defective and relies on mandatory auxiliary functions provided by HBV to spread; in fact, HDV requires an associated or pre-existing HBV infection to be infectious and multiply, in particular a viral envelope containing hepatitis B surface antigen. HDV can cause severe acute and chronic forms of liver disease associated with HBV. Hepatitis D infection or hepatitis D is very prevalent in several African countries, the Amazon River Basin and the Middle East, while the prevalence is lower in industrialized countries except the Mediterranean region.

[0289] Transmission of HDV can occur via simultaneous infection with HBV (coinfection) or superimposed chronic hepatitis B or the hepatitis B carrier state (superinfection). Superinfection and co-infection with HDV generally result in more severe complications than infection with HBV alone. These complications include a greater likelihood of liver failure and rapid progression to cirrhosis in acute infection and an increased likelihood of liver cancer in chronic infection. In combination with hepatitis B virus, hepatitis D has the highest mortality rate of all hepatitis infections at 20%.

[0290] In some embodiments, HBV related diseases are acute hepatitis B. Acute hepatitis B includes liver inflammation that lasts less than six months. Typical symptoms of acute hepatitis B are fatigue, anorexia, nausea and vomiting. Very high aminotransferase values ​​(>1000U / L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B may rapidly develop into acute liver failure, which is characterized by low liver synthetic function. This is usually defined as a prothrombin time (PT) of 16 seconds, or an international normalized ratio (INR) of 1.5 (in the absence of previous liver disease). Acute hepatitis B may evolve into chronic hepatitis B.

[0291] In some embodiments, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B have symptoms of acute hepatitis, with additional symptoms of confusion or coma (due to the liver's failure to detoxify chemicals) and bruising or bleeding (due to lack of clotting factors).

[0292] Subjects with HBV infection (e.g., chronic HBV) may develop liver fibrosis. Therefore, in some embodiments, the HBV-related disease is liver fibrosis. Liver fibrosis or cirrhosis is histologically defined as a diffuse liver process characterized by fibrosis (excess fibrous connective tissue) and transformation of normal liver structure into structural abnormal nodules.

[0293] Subjects with HBV infection (e.g., chronic HBV) may develop end-stage liver disease. Thus, in some embodiments, the HBV-related disease is end-stage liver disease. For example, liver fibrosis may develop to the extent that the body may no longer be able to compensate for, for example, reduced liver function due to liver fibrosis (i.e., liver decompensation), and lead to, for example, psychiatric and neurological symptoms and liver failure.

[0294] Subjects with HBV infection (e.g., chronic HBV) may develop hepatocellular carcinoma (HCC), also known as malignant hepatoma. Therefore, in some embodiments, the HBV-related disease is HCC. HCC usually develops in subjects with CHB and can be fibrolamellar cells, pseudoglandular tubular (adenoid) cells, pleomorphic cells (giant cells) or clear cells.

[0295] An "HDV-associated disorder" or "hepatitis D virus-associated disease" is a disorder or condition associated with HDV expression. Exemplary HDV-associated diseases 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.

[0296] As used herein, "therapeutically effective amount" is intended to include an amount of siRNA, anti-HBV antibody or other active agent (e.g., tenofovir) that, when administered to a patient for treatment of a subject suffering from HBV infection or HBV-related disease, is sufficient to achieve treatment of the disease (e.g., by alleviating or maintaining an existing disease, or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the active agent, the mode of administration of the active agent, the disease and its severity, medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by HBV gene expression, the type of previous treatment or concomitant treatment, and other individual characteristics of the patient to be treated (if any). A therapeutically effective amount may require administration of more than one dose.

[0297] "Therapeutically effective amount" also includes an amount of siRNA, anti-HBV antibody or other active agent that produces a desired effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutic agent (e.g., siRNA, anti-HBV antibody) used in the disclosed method can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0298] As used herein, the term "sample" includes a collection of similar fluids, cells or tissues separated from a subject, and fluids, cells or tissues present in a subject. Examples of biological fluids include blood, serum and serosal fluid, plasma, lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs or local areas. For example, a sample can derive from a specific organ, a part of an organ, or a fluid or cell in these organs. In certain embodiments, a sample can derive from liver (for example, the whole liver, or certain sections of the liver, or certain types of cells in the liver, such as hepatocytes). In certain embodiments, "sample derived from a subject" refers to blood, or plasma or serum obtained from the blood taken from a subject. In other embodiments, "sample derived from a subject" refers to liver tissue (or its subcomponents) or blood tissue (or its subcomponents, such as serum) derived from a subject.

[0299] In some embodiments of the methods described herein, the anti-HBV antibody is administered subcutaneously. In some embodiments, the anti-HBV antibody is administered once every 4 weeks. In some embodiments, the anti-HBV antibody is administered once every 8 weeks. In some embodiments, the anti-HBV antibody is administered at a dose of 100 mg to 300 mg. In some embodiments, the anti-HBV antibody is administered at a dose of 100 mg. In some embodiments, the anti-HBV antibody is administered at a dose of 150 mg. In some embodiments, the anti-HBV antibody is administered at a dose of 200 mg. In some embodiments, the anti-HBV antibody is administered at a dose of 250 mg. In some embodiments, the anti-HBV antibody is administered at a dose of 300 mg. In some embodiments, the anti-HBV antibody is administered to the subject for a period of 8 weeks, 24 weeks, 48 ​​weeks, or 88 weeks.

[0300] In some embodiments of the methods described herein, siRNA is administered subcutaneously. In some embodiments, siRNA is administered once every 4 weeks. In some embodiments, siRNA is administered once every 8 weeks. In some embodiments, siRNA is administered at a dosage of 20mg to 900mg. In some embodiments, siRNA is administered at a dosage of 100mg to 300mg. In some embodiments, siRNA is administered at a dosage of 20mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 400mg or 450mg. In some embodiments, siRNA is administered at a dosage of 200mg. In some embodiments, siRNA is administered to a subject for a period of 8 weeks, 24 weeks, 48 ​​weeks or 88 weeks.

[0301] In some embodiments of the methods described herein, the NRTI is administered orally. In some embodiments, the NRTI is administered daily. In some embodiments, the NRTI is administered at a dose of 300 mg.

[0302] In some embodiments of the methods described herein, the subject begins administration of the siRNA and the anti-HBV antibody on the same day.

[0303] In some embodiments of the methods described herein, the subject begins administration of the siRNA and the anti-HBV antibody on the same day and for a period of 8 weeks, 24 weeks, 48 weeks, or 88 weeks.

[0304] In some embodiments of the methods described herein, the subject achieves one or more of the following: ≥2 log10 decrease in HDV RNA compared to baseline (i.e., before treatment); at week 24, HDV RNA < LOQ; and at week 24, ALT < upper limit of normal (ULN). In some embodiments, the ALT ULN value is 34 IU / mL for females and 43 IU / mL for males.

[0305] The present disclosure also provides the antibodies, siRNAs, and / or NRTIs described herein, and pharmaceutical compositions comprising these substances for use in the methods mentioned above. The use of these antibodies, siRNAs, and / or NRTIs.

[0306] V. Kits for Combination Therapy

[0307] The present disclosure also provides kits that include components of a therapy for treating HDV infection or HDV-related diseases. The kit can include an siRNA (e.g., SIRNA01) and an anti-HBV antibody (e.g., AB01). The kit can include an siRNA (e.g., SIRNA01), an anti-HBV antibody (e.g., AB01), and an NRTI (e.g., tenofovir disoproxil fumarate, tenofovir disoproxil). The kit can optionally include instructions for preparing and / or administering the components of the HBV combination therapy.

[0308] VI. Example Implementation

[0309] In some embodiments, the present disclosure provides:

[0310] 1. A method of treating hepatitis delta virus (HDV) infection in a subject in need thereof, comprising administering to the subject:

[0311] (a) an anti-HBV antibody; and

[0312] (b) an siRNA that targets HBV mRNA.

[0313] 2. A method of treating an HDV-related disease in a subject in need thereof, comprising administering to the subject:

[0314] (a) anti-HBV antibodies; and

[0315] (b) siRNA targeting HBV mRNA.

[0316] 3. The method according to embodiment 2, wherein the HDV-related disease is chronic hepatitis; acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stage liver disease; or hepatocellular carcinoma.

[0317] 4. The method according to any one of the preceding embodiments, wherein the subject suffers from chronic hepatitis B virus (HBV) / HDV co-infection.

[0318] 5. The method according to any one of the preceding embodiments, wherein the subject has 2 positive serum HBsAg, HBV DNA or HBeAg tests at least 6 months apart before treatment.

[0319] 6. The method according to any one of the preceding embodiments, wherein the subject has tested positive for HDV antibodies or HDV RNA prior to treatment.

[0320] 7. The method according to any one of the preceding embodiments, wherein the subject has tested positive for HDV antibodies and HDV RNA prior to treatment.

[0321] 8. The method according to any one of the preceding embodiments, wherein the subject has tested positive for HDV antibodies for at least 6 months prior to treatment.

[0322] 9. The method according to any one of the preceding embodiments, wherein the subject has HDV RNA ≥ 500 IU / mL before treatment.

[0323] 10. The method according to any one of the preceding embodiments, wherein the subject has an HBsAg level > 0.05 IU / mL before treatment.

[0324] 11. The method according to any one of the preceding embodiments, wherein the subject has an HBsAg level > 10,000 IU / mL before treatment.

[0325] 12. The method according to any one of the preceding embodiments, wherein the subject suffers from chronic hepatitis.

[0326] 13. A method according to any one of the preceding embodiments, wherein the subject's alanine aminotransferase (ALT) level before treatment is > upper limit of normal (ULN) and < 5 times ULN.

[0327] 14. The method according to any one of the preceding embodiments, wherein the subject has an ALT level > ULN and an aspartate aminotransferase (AST) level > ULN before treatment.

[0328] 15. The method according to any one of the preceding embodiments, wherein the subject has an ALT level <5 times ULN and an AST level <5 times ULN before treatment.

[0329] 16. The method according to any one of the preceding embodiments, wherein the subject is non-cirrhotic.

[0330] 17. The method according to embodiment 16, wherein the subject has a viral hepatitis histology data meta-analysis (METAVIR) grade of F0-F3 on liver biopsy or liver elastic imaging within 12 months before treatment. <12 kilopascals (kPa).

[0331] 18. The method according to embodiment 16 or embodiment 17, wherein the subject's creatine clearance (CLcr) calculated by the Cockcroft-Gault formula before treatment is ≥ 30 mL / min.

[0332] 19. The method of any one of embodiments 1 to 15, wherein the subject has cirrhosis of the liver.

[0333] 20. The method according to embodiment 19, wherein the subject has a liver biopsy METAVIR grade of F4 or liver elastography within 12 months before treatment. ≥12 kilopascals (kPa).

[0334] 21. The method according to embodiment 19 or embodiment 20, wherein the subject's creatine clearance (CLcr) calculated by the Cockcroft-Gault formula before treatment is ≥ 60 mL / min.

[0335] 22. A method according to any one of embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 5 or higher before treatment.

[0336] 23. A method according to any one of embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 5 or 6 before treatment.

[0337] 24. A method according to any one of embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 7 or higher before treatment.

[0338] 25. A method according to any one of embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 7 to 9 before treatment.

[0339] 26. A method according to any one of embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score >10 before treatment.

[0340] 27. The method according to any one of the preceding embodiments, wherein the subject has not been previously administered anti-HIV antibodies.

[0341] 28. The method according to any one of the preceding embodiments, wherein the subject has not been previously administered siRNA targeting HBV mRNA.

[0342] 29. A method according to any one of the preceding embodiments, wherein the anti-HBV antibody recognizes HBV genotypes A, B, C, D, E, F, G, H, I and J.

[0343] 30. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is a human antibody.

[0344] 31. The method according to any one of the preceding embodiments, wherein the antibody is HBC34, or a non-natural variant of HBC34.

[0345] 32. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0346] (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NO:44, SEQ ID NO:45 or 46, and SEQ ID NO:47, respectively; and

[0347] (ii) the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NO: 48, SEQ ID NO: 49 or 50, and SEQ ID NO: 52, respectively.

[0348] 33. The method of embodiment 32, wherein the anti-HBV antibody comprises:

[0349] (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and

[0350] (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

[0351] 34. The method of embodiment 32, wherein the anti-HBV antibody comprises:

[0352] (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 46 and 47, respectively; and

[0353] (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 50 and 52, respectively.

[0354] 35. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0355] (a) a light chain variable domain (VL) that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:55; and (b) a heavy chain variable domain (VH) that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:53.

[0356] 36. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0357] (a) a light chain variable domain (VL) amino acid sequence according to SEQ ID NO:55; and (b) a heavy chain variable domain (VH) amino acid sequence according to SEQ ID NO:53.

[0358] 37. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0359] (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:59, and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:57.

[0360] 38. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0361] (a) the light chain amino acid sequence according to SEQ ID NO:59, and (b) the heavy chain amino acid sequence according to SEQ ID NO:57.

[0362] 39. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is a monoclonal antibody.

[0363] 40. The method according to any of the preceding embodiments, wherein the anti-HBV antibody is a bispecific antibody having a first specificity for HBsAg, and a second specificity for stimulating immune effectors.

[0364] 41. A method according to embodiment 40, wherein the second specificity stimulates cytotoxicity or a vaccine effect.

[0365] 42. The method of any one 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 about 3 mg / kg to about 30 mg / kg.

[0366] 43. The method according to any one of the preceding embodiments, wherein the siRNA inhibits the expression of HBV transcripts encoding HBsAg protein, HBcAg protein and HBx protein or HBV DNA polymerase protein.

[0367] 44. The method according to any one of the preceding embodiments, wherein the siRNA 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, wherein T is replaced by U.

[0368] 45. The method according to any one of the preceding embodiments, wherein the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotides 1579-1597 of SEQ ID NO: 1, wherein T is replaced by U.

[0369] 46. ​​A method according to any of the preceding embodiments, wherein the siRNA binds to at least 15 consecutive nucleotides of a target encoded by: the P gene, nucleotides 2309-3182 and nucleotides 1-1625 of NC_003977.2; the S gene (encoding L, M and S proteins), nucleotides 2850-3182 and nucleotides 1-837 of NC_003977.2; HBx, nucleotides 1376-1840 of NC_003977.2; or the C gene, nucleotides 1816-2454 of NC_003977.2.

[0370] 47. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA comprises at least 15 consecutive nucleotides of the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0371] 48. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA comprises at least 19 consecutive nucleotides of the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0372] 49. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA comprises the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0373] 50. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA consists of the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0374] 51. The method according to any one of the preceding embodiments, wherein the sense strand of the siRNA comprises the nucleotide sequence 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

[0375] 52. The method according to any one of the preceding embodiments, wherein the sense strand of the siRNA consists of the nucleotide sequence 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

[0376] 53. The method according to any one of the preceding embodiments, wherein at least one strand of the siRNA comprises a 3' overhang of at least 1 nucleotide.

[0377] 54. The method according to any one of the preceding embodiments, wherein at least one strand of the siRNA comprises a 3' overhang of at least 2 nucleotides.

[0378] 55. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs in length.

[0379] 56. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 17 to 23 nucleotide pairs in length.

[0380] 57. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 17 to 25 nucleotide pairs in length.

[0381] 58. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 23 to 27 nucleotide pairs in length.

[0382] 59. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 19 to 21 nucleotide pairs in length.

[0383] 60. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 21 to 23 nucleotide pairs in length.

[0384] 61. The method of any one of the preceding embodiments, wherein each strand of the RNAi agent has 15 to 30 nucleotides.

[0385] 62. The method of any one of the preceding embodiments, wherein each strand of the RNAi agent has 19 to 30 nucleotides.

[0386] 63. A method according to any of the preceding embodiments, wherein substantially all nucleotides of the sense strand of the siRNA and substantially all nucleotides of the antisense strand of the siRNA are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached to the 3'-end.

[0387] 64. A method according to embodiment 63, wherein the ligand is one or more GalNAc derivatives attached via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

[0388] 65. The method according to embodiment 63 or embodiment 64, wherein the ligand is

[0389]

[0390] 66. The method of embodiment 65, wherein the siRNA is conjugated to the ligand as shown in the following structure:

[0391] Wherein X is O or S.

[0392] 67. The method of embodiment 66, wherein X is O.

[0393] 68. A method according to any one of the preceding embodiments, wherein at least one nucleotide of the siRNA is a modified nucleotide, including: deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, configuration-restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, adenosine-diol nucleic acids, and nucleotides containing 5'-phosphate mimetics.

[0394] 69. The method according to any one of the preceding embodiments, wherein the siRNA comprises a phosphate backbone modification, a 2' ribose modification, a 5' triphosphate modification, or a GalNAc conjugation modification.

[0395] 70. A method according to any one of the preceding embodiments, wherein the phosphate backbone modification comprises a phosphorothioate bond.

[0396] 71. The method according to any one of the preceding embodiments, wherein the siRNA comprises a 2'-fluoro or 2'-O-methyl substitution.

[0397] 72. The method according to any one of the preceding embodiments, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand of the siRNA are modified nucleotides.

[0398] 73. The method of any one of the preceding embodiments, wherein the siRNA comprises: a sense strand having 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand having 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0399] 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;

[0400] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0401] (Agn) is adenosine diol nucleic acid (GNA);

[0402] s is a phosphorothioate bond; and

[0403] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0404] 74. The method of any one of the preceding embodiments, wherein the siRNA comprises: a sense strand having 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 7), and an antisense strand having 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 8),

[0405] 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;

[0406] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0407] s is a phosphorothioate bond; and

[0408] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0409] 75. The method of embodiment 67 or embodiment 68, wherein the L96 is conjugated to the sense strand as shown in the following structure:

[0410]

[0411] Where X is O.

[0412] 76. A method according to any preceding embodiment, wherein the subject is a human, and a therapeutically effective amount of siRNA is administered to the subject; and wherein the effective amount of the siRNA is about 1 mg / kg to about 8 mg / kg.

[0413] 77. A method according to any preceding embodiment, wherein the siRNA is administered to the subject twice a day, once a day, once every two days, once every three days, twice a week, once a week, once every other week, once every four weeks, or once a month.

[0414] 78. The method according to any one of the preceding embodiments, further comprising administering a nucleoside(t) reverse transcriptase inhibitor (NRTI) to the subject.

[0415] 79. The method according to any one of the preceding embodiments, wherein the subject has been previously administered an NRTI.

[0416] 80. The method of any one of Embodiments 78 or 79, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil (TD), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, dipivoxil, lobcavir, famciclovir, N-acetylcysteine ​​(NAC), PC1323, Theradigm-HBV, thymosin alfa and ganciclovir, besifovir (ANA-380 / LB-80380), or tenofovir-exaliade (TLX / CMX157).

[0417] 81. The method of any one of embodiments 78 to 80, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), or tenofovir disoproxil (TD).

[0418] 82. The method of any one of embodiments 78 to 81, wherein the NRTI is tenofovir disoproxil fumarate (TDF).

[0419] 83. The method according to any one of the preceding embodiments, wherein:

[0420] (a) the anti-HBV antibody comprises or consists of: a light chain amino acid sequence according to SEQ ID NO: 59 and a heavy chain amino acid sequence according to SEQ ID NO: 57; and

[0421] (b) the siRNA comprises or consists of the following parts: a sense strand comprising or consisting of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand comprising or consisting of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0422] 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;

[0423] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0424] (Agn) is adenosine diol nucleic acid (GNA);

[0425] s is a phosphorothioate bond; and

[0426] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol. 84. The method according to any one of embodiments 72 to 76, wherein:

[0427] (a) the anti-HBV antibody comprises or consists of: a light chain amino acid sequence according to SEQ ID NO: 59 and a heavy chain amino acid sequence according to SEQ ID NO: 57;

[0428] (b) the siRNA comprises or consists of the following parts: a sense strand comprising or consisting of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand comprising or consisting of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0429] 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;

[0430] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0431] (Agn) is adenosine diol nucleic acid (GNA);

[0432] s is a phosphorothioate bond; and

[0433] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol;

[0434] and

[0435] (c) The NRTI is tenofovir disoproxil fumarate (TDF) or entecavir.

[0436] 85. The method according to any one of the preceding embodiments, wherein:

[0437] (a) the anti-HBV antibody comprises: a light chain amino acid sequence according to SEQ ID NO: 59 and a heavy chain amino acid sequence according to SEQ ID NO: 57; and

[0438] (b) the siRNA comprises: a sense strand having 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand having 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0439] 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;

[0440] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0441] (Agn) is adenosine diol nucleic acid (GNA);

[0442] s is a phosphorothioate bond; and

[0443] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol. 86. The method according to any one of the preceding embodiments, wherein:

[0444] (a) the anti-HBV antibody consists of: a light chain amino acid sequence according to SEQ ID NO: 59 and a heavy chain amino acid sequence according to SEQ ID NO: 57; and

[0445] (b) the siRNA consists of the following parts: a sense strand consisting of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand consisting of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[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 phosphorothioate bond; and

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

[0451] 87. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody and the siRNA are according to Figures 1 to 7C and FIG. 12A to FIG. 19D Any one of the procedures described above is applied to the subject.

[0452] 88. A method according to any of the preceding embodiments, wherein the anti-HBV antibody is administered subcutaneously.

[0453] 89. A method according to any one of the preceding embodiments, wherein the siRNA is administered subcutaneously.

[0454] 90. The method of any one of embodiments 78 to 89, wherein the NRTI is administered orally.

[0455] 91. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered once every 2 weeks.

[0456] 92. A method according to any of the preceding embodiments, wherein the anti-HBV antibody is administered once every 4 weeks.

[0457] 93. A method according to any of the preceding embodiments, wherein the anti-HBV antibody is administered once every 8 weeks.

[0458] 94. A method according to any one of the preceding embodiments, wherein the siRNA is administered once every 4 weeks.

[0459] 95. A method according to any one of the preceding embodiments, wherein the siRNA is administered once every 8 weeks.

[0460] 96. The method of any one of embodiments 78 to 95, wherein the NRTI is administered daily.

[0461] 97. A method according to any of the preceding embodiments, wherein the anti-HBV antibody is administered at a dose of 300 mg.

[0462] 98. The method according to any one of the preceding embodiments, wherein the siRNA is administered at a dose of 200 mg.

[0463] 99. The method of any one of embodiments 78 to 98, wherein the NRTI is administered at a dose of 300 mg.

[0464] 100. The method of any one of embodiments 78 to 98, wherein the NRTI is administered at a dose of 245 mg.

[0465] 101. The method according to any one of the preceding embodiments, wherein the subject begins administration of the siRNA and the anti-HBV antibody on the same day.

[0466] 102. The method of any one of the preceding embodiments, wherein the subject is human.

[0467] 103. A method according to any of the preceding embodiments, wherein the subject is administered the siRNA and the anti-HBV antibody for up to 96 weeks, 96 weeks, at least 96 weeks, or 96 weeks or longer.

[0468] 104. An anti-HBV antibody; and an siRNA targeting HBV mRNA; for use in a method according to any one of embodiments 1 to 103.

[0469] 105. Use of an anti-HBV antibody; and an siRNA targeting HBV mRNA; in the manufacture of a medicament for use in the method according to any one of embodiments 1 to 103.

[0470] 106. Use of an anti-HBV antibody in the manufacture of a first drug; and use of an siRNA targeting HBV mRNA in the manufacture of a second drug; wherein the first drug and the second drug are to be used in a combination therapy according to the method described in any one of embodiments 1 to 103.

[0471] 107. An anti-HBV antibody; an siRNA targeting HBV mRNA; and an NRTI; for use in a method according to any one of embodiments 78 to 103.

[0472] 108. An anti-HBV antibody; an siRNA targeting HBV mRNA; and a NRTI; for use in the manufacture of a medicament for use in a method according to any one of embodiments 78 to 103.

[0473] 109. Use of an anti-HBV antibody in the manufacture of a first drug; use of an siRNA targeting HBV mRNA in the manufacture of a second drug; and use of an NRTI in the manufacture of a third drug; wherein the first drug, the second drug and the third drug are to be used in a combination therapy according to any one of embodiments 78 to 103.

[0474] 110. A kit comprising:

[0475] A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient;

[0476] as well as

[0477] A pharmaceutical composition comprising siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient.

[0478] 111. The kit of embodiment 110, further comprising instructions for performing the method of any one of embodiments 1 to 103.

[0479] 112. A kit comprising:

[0480] A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient;

[0481] A pharmaceutical composition comprising siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient; and

[0482] A pharmaceutical composition comprising a NRTI and a pharmaceutically acceptable excipient.

[0483] 113. The kit according to embodiment 104 further comprises a method for completing the

[0484] Instructions for the method of any one of 78 to 103.

[0485] Example

[0486] Example 1

[0487] Clinical evaluation of monotherapy or combination therapy for the treatment of chronic HDV infection

[0488] The safety, efficacy, and tolerability of anti-HBV antibodies combined with anti-HBV siRNA were evaluated in a phase 2, multicenter, open-label clinical study in human patients with non-cirrhotic and compensated cirrhosis (CPT-A to CPT-C) with chronic HBV / HDV co-infection who were not currently receiving HDV treatment.

[0489] background

[0490] Hepatitis delta virus (HDV) is the only virus in the genus Deltaviridae that requires hepatitis B virus (HBV) co-infection to enter hepatocytes, spread within the liver, and disseminate. HBV / HDV co-infection is associated with a more rapid and severe course of liver disease compared with other forms of viral hepatitis (Lempp 2016; Lucifora 2020). Based on sequence variation among HDV isolates, HDV has been classified into eight genotypes (Le Gal 2017). Genotype 1 is distributed worldwide, with infections ranging from fulminant hepatitis to asymptomatic chronic liver disease. HDV genotypes 2–8 have limited geographic distribution (Niro 2012). The median HDV prevalence among hepatitis B surface antigen (HBsAg) carriers is estimated to be approximately 5%; however, in some regions, up to 80% of HBsAg carriers have laboratory evidence of current or past HDV infection (Rizzetto 2021). Due to large knowledge gaps, epidemiological data on the prevalence of HDV may be severely underestimated, ranging from 12 million to 72 million people (Chen 2019; Miao 2020; Stockdale 2020).

[0491] HDV is a defective RNA satellite virus that does not encode envelope proteins and is dependent on HBsAg to complete its life cycle. Therefore, HDV must co-infect or superinfect HBV-infected hepatocytes. HDV virions are roughly spherical structures of 35nm to 43nm without a distinct nucleocapsid. The ribonucleoprotein, which contains a total of 60 large and small delta antigens, is the only protein encoded by HDV. Extracellular HDV virions contain genomic HDV RNA, which is a single-stranded, antisense, covalently closed circular RNA molecule containing 1,668 to 1,697 nucleotides depending on the genotype (Le Gal 2017). HDV does not encode an RNA-dependent RNA polymerase, but relies on the host’s DNA-dependent RNA polymerase to promote RNA-directed RNA synthesis to transcribe and replicate its genome in the hepatocyte nucleus using a double rolling circle mechanism (Chang 2008; Modahl 2000; Sureau 2016; Urban 2021).

[0492] HDV virions can be assembled in hepatocytes using all forms of HBsAg derived from cccDNA (in cases where HBV co-infects hepatocytes) and HBsAg derived from integrated HBV. Virion assembly depends on the interaction between HBsAg and the farnesylated N-terminus of the large form of HDV delta antigen (L-HDAg) (Freitas 2014; Shirvani-Dastgerdi 2015). The large form of HBsAg (L-HBsAg) is required for the formation of infectious virions because the preSl domain of L-HBsAg mediates the interaction between HBsAg and NTCP. NTCP is a basolateral Na-dependent bile salt transporter that is exclusively localized at the basolateral membrane of differentiated mammalian hepatocytes and is hepatocyte-specific. Therefore, although HDV can efficiently replicate its genome and express hepatitis delta antigens, L-HBsAg is required for the formation of infectious progeny virions.

[0493] HDV infection can occur in two ways: HBV-HDV co-infection and HDV super-infection. Co-infection occurs when HBV and HDV are transmitted simultaneously to HBV-susceptible individuals, while super-infection occurs when an HBsAg-positive person (typical of chronic HBV infection) acquires HDV. HBV / HDV co-infection occurs in 5% to 15% of HDV infection cases and frequently causes acute hepatitis, which is usually more severe than acute HBV; however, progression to chronic HDV infection occurs in only 2% to 5% (Bahcecioglu 2017; Raimondo 1982; Romeo 2009; Vlachogiannakos 2020). In contrast, HDV super-infection in HBV carriers occurs in approximately 75% of cases and often exacerbates existing liver disease, progressing to fulminant hepatitis in 7% to 15% of cases (Farci 1983).

[0494] Chronic HDV infection is defined as the persistence of HDV RNA or hepatitis D antigen (HDAg) in the serum for at least 6 months after HDV infection. Chronic HBV / HDV infection leads to more severe disease than chronic HBV, with a more rapid progression of fibrosis (Mathurin 2000; Sagnelli 1989). In addition, patients with chronic HDV infection are twice as likely to develop and die from liver decompensation or HCC as those with HBV monoinfection (Fattovich 2000; Niro 2010; Romeo 2009). Persistent HDV replication is the only factor associated with an increased risk of death (Romeo 2009).

[0495] Given the dependence of HDV on HBV, strategies to prevent HBV infection (e.g., prophylactic vaccines) also prevent HDV infection. However, HBV vaccines are difficult to obtain in some parts of the world (WHO 2021, Hepatitis D). In addition, vaccine non-responders, immunocompromised patients, and immunocompromised patients constitute a group that remains susceptible to chronic HDV infection. Currently, the only available curative treatment for HDV infection is pegylated interferon alpha (PEG-IFNa). Current guidelines from the American Association for the Study of Liver Diseases (AASLD), the Asia-Pacific Association for the Study of the Liver (APASL), and the European Association for the Study of the Liver (EASL) recommend the administration of PEG-IFNa for at least 48 weeks to patients with chronic HDV infection (EASL 2017; Sarin 2016; Terrault 2018). PEG-INFa treatment establishes a sustained virologic response (SVR, serum HDV clearance maintained for 6 months after cessation of treatment) in approximately 25% to 30% of patients, however relapses occur in -50% of patients after long-term follow-up (Heidrich 2014). In addition to low SVR rates, adverse effects associated with PEG-INFa therapy are well described, and PEG-INFa is contraindicated in patients with autoimmune diseases, some psychiatric syndromes, and Child-Pugh-Turcotte (CPT)-B or CPT-C cirrhosis (Rizzetto 2015; Sleijfer 2005). Reduced efficacy of PEG-INFa has been observed in the treatment of cirrhotic patients with chronic HDV, further limiting the usefulness of this therapy (Gunsar 2005). The limitation of not being able to use peg-INFa in cirrhotic HDV patients is particularly evident because 50% of HDV-infected patients have cirrhosis at the time of diagnosis (Fattovich 1987). NRTIs with anti-HBV activity, such as adefovir, entecavir, famciclovir, and tenofovir disoproxil fumarate (TDF), do not affect HDV when used alone or in combination with PEG-INFa (Wedemeyer 2011). Despite this limitation, nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are used in HBV / HDV co-infected patients due to their ability to modulate HBV replication.

[0496] BLV, an entry inhibitor targeting the NT CP receptor, recently received conditional marketing authorization from the European Commission and status under the European Medicines Agency’s Priority Medicines (PRIME) program, becoming the first treatment approved in Europe for adults with chronic HDV and compensated liver disease. Interim results from the Phase 3 MYR301 study showed that after 24 weeks of daily subcutaneous (SC) doses of 2 mg BLV, 36.7% of participants with chronic HDV achieved combined virological (HDV RNA undetectable (< limit of detection (LOD)) or decrease ≥2 log10 IU / ml from baseline) and biochemical responses (ALT normalization). Additional planned follow-up studies will examine the durability of these responses. No participant demonstrated HBsAg loss, and therefore, lifelong treatment with BLV may be required to successfully control HDV. In addition, BLV has not been studied in, and cannot be administered to, patients with CPT-B or CPT-C liver impairment (Hepcludex Summary of Product Characteristics 2020), which make up a large proportion of patients with HDV infection.

[0497] Despite the recent introduction of BLV, treatment options for chronic HDV remain unsatisfactory for several reasons. The efficacy of PEG-IFNa or BLV is incomplete, with only -17% of PEG-IFNa-treated individuals achieving a long-term virologic response, while BLV, while better, still only -37% of participants reaching the endpoints of HDV RNA suppression and ALT normalization (Wedemeyer 2021). PEG-IFN-a and BLV require weekly and daily subcutaneous administration, respectively, which affects treatment adherence. PEG-IFN-a is often associated with several adverse reactions that further limit adherence, as well as drug-related toxicities that often require intensive laboratory monitoring and dose adjustments. In real-world studies, BLV has been associated with 10% of patients discontinuing treatment due to adverse events (de Ledinghen 2021). In addition, several potential drug interactions have been identified for BLV based on its interactions with the NTCP receptor and the liver transporter OATP1B1 / 3 (Hepcludex Summary of Product Characteristics 2020).

[0498] Finally, another area that is not addressed by current treatments is the treatment of patients with moderate to severe liver damage. This group includes a large proportion of chronic HDV patients. PEG-IFN-a or BLV cannot be safely used in these patients, so they have no treatment options.

[0499] The goals of current and emerging anti-HDV therapeutic strategies include inhibiting HDV RNA transcription, preventing HBsAg production, or blocking infection of susceptible hepatocytes (Lok 2021; Yurdaydin 2019). In ongoing studies, small interfering RNA (siRNA) targeting HBV (SIRNA01) and monoclonal antibody (mAb) targeting HBsAg (AB01) have demonstrated the ability to suppress HBsAg in HBV mono-infected individuals. In addition, in preclinical models, reducing HBsAg with either siRNA (SIRNA01) or monoclonal antibody targeting HBsAg (AB01) has resulted in a reduction in HDV viremia (Lempp 2021).

[0500] SIRNA01 was well tolerated in healthy volunteers as a single dose up to 900 mg and in participants with chronic HBV infection as six doses of 200 mg every 4 weeks and demonstrated a safety profile that supports continued clinical development. SIRNA01 was associated with substantial reductions in HBsAg (up to 2log10 IU / mL) regardless of hepatitis B e antigen (HBeAg) status, but did not induce seroclearance of HBsAg (Gane 2021). As HDV replication is dependent on HBsAg, SIRNA01 is expected to reduce (or potentially eliminate) HDV viremia while reducing HBsAg. Independent of the antiviral activity of SIRNA01, AB01 has the potential to further reduce HBsAg, thereby further deepening the suppression of HDV viremia. AB01 is being evaluated in participants with chronic HBV in an ongoing Phase 1 study. Participants receive a single dose of AB01. The greatest and most sustained HBsAg reductions were observed in the 300 mg dose cohort (mean change from baseline HBsAg -2.42 log10 IU / mL).By neutralizing HDV virions, AB01 will inhibit new hepatocyte infection, and engineering modifications to this mAb designed to recruit immune effector cells should accelerate clearance of HBV / HDV co-infected hepatocytes.

[0501] The objectives of this study are to evaluate the safety of SIRNA01 and AB01 in participants with HBV / HDV co-infection and to assess whether monotherapy or combination therapy with investigational agents can durably block HDV replication and normalize ALT when given on a monthly or bimonthly schedule in participants of all liver disease severities.

[0502] Study Design

[0503] Table 4 shows the treatment groups for this study.

[0504] Table 4. Treatment groups in clinical studies.

[0505]

[0506] SC = subcutaneous

[0507] a Participants who reach the primary endpoint after receiving 3 doses over 4 weeks will receive 10 additional doses x 8 weeks.

[0508] b Participants who reach the primary endpoint after receiving 7 doses over 4 weeks will receive 8 additional doses × 8 weeks.

[0509] c After 7 doses over 4 weeks No Participants who reach the primary endpoint will receive 5 additional doses x 4 weeks.

[0510] d Participants who reach the primary endpoint after receiving 13 doses over 4 weeks will receive 5 additional doses × 8 weeks.

[0511] The study protocol for cohort 1 is Figure 1 As shown in the study plan for cohorts 2 to 4, Figure 2 Shown in.

[0512] Up to 58 participants will be enrolled in the study. In Cohorts 1a and 1b, a total of approximately 10 participants with HBV / HDV co-infection and Meta-Analysis of Viral Hepatitis Histological Data (METAVIR) F0-F3 will be enrolled, with 5 participants in each cohort. In Cohort 2, a minimum of 12 and a maximum of 22 participants will be enrolled, including the following 3 groups: (i) HBV / HDV co-infection and METAVIR F0-F3 fibrosis from Cohort 1 (up to 10 participants); (ii) newly enrolled participants with METAVIR F0-F3; and (iii) newly enrolled participants with METAVIR F4 and mild liver injury (CPT-A). In addition, a total of up to 8 floating participants may be added to Cohorts 1 or 2. In Cohort 3, a total of 12 participants with HBV / HDV co-infection and liver fibrosis stage METAVIR F4 and moderate liver damage (CPT-B) will be enrolled. In Cohort 4, a total of 6 participants with HBV / HDV co-infection and liver fibrosis stage METAVIR F4 and severe liver damage (CPT-C) will be enrolled.

[0513] Cohorts 1, 2, 3, and 4 will be enrolled sequentially based on a review of safety data. Cohorts may be closed or discontinued at the sole discretion of the Sponsor. Cohort 2 will begin enrolling participants from Cohort 1 after review of 12-week monotherapy safety data, discussion with the Investigator, and re-consent from the participant. Participants in Cohort 1 will be eligible to enter Cohort 2 after (or at) Week 16 of their initial study. Cohort 2 will also enroll additional participants after a safety review of the 12-week monotherapy safety data for the first 10 participants in Cohort 1. Cohort 3 will begin enrolling after (1) review of safety results from other studies and (2) review of safety data for the first 5 participants in Cohort 2 who complete Week 12 of the study. Cohort 4 will begin enrolling after (1) review of Cohort 3 safety results from other studies and (2) review of safety data for the first 5 participants in Cohort 3 who complete Week 12 of the study.

[0514] The total study duration is planned to be up to 102 weeks. For all cohorts, the intervention period consists of 2 periods: an induction period and a maintenance period. For Cohort 1, this includes a screening period (up to 6 weeks), an induction period (12 weeks), and a maintenance period (84 weeks). At the end of the induction period, participants who did not transition to the maintenance period and did not enter Cohort 2 will enter the follow-up period (48 weeks). Participants who prematurely discontinued study treatment during the induction period will enter the follow-up period (48 weeks). Participants who prematurely discontinued study treatment during the maintenance period will enter a 48-week follow-up period after the last dose of the study intervention or until Week 96, whichever comes first. For Cohorts 2 to 4, this includes a screening period (up to 6 weeks), an induction period (24 or 48 weeks), and a maintenance period (72 or 48 weeks). At the end of the induction period, participants who did not transition to the maintenance period will enter the follow-up period (48 weeks). Participants who prematurely discontinued study treatment during the induction period will enter the follow-up period (48 weeks). Participants who prematurely discontinue study treatment during the maintenance period will enter a 48-week follow-up period after the last dose of study intervention or until Week 96, whichever comes first.

[0515] All participants will be screened for up to 42 days.

[0516] The treatment period will consist of 2 phases: an induction phase and a maintenance phase. Participants in cohorts 1a and 1b will receive 3 doses of the study treatment during the induction phase (Day 1, Week 4, and Week 8). If at the Week 12 visit, HDV RNA has decreased by ≥2 log10 compared to baseline or HDV RNA < LOQ and ALT < upper limit of normal (ULN) (combined endpoint), they may proceed to the maintenance phase for an additional 84 weeks. Participants who do not meet the combined endpoint at Week 12 may enter cohort 2 or the follow-up period. Participants in cohorts 2 to 4 will receive treatment for 24 weeks or 48 weeks during the induction phase. Participants who meet the combined endpoint at the Week 24 visit will transition to a 72-week maintenance phase and receive the next dose of the drug at Week 32. Participants who do not meet the combined endpoint at the Week 24 visit will continue to receive the drug during the induction phase until Week 48. Participants who meet the combined endpoint at the Week 48 visit will transition to a 48-week maintenance phase and receive the next dose of the drug at Week 56. Participants who do not meet these criteria at Week 48 will end the treatment period and enter the follow-up period. Participants whose HDV RNA rebounds within 1 log10 IU / mL of its baseline during the maintenance phase will return to receive SIRNA01 + AB01 every 4 weeks until Week 88. In these cases, the next dose of the drug should be administered at the next scheduled visit according to the schedule of activities (SoA). If the next scheduled visit will not occur within 8 weeks, the next dose should be administered within 4 weeks through an unscheduled visit, and then a dose should be administered every 4 weeks until Week 88.

[0517] The maximum duration of the follow-up period is 48 weeks or Week 96, whichever is earlier, after the last dose of the study intervention. Participants will enter the follow-up period when they meet one of the following conditions: (1) are enrolled in cohort 1 and do not transition to the maintenance phase after meeting the combined endpoint at Week 12; (2) are enrolled in cohort 1 and do not enter cohort 2 after failing to meet the combined endpoint at Week 12; (3) are enrolled in cohorts 2 to 4 and do not transition to the maintenance phase at Week 48; or (4) are enrolled in any cohort and prematurely discontinue the study drug.

[0518] AB01 is provided as a reconstituted lyophilized powder to be administered subcutaneously (SC) at 300 mg every 4 weeks or every 8 weeks (see Table 4). AB01 (HBC34v35-MLNS-GAALIE) contains the light chain amino acid sequence SEQ ID NO:59 and the heavy chain amino acid sequence SEQ ID NO:57. SIRNA01 is provided as a liquid to be administered SC at 200 mg every 4 weeks or every 8 weeks (see Table 4). SIRNA01 has a sense strand containing the nucleotide sequence SEQ ID NO:5 and an antisense strand containing the nucleotide sequence SEQ ID NO:6.

[0519] The study objectives and related endpoints are shown in Table 5.

[0520] Table 5. Study objectives and relevant endpoints.

[0521]

[0522]

[0523] The Child-Pugh Turcotte (CPT) score estimates for liver disease are shown in Table 6.

[0524] Table 6. Child-Pugh Turcotte (CPT) score estimates for liver disease.

[0525]

[0526] *Level 0: Consciousness, personality, neurological examination, and EEG are all normal

[0527] Grade 1: restlessness, sleep disturbance, irritability / agitation, tremor, impaired writing ability, EEG waveform 5 cps

[0528] Grade 2: Lethargy, time disorientation, abnormal behavior, asterixis, ataxia, slow triphasic EEG waves

[0529] Grade 3: drowsiness, coma, disorientation, hyperreflexia, rigidity, slow EEG waves

[0530] Grade 4: Coma, unable to be awakened, no personality / action, no brain reflexes, slow 2-3 cps delta wave activity on EEG Source: FDA 2003

[0531] Assessment: If the score is 5 or 6, it is Class A (mild liver damage); if the score is 7 to 9, it is Class B (moderate liver damage); if the score is 10 to 15, it is Class C (severe liver damage).

[0532] The ULN value of ALT may be, for example, 34 IU / mL for females and 43 IU / mL for males.

[0533] PK parameters (free PK and total PK, where applicable) for SIRNA01 and AB01 will be calculated using standard non-compartmental methods, where applicable.

[0534] Including but not limited to C max , C last , T max , T last , AUC inf , AUC last , %AUC exp ,t 1 / 2 ,λz,V z / F and CL / F.

[0535] Immunogenicity data may include, but are not limited to, incidence, titer, and neutralization data.

[0536] FIG. 3A to FIG. 8C The schedule of activities (“SoA”) for the cohort and follow-up period is provided in .

[0537] Participant Groups

[0538] Participants were male and female participants aged 18 to 70 years with chronic HBV / HDV co-infection with all degrees of liver damage currently receiving NRTI therapy. For the purposes of this study, “chronic HBV infection” was defined as 2 positive serum HBsAg, HBV DNA, or HBeAg at least 6 months apart based on previous (within the past 12 months) or current laboratory records (any combination of these tests performed 6 months apart was acceptable). Enrollment in each cohort will aim to screen approximately 40% of participants with baseline HBsAg >10,000 IU / mL. Participants will receive locally approved NRTI therapy for at least 12 weeks prior to Day 1. In addition, participants will have HBsAg greater than 0.05 IU / mL at screening, clinical evidence of chronic hepatitis and positive HDV antibodies for at least 6 months, and HDV RNA remaining positive for at least 3 months prior to screening. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) >ULN and <5×ULN.

[0539] Participants were aged >18 years (or the legal age of consent, whichever is greater) to <70 years at screening. Participants had a body mass index (BMI) ≥18 kg / m 2 To ≤40kg / m2.

[0540] Additional inclusion criteria included the following:

[0541] ●Female participants must have a negative pregnancy test or confirmed postmenopausal status. Postmenopausal status is defined as 12 months without menstruation and no alternative medical reason. Confirmation of a negative follicle-stimulating hormone [FSH] test is required to confirm postmenopausal status. Women of childbearing potential (WOCBP) must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, must not be breastfeeding, and must be willing to use a highly effective contraceptive method from 14 days prior to study drug administration until 48 weeks after the last dose of SIRNA01 or AB01. Female participants must also agree not to donate eggs and undergo in vitro fertilization from the time of study drug administration until 48 weeks after the last dose of SIRNA01 or AB01.

[0542] ● Male participants with female partners of reproductive potential must agree to meet one of the following contraceptive requirements from the time of study drug administration until 48 weeks after the last dose of SIRNA01 or AB01: vasectomy or documentation of azoospermia, or use of male condoms with their partner using one of the contraceptive options listed for WOCBP contraception. Male participants must also agree not to donate sperm from the time of the first study drug administration until 48 weeks after the last dose of SIRNA01 or AB01.

[0543] ●Able to sign informed consent

[0544] ●12-lead electrocardiogram (ECG) within normal limits; or, as determined by the clinical investigator, no clinically significant abnormalities at screening.

[0545] ●Agree not to donate blood during the duration of the study and for 3 months after the last dose of study drug.

[0546] Specific inclusion criteria for cohorts 1 and 2

[0547] ○Non-cirrhotic

[0548] ■ Liver biopsy with METAVIR grade F0-F3 within 12 months before screening or liver elastography <12 kilopascals (kPa) (cohort 1 and cohort 2)

[0549] ■Creatinine clearance (CLcr) calculated by the Cockcroft-Gault formula at screening ≥ 30 mL / min;

[0550] Cirrhosis

[0551] ■ METAVIR grade F4 on liver biopsy within 12 months before screening or liver elastography ≥12kPa (cohort 2)

[0552] ■CLcr ≥ 60 mL / min calculated by the Cockcroft-Gault formula at screening

[0553] CPT score of 5 or 6, both at screening and at study entry

[0554] ● Specific inclusion criteria for Cohort 3 (moderate liver impairment) o In addition to liver impairment, participants must be deemed by the investigator to be healthy enough to participate in the study based on medical history, physical examination, vital signs, and screening laboratory evaluations

[0555] ○ Liver biopsy with METAVIR grade F4 within 12 months before screening or liver elastography ≥12kPa

[0556] ○ CLcr ≥ 60 mL / min calculated by the Cockcroft-Gault formula at screening

[0557] ○ CPT score of 7 to 9, both at screening and at study entry

[0558] Specific inclusion criteria for cohort 4 (severe liver impairment)

[0559] ○ Except for liver dysfunction, participants must be deemed by the investigator to be healthy enough to participate in the study based on medical history, physical examination, vital signs, and screening laboratory evaluation results

[0560] ○ Liver biopsy with METAVIR grade F4 or liver elastography Readout ≥12 kPa

[0561] ○ CLcr ≥ 60 mL / min calculated by the Cockcroft-Gault formula at screening and study entry with a screening CPT score > 10

[0562] Exclusion criteria included the following:

[0563] ●A history of clinically significant liver disease of non-HBV and non-HDV etiology as determined by the investigator.

[0564] History of immune complex disease

[0565] History of autoimmune disease

[0566] History of HBV-related extrahepatic disease, including but not limited to HBV-related rash, arthritis, or glomerulonephritis

[0567] ● History of allergic reaction, hypersensitivity, or intolerance to the study drug, its metabolites, or excipients

[0568] Anti-HB >10 IU / L at screening

[0569] Corrected QT interval (QTc) > 450 milliseconds

[0570] AST or ALT >5× ULN

[0571] Total bilirubin >3×ULN (cohorts 1 to 3), >5×ULN (cohort 4)

[0572] Serum albumin <28 g / L (cohorts 1 to 3), <25 g / L (cohort 4)

[0573] ●Absolute neutrophil count <1000 / mm3

[0574] Platelets <20,000 / mm

[0575] Hemoglobin <8 g / dL

[0576] History of allergic reactions

[0577] ●History of malignancy diagnosed or treated within 5 years (topically treated squamous or noninvasive basal cell skin cancer permitted; cervical cancer in situ permitted if appropriately treated prior to screening); participants who underwent evaluation for malignancy were ineligible

[0578] ● History of bone marrow or solid organ transplantation, or listed as having undergone a bone marrow or solid organ transplant ● Known active infection other than chronic HBV and HDV infection within 7 days prior to Day 1, or any clinically significant acute condition such as fever (>38°C) or acute respiratory illness

[0579] ● Co-infection with human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), or hepatitis E virus (HEV). Participants who are HCV antibody positive and HCV RNA negative are eligible. Participants who are HAV or HEV immunoglobulin M antibody (IgM) positive are eligible. No Eligibility. Participants who were asymptomatic and positive for HAV or HEV immunoglobulin G antibodies (IgG) were eligible.

[0580] - Any clinically significant medical or psychiatric condition that may interfere with study interventions, assessments, or compliance with the protocol, or otherwise render the participant unsuitable for participation in this study, as determined by the Investigator. Participants with controlled diabetes are eligible.

[0581] ●Acute or worsening chronic hepatitis, fluctuations or rapid worsening of liver function, or use of any therapy that the investigator believes has exacerbated liver dysfunction.

[0582] ●Treatment with immunomodulators, interferon-alpha (eg, IFN-α-2a or IFN-α-2b, or pegylated IFN-α-2a or α-2b), cytotoxic or chemotherapeutic agents, or chronic systemic corticosteroids within 6 months of screening.

[0583] ● Received HDV active agents (including lonafamib and brevitide) within 90 days or 5 half-lives (if known) before administration of study drug (whichever is longer), or HDV active agents are active during the follow-up period of another clinical study involving interventional treatment. Participants must also agree not to participate in any other interventional studies at any time during participation in this study (including the follow-up period).

[0584] ●Receipt of oligonucleotides with anti-HBV activity (eg, siRNA, antisense oligonucleotides) within 48 weeks prior to study drug administration

[0585] ● For newly enrolled participants: Receipt of AB01 within 24 weeks prior to Study Day 1.

[0586] ●History or clinical evidence of alcohol or drug abuse within 12 months prior to screening, or a positive drug screen result at screening, unless it can be explained by prescription medication (diagnosis and prescription must be approved by the investigator). Marijuana use is permitted.

[0587] ●Additional exclusion criteria for participants with impaired hepatic function

[0588] ○ Participants need piercing more than once a month.

[0589] ○ Participants have refractory encephalopathy or severe central nervous system disease as determined by the investigator.

[0590] ○ History of gastric or esophageal varicose bleeding within the past 6 months.

[0591] ○ Participants underwent transjugular intrahepatic portosystemic shunt (TIPS).

[0592] ○Presence of hepatopulmonary or hepatorenal syndrome,

[0593] ○Presence of primary cholestatic liver disease.

[0594] ○ Inability or unwillingness to adhere to the investigator-suggested dietary recommendations for cirrhosis and liver damage, and the lifestyle considerations outlined in that regimen.

[0595] Concomitant therapy

[0596] ●No concomitant therapy was allowed during the study

[0597] ● Participants must avoid taking prescription medications other than those ordered by their prescribing physician within 7 days (14 days if the drug is a potential enzyme inducer) or 5 half-lives (whichever is longer) before the start of the study intervention and over-the-counter medications (including vitamins, recreational drugs, and dietary or herbal supplements) until the end of follow-up unless the investigator and sponsor determine that the medication will not interfere with the study.

[0598] ● Participants with liver impairment who have comorbid conditions requiring medication must have been taking medications without dose changes for more than 3 months prior to screening. All concomitant medications must be approved by the medical monitor prior to study enrollment.

[0599] ● Use of any of the following systemic medications is prohibited within 14 days prior to study drug administration and throughout the study:

[0600] ■Chronic systemic steroids (prednisone equivalents > 10 mg / day) or other immunosuppressants (note: corticosteroids are allowed for immune-mediated AEs, and short-term corticosteroids are allowed for COPD or asthma exacerbations)

[0601] ■ Paracetamol (acetaminophen) ≥ 3 g / day

[0602] Isoniazid

[0603] ●In addition, any potentially hepatotoxic medications should be considered for administration during the study only if no therapeutic alternatives can be identified and after careful consideration of the potential risks and benefits to the participant. Medications that are potentially hepatotoxic or have been associated with drug-induced liver injury include, but are not limited to, the following (Bjornsson 2016): aspirin > 3 g / day or ibuprofen > 1.2 g / day, tricyclic antidepressants, valproate, phenytoin, amiodarone, anabolic steroids, allopurinol, amoxicillin-clavulanate, minocycline, nitrofurantoin, sulfamethoxazole / trimethoprim, erythromycin, rifampin, azole antifungals, and herbal or natural remedies.

[0604] References

[0605] Abbas Z,Khan MA,Salih M,Jafri W.Interferon alpha for chronichepatitis D.

[0606] Cochrane Database Syst Rev.2011Dec 7;2011(12):CD006002.doi:

[0607] 10.1002 / 14651858.CD006002.pub2.PMID:22161394; PMCID:PMC6823236.

[0608] Agarwal K,Yuen MF,Wedemeyer H,Arizpe A,Shen L,Cloutier D,Tay C,GuptaSV,Fanget M,Dholakiya SL,Seu L,Cathcart AL,Hwang C,Pang PS,Gane E.Rapid hbsagreduction in chronic hepatitis B virus infection:preliminary results from aPhase 1Study evaluating a singledose of VIR-3434,a novel neutralizing,vaccinal monoclonal antibody.AASLD:The Liver Meeting 2021,November 12-15.

[0609] Bahcecioglu IH,Sahin A.Treatment of Delta Hepatitis:Today and in theFuture-A review.Infect Dis(Lond).2017Apr;49(4):241-250.doi:

[0610] 10.1080 / 23744235.2016.1271998.Epub 2017Jan 16.PMID:28092214.

[0611] Bjornsson ES.Hepatotoxicity by Drugs:The Most Common ImplicatedAgents.Int J Mol Sci.2016Feb 6;17(2):224.doi:10.3390 / ijmsl7020224.PMID:26861310;

[0612] PMCID:PMC4783956.

[0613] Chang J,Nie X,Chang HE,Han Z,Taylor J.Transcription of hepatitisdelta virus RNA by RNA polymerase II.J Virol.2008Feb;82(3):1118-27.doi:

[0614] 10.1128 / JVI.01758-07.Epub 2007 Nov 21.PMID:18032511;PMCID:PMC2224410.

[0615] Chen HY,Shen DT,Ji DZ,Han PC,Zhang WM,Ma JF,Chen WS,Goyal H,Pan S,XuHG.Prevalence and burden of hepatitis D virus infection in the globalpopulation:a systematic review and meta-analysis.Gut.2019Mar;68(3):512-521.doi:

[0616] 10.1136 / gutjnl-2018-316601.Epub 2018 Sep 18.PMID:30228220.

[0617] de Ledinghen V et.al.Safety and Efficacy of 2mg Bulevirtide inPatients with Chronic HBV / HDV Co-Infection:First Real-World Results AASLD2021Nov 12-15.

[0618] European Association for the Study of the Liver.European Associationfor the Study of the Liver.EASL 2017 Clinical Practice Guidelines on themanagement of hepatitis B virus infection.J Hepatol.2017 Aug;67(2):370-398.doi:10.1016 / j.jhep.2017.03.021.Epub 2017 Apr 18.PMID:28427875.

[0619] Farci P,Smedile A,Lavarini C,Piantino P,Crivelli O,Caporaso N,Toti M,Bonino

[0620] F,Rizzetto M.Delta hepatitis in inapparent carriers of hepatitis Bsurface antigen.A disease simulating acute hepatitis B progressive to chronicity.Gastroenterology.1983 Sep;85(3):669-73.PMID:6873613.

[0621] Fattovich G,Boscaro S,Noventa F,Pornaro E,Stenico D,Alberti A,Ruol A,Real di

[0622] G.Influence of hepatitis delta virus infection on progression tocirrhosis in chronic hepatitis type B.J Infect Dis.1987 May;155(5):931-5.doi:10.1093 / infdis / l 55.5.931.PMID:3559292.

[0623] Fattovich G,Giustina G,Christensen E,Pantalena M,Zagni I,Real di G,Schalm SW.Influence of hepatitis delta virus infection on morbidity andmortality in compensated cirrhosis type B.The European Concerted Action onViral Hepatitis(Eurohep).Gut.2000 Mar;46(3):420-6.doi:10.1136 / gut.46.3.420.PMID:10673308;PMCID:PMC1727859.

[0624] FDA:Pharmacokinetics in Patients with Impaired Hepatic Function:StudyDesign,Data Analysis,and Impact on Dosing and Labeling.Center for DrugEvaluation and Research,Center for Biologies Evaluation andResearch.May2003.https: / / www.fda.gov / media / 71311 / download Freitas N,Abe K,Cunha C,Menne S,Gudima SO.Support of the infectivity of hepatitis delta virusparticles by the envelope proteins of different genotypes of hepatitis Bvirus.J Virol.2014Jun;88(l l):6255-67.doi:10.1128 / JVI.00346-14.Epub 2014 Mar19.PMID:24648462;PMCID:PMC4093875.

[0625] Galun E,Eren R,Safadi R,Ashour Y,Terrault N,Keeffe EB,Matot E,Mizrachi S,Terkieltaub D,Zohar M,Lubin I,Gopher J,Shouval D,Dagan S.Clinicalevaluation(phase I)of a combination of two human monoclonal antibodies toHBV:safety and antiviral properties.Hepatology.2002Mar;35(3):673-9.doi:10.1053 / jhep.2002.31867.Erratum in:Hepatology 2002Apr;35(4):986.PMID:11870383.

[0626] Gane E,Lim YS,Yuen MF,Cloutier D,Thanawala V et al.Longer treatmentduration of monthly VIR-2218 results in deeper and more sustained reductionsin hepatitis B surface antigen in participants with chronic hepatitis Binfection.Abstract submitted at the EASL International Liver Conference,June22-26,202.

[0627] Gane E,Lim YS,Cloutier D,Shen L,et al.Safety and Antiviral Activityof VIR-2218,An XTargeting RNAi Therapeutic,In Participants With ChronicHepatitis B Infection:Week 48 Follow-Up Results.Oral Presentation.Presentedat the EASL Digital International Liver Conference,June 23-26,2021.

[0628] Gunsar F,Akarca US,Ersoz G,Kobak AC,Karasu Z,Yuce G,liter T,BaturY.Two-year interferon therapy with or without ribavirin in chronic deltahepatitis.Antivir Ther.2005;10(6):721-6.PMID:16218171.

[0629] Gupta SV,Fanget MC,MacLauchlin C,Clausen VA,Li J,Cloutier D,Shen L,Robbie GJ,Mogalian E.Clinical and Preclinical Single-Dose Pharmacokinetics ofVIR-2218,an RNAi Therapeutic Targeting HBV Infection.Drugs R D.2021 Dec;21(4):455-465.doi:10.1007 / s40268-021-00369-w.Epub2021 Nov 6.PMID:34741731;PMCID:PMC8602582.

[0630] Gupta SV,Arizpe A,Fanget M,Dholakiya SL,et al.PreliminaryPharmacokinetics and Safety in Healthy Volunteers of VIR-3434,a MonoclonalAntibody for the Treatment of Chronic Hepatitis

[0631] B Infection.Poster.Presented at the EASL Digital International LiverConference,June 23-26,2021.

[0632] Heidrich B,Yurdaydin C,Kabagam G,Ratsch BA,Zachou K,Bremer B,etal.Late HDV RNA relapse after peginterferon alpha-based therapy of chronichepatitis delta.Hepatology 2014;60:87-97.Hepcludex Summary of ProductCharacteristics.

[0633] Hepcludex,2mg powder for solution for injection.Ireland:GileadSciences Ireland UC;2020.Available from:http: / / www.ema.europa.eu.

[0634] Kamili S,Drobeniuc J,Mixson-Hayden T,Kodani M.Delta hepatitis:Towardimproved diagnostics.Hepatology.2017 Dec;66(6):1716-1718.doi:

[0635] 10.1002 / hep.29564.Epub 2017 Oct 30.PMID:28961326.

[0636] Le Gal F,Brichler S,Drugan T,Alloui C,Roulot D,Pawlotsky JM,Deny P,Gordien E.Genetic diversity and worldwide distribution of the deltavirusgenus:A study of 2,152 clinical strains.Hepatology.2017 Dec;66(6):1826-1841.doi:10.1002 / hep.29574.Epub 2017 Oct 30.PMID:28992360.

[0637] Lee AU,Lee C.Hepatitis D Review:Challenges for the Resource-PoorSetting.Viruses.2021 Sep 23;13(10):1912.doi:10.3390 / vl3101912.PMID:34696341;

[0638] PMCID:PMC8538672.

[0639] Lempp FA,Ni Y,Urban S.Hepatitis delta virus:insights into a peculiarpathogen and novel treatment options.Nat Rev Gastroenterol Hepatol.2016Oct;13(10):580-9.doi:10.1038 / nrgastro.2016.126.Epub 2016 Aug 18.PMID:27534692.

[0640] Lempp FA,Vincenzetti L,Volz T,et al.,editors.PreclinicalCharacterization of VIR-3434,a Monoclonal Antibody Neutralizing Hepatitis BVirus That Facilitates FcyR-Mediated Elimination of HBsAg.AmericanAssociation for the Study of Liver Diseases(AASLD);2021 November 12-15,2021;Anaheim,CA,PO-838.

[0641] Lok AS,Negro F,Asselah T,Farci P,Rizzetto M.Endpoints and New Optionsfor Treatment of Chronic Hepatitis D.Hepatology.2021Dec;74(6):3479-3485.doi:

[0642] 10.1002 / hep.32082.Epub 2021 Sep 16.PMID:34331781.

[0643] Lucifora J,Delphin M.Current knowledge on Hepatitis Delta Virusreplication.

[0644] Antiviral Res.2020 Jul;179:104812.doi:10.1016 / j.antiviral.2020.104812.Epub 2020 Apr 29.PMID:32360949.

[0645] Mathurin P,Thibault V,Kadidja K,Ganne-Carrie N,Moussalli J,El YounsiM,Di Martino V,Lunel F,Charlotte F,Vidaud M,Opolon P,Poynard T.Replicationstatus and histological features of patients with triple(B,C,D)and dual(B,C)hepatic infections.J Viral Hepat.2000 Jan;7(l):15-22.doi:10.1046 / j.l365-2893.2000.00195.x.PMID:10718938.

[0646] Miao Z,Zhang S,Ou X,Li S,Ma Z,Wang W,Peppelenbosch MP,Liu J,PanQ.Estimating the Global Prevalence,Disease Progression,and Clinical Outcomeof Hepatitis Delta Virus Infection.J Infect Dis.2020 Apr 27;221(10):1677-1687.doi:10.1093 / infdis / jiz633.PMID:31778167;PMCID:PMC7184909.

[0647] Modahl LE,Macnaughton TB,Zhu N,Johnson DL,Lai MM.RNA-Dependentreplication and transcription of hepatitis delta virus RNA involve distinctcellular RNA polymerases.Mol Cell Biol.2000 Aug;20(16):6030-9.doi:10.1128 / MCB.20.16.6030-6039.2000.PMID:10913185;PMCID:PMC86079.

[0648] National Institute for Health.National Institute of Diabetes andDigestive and Kidney Diseases.United States 2017.Department of Health andHuman Services.Accessed April 10,2020.

[0649] NCI Common Terminology Criteria for Adverse Events(CTCAE)Version5.0Published:November 27,2017,U.S.Department of Health and Human Services,National Institutes of Health.National Cancer Institute.https: / / ctep.cancer.gov / protocoldevelopment / electronic_applications / docs / CTCA E_v5_Quick_Reference_8.5xl 1.pdf.

[0650] Niro GA,Smedile A.Current concept in the pathophysiology of hepatitisdelta infection.Curr Infect Dis Rep.2012 Feb;14(1):9-14.doi:10.1007 / sl 1908-011-0233-5.PMID:22161240.

[0651] Niro GA,Smedile A,Ippolito AM,Ciancio A,Fontana R,Olivero A,ValvanoMR,Abate ML,Gioffreda D,Caviglia GP,Rizzetto M,Andriulli A.Outcome of chronicdelta hepatitis in Italy:a long-term cohort study.J Hepatol.2010 Nov;53(5):834-40.doi:10.1016 / j.jhep.2010.06.008.Epub 2010 Jul 29.PMID:20800919.

[0652] Petrillo R,Buti M,Durand F,Charlton M,Gadano A,Cantisani G,etal.Entecavir and hepatitis B immune globulin in patients undergoing livertransplantation for chronic hepatitis B.Liver Transpl.2013 Aug;19(8):887-95.

[0653] Polaris Observatory Collaborators.Global prevalence,treatment,andprevention of hepatitis B virus infection in 2016:a modelling study.LancetGastroenterol Hepatol.2018 Jun;3(6):383-403.doi:10.1016 / S2468-1253(18)30056-6.Epub 2018 Mar 27.PMID:29599078.

[0654] Raimondo G,Smedile A,Gallo L,Balbo A,Ponzetto A,RizzettoM.Multicentre study of prevalence of HBV-associated delta infection and liverdisease in drug-addicts.Lancet.1982 Jan 30;l(8266):249-51.doi:10.1016 / s0140-6736(82)90976-x.PMID:6120275.

[0655] Reed WD,Eddleston AL,Cullens H,Williams R,Zuckerman AJ,Peters DK,Williams DG,May cock WA.Infusion of hepatitis-B antibody in antigen-positiveactive chronic hepatitis.Lancet.1973 Dec 15;2(7842):1347-51.doi:10.1016 / s0140-6736(73)93321-7.PMID:4128052.

[0656] Reilly MC,Zbrozek AS,Dukes EM.The validity and reproducibility of awork productivity and activity impairment instrument.Pharmacoeconomics.1993Nov;4(5):353-65.doi:10.2165 / 00019053-199304050-00006.PMID:10146874.

[0657] Rizzetto M.Hepatitis D Virus:Introduction and Epidemiology.ColdSpring Harb Perspect Med.2015 Jul l;5(7):a021576.doi:10.1101 / cshperspect.a021576.PMID:26134842;PMCID:PMC4484953.

[0658] Rizzetto M,Hamid S,Negro F.The changing context of hepatitis D.JHepatol.2021 May;74(5):1200-1211.doi:10.1016 / j.jhep.2021.01.014.Epub2021 Jan20.PMID:33484770.

[0659] Romeo R,Del Ninno E,Rumi M,Russo A,Sangiovanni A,de Franchis R,RonchiG,Colombo MA 28-year study of the course of hepatitis Delta infection: a risk factor for cirrhosis and hepatocellular carcinoma.Gastroenterology.2009May;136(5):1629-38.doi:10.1053 / j.gastro.2009.01.052.Epub 2009 Jan 29.PMID:19208358.

[0660] Sagnelli E, Felaco FM, Filippini P, Pasquale G, Peinetti P, Buonagurio E, Aprea L, Pulella C, Piccinino F, Giusti G. Influence of HDV infection on clinical, biochemical and histological presentation of HBsAg positive chronichepatitis. Liver. 1989 Aug;9(4):229-34. doi:10.1111 / jl 600-0676.1989.tb00404.x. PMID:2770435.

[0661] Sarin SK,Kumar M,Lau GK,Abbas Z,Chan HL,Chen CJ,Chen DS,Chen HL,ChenPJ,Chien RN,Dokmeci AK,Gane E,Hou JL,Jafri W,Jia J,Kim JH,Lai CL,Lee HC,LimSG,Liu CJ,Locarnini S,Al Mahtab M,Mohamed R,Omata M,Park J,Piratvisuth T,Sharma BC,Sollano J,Wang FS,Wei L,Yuen MF,Zheng SS,Kao JH.Asian-Pacificclinical practice guidelines on the management of hepatitis B:a 2015update.Hepatol Int.2016 Jan;10(1):1-98.doi:10.1007 / sl2072-015-9675-4.Epub2015 Nov 13.PMID:26563120;PMCID:PMC4722087.

[0662] Shirvani-Dastgerdi E,Tacke F.Molecular interactions between hepatitisB virus and delta virus.World J Virol.2015 May 12;4(2):36-41.doi:10.5501 / wjv.v4.i2.36.PMID:25964870;PMCID:PMC4419120.

[0663] Sleijfer S,Bannink M,Van Gool AR,Kruit WH,Stoter G.Side effects ofinterferonalpha therapy.Pharm World Sci.2005 Dec;27(6):423-31.doi:10.1007 / s11096-005-1319-7.PMID:16341948.

[0664] Stockdale AJ,Kreuels B,Henrion MYR,Giorgi E,Kyomuhangi I,de Martel C,Hutin Y,Geretti AM.The global prevalence of hepatitis D virus infection:Systematic review and meta-analysis.J Hepatol.2020 Sep;73(3):523-532.doi:10.1016 / j.jhep.2020.04.008.Epub 2020 Apr 23.PMID:32335166;PMCID:

[0665] PMC7438974.

[0666] Sureau C,Negro F.The hepatitis delta virus:Replication andpathogenesis.J Hepatol.2016 Apr;64(l Suppl):S102-S116.doi:10.1016 / j.jhep.2016.02.013.PMID:27084031.

[0667] Terrault NA,Lok ASF,McMahon BJ,Chang KM,Hwang JP,Jonas MM,Brown RSJr,Bzowej NH,Wong JB.Update on prevention,diagnosis,and treatment of chronichepatitis B:AASLD 2018 hepatitis B guidance.Hepatology.2018 Apr;67(4):1560-1599.doi:10.1002 / hep.29800.PMID:29405329;PMCID:PMC5975958.

[0668] Tsuge M,Hiraga N,Uchida T,Kan H,Miyaki E,Masaki K,Ono A,Nakahara T,Abe-Chayama H,Zhang Y,Naswa MG,Kawaoka T,Miki D,Imamura M,Kawakami Y,AikataH,Ochi H,Hayes CN,Chayama K.Antiviral effects of anti-HBs immunoglobulin andvaccine on HBs antigen seroclearance for chronic hepatitis B infection.JGastroenterol.2016 Nov:51(11):1073-1080.doi:10.1007 / s00535-016-1189-x.Epub2016 Mar 4.PMID:26943168.

[0669] Urban S,Neumann-Haefelin C,Lampertico P.Gut.2021 Sep170(9):1782-1794.doi:10.1136 / gutjnl-2020-323888.Epub 2021Jun 8.PMID:34103404;PMCID:PMC8

[0670] Vlachogiannakos J,Papatheodoridis GV.Liver Int.2020 Feb:40 Suppl 1:48-53.doi:10.111 l / liv.14357.PMID:3

[0671] Wedemeyer H,et al.Treatment with bulevirtide improves patientreported outcomes in patients with chronic hepatitis delta(CHD):an interimexploratory analysis at week 24.The Liver Meeting,abstract 680,2021.

[0672] Wedemeyer H,Yurdaydin C,Dalekos GN,Erhardt A,Qakaloglu Y,DegertekinH,Gurel S,Zeuzem S,Zachou K,Bozkaya H,Koch A,Bock T,Dienes HP,Manns MP;HID ITStudy Group.Peginterferon plus adefovir versus either drug alone forhepatitis delta.N Engl J Med.2011 Jan 27;364(4):322-31.doi:10.1056 / NEJMoa0912696.

[0673] PMID:21268724.

[0674] WHO(World Health Organization).Hepatitis D.https: / / www.who.int / news-room / factsheets / detail / hepatitis-d.Published July2021.Accessed April 10,2022.

[0675] WHO(World Health Organization).Hepatitis B.https: / / www.who.int / news-room / factsheets / detail / hepatitis-b.Published July2021.Accessed April 10,2022.

[0676] Ye X,Tateno C,Thi EP,Kakuni M,Snead NM,Ishida Y,Barnard TR,Sofia MJ,Shimada T,Lee ACH.Hepatitis B Virus Therapeutic Agent ARB-1740 Has InhibitoryEffect on Hepatitis Delta Virus in a New Dually-Infected Humanized MouseModel.ACS Infect Dis.2019 May 10;5(5):738-749.doi:10.1021 / acsinfecdis.8b00192.Epub 2018 Nov 20.PMID:30408957.

[0677] Younossi ZM,Guyatt G,Kiwi M,Boparai N,King D.Development of a diseasespecific questionnaire to measure health related quality of life in patientswith chronic liver disease.Gut.1999 Aug;45(2):295-300.doi:10.1136 / gut.45.2.295.PMID:

[0678] 10403745;PMCID:PMC1727607.

[0679] Younossi ZM,Stepanova M,Younossi I,Racila A.Development andvalidation of a hepatitis B specific health-related quality-of-lifeinstrument:CLDQ-HBV.J Viral Hepat.2021 Mar;28(3):484-492.doi:10.1111 / jvh.13451.Epub 2020 Dec 20.PMID:33306234.

[0680] Yuen MF,Lim YS,Cloutier D,Thanawala V,et al.Preliminary Results Froma Phase 2 Study Evaluating VIR-2218 Alone and in Combination With PegylatedInterferon Alfa-2a in Participants With Chronic Hepatitis B Infection.OralPresentation.Presented at AASLD:The Liver Meeting(Virtual),Novemberl2-15,2021.

[0681] Yurdaydin C,Abbas Z,Buti M,Cornberg M,Esteban R,Etzion O,Gane EJ,GishRG,Glenn JS,Hamid S,Heller T,Koh C,Lampertico P,Lurie Y,Manns M,Parana R,Rizzetto M,Urban S,Wedemeyer H;Hepatitis Delta International Network(HDIN).Treating chronic hepatitis delta:The need for surrogate markers of treatmentefficacy.J Hepatol.2019May;70(5):1008-1015.doi:10.1016 / jjhep.2018.12.022.Epub 2018Dec 27.PMID:30982526.

[0682] Zachou K, Yurdaydin C, Drebber U, Dalekos GN, Erhardt A, Cakaloglu Y, Degertekin H, Gurel S, Zeuzem S, Bozkaya H, Schlaphoff V, Dienes HP, Bock TC, MannsMP, Wedemeyer H; HIDT-1Study Group. Quantitative HBsAg and HDV-RNA levels inchronic delta hepatitis. Liver Int.2010Mar;30(3):430-7.doi:10.1111 / j.1478-3231.2009.02140.x.Epub 2009Oct 16.PMID:19840253.

[0683] Example 2

[0684] Neutralization of HDV by antibodies targeting HBsAg

[0685] background

[0686] Chronic hepatitis B virus (HBV) infection is a major global public health burden, affecting approximately 296 million people worldwide and causing an estimated 820,000 deaths each year (Polaris Observatory Collaborators, 2018; WHO, Hepatitis B, 2021).

[0687] SIRNA01 is an experimental siRNA therapeutic that targets the HBx region of the HBV genome and exhibits strong antiviral activity in vitro and in vivo. The sense strand of SIRNA01 is conjugated to an N-acetylgalactosamine (GalNAc) ligand, enabling targeted delivery to the liver. AB01 is an experimental neutralizing monoclonal antibody that targets the antigenic loop of HBsAg and has pan-genotype neutralizing activity in vitro. Treatment with murinized AB01 inhibits viral dissemination and causes elimination of HBsAg in vivo. The mAb carries an engineered Fc that prolongs serum half-life (LS mutation) and increases binding to activating FcgR (FcgRIIa and Illa), but reduces binding to inhibitory FcgRIIb (XX2 / GAALIE mutation).

[0688] method

[0689] Primary human hepatocytes (PHH) infected with HBV (genotype D) were used to demonstrate HBV entry inhibition after AB01 treatment. HBeAg in cell culture supernatants was quantified by chemiluminescent immunoassay (CLIA) as a marker of infection.

[0690] To evaluate the in vitro activity of AB01 against eight different HBV genotypes, hepatitis D virus (HDV), a HBV satellite virus found in 5% of chronic HBV patients (WHO, Hepatitis D, 2021), was used. Since HDV requires HBV envelope proteins to enter and be secreted from hepatocytes, it can be used as a tool to study HBsAg from different HBV genotypes (Wang et al., 2021). Here, the neutralization effect of AB01 was evaluated in Huh7-NTCP cells infected with HDV pseudotyped with HBsAg from eight different HBV genotypes (AH).

[0691] To evaluate the in vitro antiviral activity of SIRNA01 against representative HBV genotypes A to D, the HBV1.3 ultra-long genome system was used, in which all viral RNAs were transcribed under the regulation of the authentic HBV promoter. Huh7 cells were transfected with plasmids containing HBV1.3 genome sequences from 13 HBV isolates representing genotypes A to D, followed by SIRNA01 or control siRNA. HBsAg was used as a readout.

[0692] To evaluate the combined activity of SIRNA01 and AB01 versus monotherapy, two in vivo studies were conducted using the following well-established mouse models of HBV infection: C57BL / 6 mice transduced with AAV8-HBV (genotype D) or human liver chimeric PXB mice infected with HBV (genotype C). Mice were treated with SIRNA01, HBC34-mu (a murinized version of AB01), entecavir (ETV, used only in AAV8-HBV studies), or a combination of agents at varying concentrations. Antiviral activity was determined by evaluating viral serum / plasma markers, including HBV DNA, HBsAg, and HBeAg.

[0693] result

[0694] AB01 and SIRNA01 showed potent activity against viruses containing all tested HBsAg genotypes. Primary human hepatocytes (PHH) were infected with HBV (genotype D) in the presence of AB01, Mai 8 / 7 mAb targeting pre-S1, or polyclonal hepatitis B immune globulin (HBIG). HBV neutralization activity was assessed 7 days after infection by quantification of secreted HBeAg as a marker of infection ( Fig. 9To evaluate the neutralization capacity of AB01 against different HBV genotypes in the context of infection, HDV encapsulated with HBsAg from different HBV genotypes was utilized. The neutralization effect of AB01 was evaluated in Huh7-NTCP cells infected with HDV pseudotyped with HBsAg from eight different HBV genotypes (AH). Fig.10 ).

[0695] The activity of SIRNA01 against HBV genotypes A to D was evaluated in human hepatoma cells (Huh7) transfected with HBV genome. Fig.11 ).

[0696] References

[0697] Polaris Observatory Collaborators.Global prevalence,treatment,andprevention of hepatitis B virus infection in 2016:a modeling study.LancetGastroenterol Hepatol 2018;3(6):383-403.

[0698] Hepatitis B. World Health Organization. July 27, 2021. Accessed April 14, 2022. https: / / www.who.int / news-room / fact-sheets / detail / hepatitis-b.

[0699] Hepatitis D.World Health Organization.July 28,2021.Accessed April 29,2022.https: / / www.who.int / news-room / fact-sheets / detail / hepatitis-d

[0700] Wang W.et.al.:Assembly and infection efficacy of hepatitis B virussurface protein exchanges in 8hepatitis D virus genotype isolates; JHepatol.2021Aug;75(2):311-323.doi:10.1016 / j.jhep.2021.03.025

[0701] Example 3

[0702] Clinical evaluation of monotherapy or combination therapy for the treatment of chronic HDV infection

[0703] The safety, efficacy, and tolerability of anti-HBV antibodies combined with anti-HBV siRNA were evaluated in a phase 2, multicenter, open-label clinical study conducted in human patients with chronic HBV / HDV co-infection, non-cirrhotic and compensated cirrhosis (CPT-A) receiving nucleoside reverse transcriptase inhibitor (NRTI) therapy.

[0704] The objectives of this study are to evaluate the safety of SIRNA01 and AB01 in participants with chronic HBV / HDV co-infection and to assess whether monotherapy or combination therapy with investigational agents can durably block HDV replication and normalize ALT when administered on a biweekly, monthly, or bimonthly schedule in participants with various degrees of liver fibrosis and compensated cirrhosis.

[0705] Study Design

[0706] Table 6 shows the treatment groups for this study. This study will consist of cohorts receiving SIRNA01 or AB01 monotherapy or combination therapy. For cohorts 1a and 1b, the intervention period using SIRNA01 and AB01 monotherapy consists of 2 periods: an induction period (12 weeks) and a maintenance period (up to 84 weeks). For cohorts 2a, 2b1, 2b2, 2c and 3, the intervention period using SIRNA01 and / or AB01 consists only of a treatment period (up to 96 weeks). Cohort 4 participants will delay treatment for 12 weeks before starting combination therapy with SIRNA01 and AB01 for up to 96 weeks. After reviewing the 12-week safety and efficacy data from cohorts 1a and 1b, cohorts 2 to 4 will be opened. This study also includes 3 optional sub-studies that collect (1) liver tissue, (2) liver fine needle aspirates, and (3) blood samples for pharmacokinetic (PK) studies.

[0707] Table 6. Treatment groups in clinical studies.

[0708]

[0709] SC = Subcutaneous

[0710] *The composite endpoint was defined as undetectable HDV RNA (<LOD) or a ≥2 log decrease in HDV RNA from baseline 10 and ALT < ULN.

[0711] a Participants who achieved the composite endpoint at Week 12 will receive 10 additional doses every 8 weeks.

[0712] b Participants who did not achieve the composite endpoint at Week 12 may enter Cohort 2c on Day 1 or enter the follow-up period. c Participants who achieved the composite endpoint at Week 48 will continue with monotherapy until Week 96.

[0713] d Participants who did not achieve the composite endpoint at Week 48 may enter the follow-up period or initiate combination therapy with SIRNA01 + AB01 and follow the investigational product (IP) administration protocol in the Cohort 2c activity schedule (SoA) from Week 52 to Week 96.

[0714] e Participants who met the virological non-response criteria at Week 24 may initiate combination therapy with SIRNA01 and follow the IP administration protocol in the Cohort 2c SoA from Week 28 to Week 96.

[0715] f Treatment-naïve participants who achieved the composite endpoint at Week 48, including those who entered from Cohort 4, will continue with combination therapy until Week 96.

[0716] g Treatment-naïve participants who did not achieve the composite endpoint at Week 48, including those who entered from Cohort 4, will enter the follow-up period.

[0717] h Participants will delay treatment for 12 weeks prior to Day 1 of transitioning to Cohort 2c.

[0718] The study protocol for Cohort 1 is shown in Fig. 12A and the study protocols for Cohorts 2, 3, and 4 are shown in Fig. 12B respectively. FIG. 13A to FIG. 20B The activity schedule (“SoA”) for the cohorts and follow-up period is provided in

[0719] Approximately up to 118 participants will be enrolled. This includes a pool of up to 24 floating participants who can be added to any arm. Participants are planned to be enrolled at study centers around the world. The participants in the cohort are as follows:

[0720] ●Cohorts 1a and 1b: approximately 10 participants with HBV / HDV co-infection and Meta-Analysis of Histological Data in Viral Hepatitis with Fibrosis Stage (METAVIR) grade F0-F3: approximately 5 participants per cohort.

[0721] Cohort 2 consists of 4 arms and will enroll up to approximately 78 participants with METAVIR F0-F3 or METAVIR F4, CPT-A liver disease. Up to approximately 50% of participants in each cohort will have METAVIR F4, CPT-A liver disease. Each cohort will enroll approximately 50% of participants with baseline HBsAg <5000 IU / mL.

[0722] ■Cohort 2a: Planned enrollment of approximately 12 participants, maximum 36 (including floating participants).

[0723] ■ Cohort 2b1 (or Cohort 2b2): Approximately 12 participants are planned to be selected, with a maximum of 36 participants (including floating participants).

[0724] ■ Cohort 2b2 (or cohort 2b1): approximately 12 participants are planned to be enrolled, with a maximum of 36 participants (including floating participants).

[0725] ■ Cohort 2c: Up to approximately 30 de novo participants are planned to be enrolled, including participants entering from Cohort 4, if applicable. Participants entering from Cohort 1a or Cohort 1b will not contribute to planned enrollment. The total cohort size (including floating participants) is a maximum of 54.

[0726] - Cohort 3: Up to approximately 30 participants are planned to be enrolled, with a maximum of 54 (including floater participants). Up to approximately 50% of participants in this cohort will have METAVIR F4, CPT-A liver disease.

[0727] Cohort 4: Up to approximately 12 participants are planned to be enrolled. Up to approximately 50% of participants in this cohort will have METAVIR F4, CPT-A liver disease.

[0728] The study will start with Cohort 1. Cohort 1 participants who do not meet the combined endpoint at Week 12 may proceed to Day 1 of Cohort 2c pending a decision by the investigator and participant at their initially scheduled Week 16 visit as to whether the participant's Week 12 data meet the inclusion / exclusion (I / E) criteria for Cohort 2. Arms within Cohort 2 may be enabled individually based on available safety and antiviral data in Cohort 1. Cohort 3 will be enabled after review of the 12-week safety and available antiviral data in Cohort 1b. Enrollment of de novo non-cirrhotic participants into Cohorts 2c and 4 will only begin after review of a minimum of 12 weeks of safety and antiviral data from Cohorts 1a (n approximately equals 5) and 1b (n approximately equals 5). Enrollment of participants with CPT-A cirrhosis into Cohorts 2a, 2b1, or 2b2 and 3 will only begin after review of a minimum of 12 weeks of safety and antiviral data from approximately 10 participants who received SIRNA01 monotherapy (n approximately equals 5) or AB01 monotherapy (n approximately equals 5) and other available safety data. Enrollment of participants with CPT-A cirrhosis into Cohorts 2c and 4 will begin only after review of a minimum of 12 weeks of safety data and antiviral data from approximately 5 non-cirrhotic participants in Cohort 2c and other available safety data. Cohorts may be paused, closed, or interrupted.

[0729] The maximum total study duration is planned to be up to 118 weeks. For Cohort 1, this includes the Screening Period (up to 6 weeks), the Induction Period (12 weeks), and the Maintenance Period (84 weeks). At the end of the Induction Period, participants may (1) transition to the Maintenance Period, (2) enter Cohort 2c, or (3) enter the Follow-up Period at the next visit (Week 16). Participants who prematurely discontinue study treatment during the Induction Period will have an Early Termination (ET) Visit and then enter the Follow-up Period 4 weeks later. Participants who prematurely discontinue study treatment during the Maintenance Period will have an ET Visit after the last dose of study intervention or until Week 96, whichever is earlier, and then enter a 48-week Follow-up Period. For Cohorts 2a, 2b1, 2b2, 2c, and 3, this includes the Screening Period (up to 6 weeks) and the Treatment Period (Weeks 48 to 96). Participants who prematurely discontinue study treatment will have an ET Visit after the last dose of study intervention or until Week 96, whichever is earlier, and then enter a 48-week Follow-up Period. For cohort 4, this included a screening period (up to 6 weeks) and a 12-week treatment delay (while continuing to receive NRTIs).

[0730] All participants will be screened for up to 42 days.

[0731] The treatment period will consist of 2 phases: an induction phase and a maintenance phase. Participants in cohorts 1a and 1b will receive 3 doses of the study treatment during the induction phase (Day 1, Week 4, and Week 8). If the combined endpoint is reached at the Week 12 visit (HDV RNA < LOD or HDV RNA decreased by ≥2 log 10 from baseline and ALT < ULN), they may proceed to the maintenance phase for an additional 84 weeks. Participants who do not meet the combined endpoint at Week 12 may enter the follow-up period at Week 16 or Day 1 of cohort 2c (must meet the I / E criteria for cohort 2 at Week 12). Cohort 1a and 1b participants whose HDV RNA rebounds within 2 log10 IU / mL of its baseline during the maintenance phase will receive SIRNA01 or AB01 every 4 weeks until Week 92. In these cases, participants should follow the activities in the SoA for cohort 2a or 2b (see FIG. 15A to FIG. 15D , as well as FIG. 16A to FIG. 16D ), where the next dose of the drug will be given within 4 weeks. Participants in cohort 2a (SIRNA01 monotherapy), cohort 2b1 or 2b2 (AB01 monotherapy), and cohort 2c (SIRNA01 + AB01 combination therapy) will receive the study treatment once monthly during the treatment period for up to 96 weeks. Cohort 2a participants who do not reach the combined endpoint at Week 48 may enter the follow-up period or start the combination therapy with AB01 added to SIRNA01 at the Week 52 visit and follow the investigational product (IP) administration protocol in the SoA for cohort 2c from Week 52 to Week 96 ( FIG. 16A to FIG. 16D ). Cohort 2b1 or 2b2 participants who do not reach the combined endpoint at Week 48 may enter the follow-up period or start the combination therapy with SIRNA01 added to AB01 at the Week 52 visit and follow the IP administration protocol in the SoA for cohort 2c from Week 52 to Week 96 ( FIG. 16A to FIG. 16D ). Cohort 2b1 participants who do not achieve at least a 1 log 10 reduction in HDV RNA at Week 24 may start the combination therapy and follow the IP administration protocol in the SoA for cohort 2c from Week 28 to Week 96 ( FIG. 15A to FIG. 15D , as well as FIG. 16A to FIG. 16D ). Enrolled de novo participants in cohort 2c who do not meet the combined endpoint at Week 48 will discontinue the study treatment and enter the follow-up period. Participants in cohort 3 will receive AB01 monotherapy every two weeks during the treatment period until Week 96. Participants who do not reach the combined endpoint at Week 48 may enter the follow-up period or start the combination therapy with SIRNA01 added to AB01 and follow the IP administration protocol in the SoA for cohort 2c from Week 52 to Week 96 ( FIG. 16A to FIG. 16D ). Participants who do not achieve at least a 1 log reduction in HDV RNA at Week 2410 IU / mL, could start combination therapy and follow the IP dosing schedule in the Cohort 2c SoA from Week 28 to Week 96 ( FIG. 15A to FIG. 15D ,as well as FIG. 16A to FIG. 16D Participants in cohort 4 will have treatment delayed for 12 weeks and continue with standard NRTI therapy, after which they will be reassigned to cohort 2c.

[0732] The maximum duration of the follow-up period is 48 weeks or week 96 after the last dose of study intervention, whichever is earlier. Participants will enter the follow-up period if they meet the following criteria: are enrolled in Cohort 1 and do not cross over to the maintenance phase after meeting the combined endpoint at Week 12; are enrolled in Cohort 1 and do not cross over to Cohort 2c after failing to meet the combined endpoint at Week 12; are enrolled in Cohorts 2a, 2b1, or 2b2 and 3 and do not cross over to combination therapy after failing to meet the combined endpoint at Week 48; are enrolled in Cohorts 2b1 or 3 and do not cross over to combination therapy after meeting virologic nonresponse criteria at Week 24; are enrolled in Cohort 2c and do not meet the combined endpoint at Week 48; fail to meet the combined endpoint at Week 48 after crossing over from Cohort 4 to Cohort 2c; or are enrolled in any cohort and prematurely discontinue study medication. Participants who continue on the study until Week 96 will not receive additional follow-up.

[0733] The optional liver biopsy substudy will be conducted in selected countries and research centers (at available locations and times). All participants enrolled in Cohort 2 from the beginning at the selected research center will be eligible to participate in this substudy. The target enrollment is approximately 18 participants across all arms in Cohort 2. Pretreatment liver tissue samples will be collected during the screening window or until Week 2 of the study. If the participant has had a liver biopsy within the previous 12 months, a tissue block is available for research purposes and is considered able to be used, the sample can be used as a pretreatment sample. Follow-up liver tissue samples will be collected at approximately Week 48 ± 2 weeks and / or Week 96 ± 2 weeks of treatment. This tissue sample will be used to directly assess changes in liver fibrosis during treatment with study drugs. The sample will also be used to assess assays for HBV and HDV replication in hepatocytes, as well as exploratory studies.

[0734] Participants in Cohorts 2b1 or 2b2 and 2c with baseline HBsAg > 3000 IU / mL may participate in the optional AB01 PK substudy. This substudy will include up to 2 additional study visits to collect AB01 PK samples. The first visit will be 5 to 7 days after the third, fourth, or fifth dose of AB01, and the second optional visit will be 5 to 7 days after the seventh, eighth, or ninth dose of AB01. Participants who enter Cohort 2c from Cohorts 1a or 1b will be excluded from this substudy. In addition to PK, HBsAg, HDV RNA, HBV DNA, and liver function tests will also be collected at the same visits. This optional PK substudy will be closed after 30 participants are enrolled in this substudy.

[0735] The optional liver fine needle aspirate substudy will be conducted at selected UK sites. All participants enrolled de novo in cohorts 2bl / 2b2, 2c and 3 at the selected sites will be eligible for this substudy. The target enrollment is approximately up to 10 participants in total. Liver FNA and peripheral blood mononuclear cell (PBMC) samples will be collected at pretreatment, week 24 ± 2 weeks, and optionally at any visit between week 48 and week 96. This FNA substudy will provide information on changes in the intrahepatic environment of participants receiving a combination of siRNA and mAb targeting HDV replication, as well as detailed immunological and virological data.

[0736] AB01 is provided as a reconstituted lyophilized powder administered subcutaneously (SC) at 300 mg every 4 weeks or every 8 weeks (see Table 6). AB01 (HBC34v35-MLNS-GAALIE) comprises a light chain amino acid sequence of SEQ ID NO: 59 and a heavy chain amino acid sequence of SEQ ID NO: 57. SIRNA01 is provided as a liquid administered SC at 200 mg every 4 weeks or every 8 weeks (see Table 6). SIRNA01 has a sense strand comprising the nucleotide sequence of SEQ ID NO: 5 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 6.

[0737] The study objectives and relevant endpoints are shown in Table 7.

[0738] Table 7. Study objectives and relevant endpoints.

[0739]

[0740]

[0741]

[0742] The Child-Pugh Turcotte (CPT) score estimates for liver disease are shown in Table 8.

[0743] Table 8. Child-Pugh Turcotte (CPT) score estimates for liver disease.

[0744]

[0745] *Level 0: Consciousness, personality, neurological examination, and EEG are all normal

[0746] Grade 1: restlessness, sleep disturbance, irritability / agitation, tremor, impaired writing ability, EEG waveform 5 cps

[0747] Grade 2: Lethargy, time disorientation, abnormal behavior, asterixis, ataxia, slow triphasic EEG waves

[0748] Grade 3: drowsiness, coma, disorientation, hyperreflexia, rigidity, slow EEG waves

[0749] Grade 4: Coma, unable to be awakened, no personality / action, no brain reflexes, slow 2-3 cps delta wave activity on EEG Source: FDA 2003

[0750] Assessment: If the score is 5 or 6, it is Class A (mild liver damage); if the score is 7 to 9, it is Class B (moderate liver damage); if the score is 10 to 15, it is Class C (severe liver damage).

[0751] The ULN value of ALT may be, for example, 34 IU / mL for females and 43 IU / mL for males.

[0752] Will be like FIG. 21A to FIG. 21G PK samples will be collected as in the above method, and then the PK parameters (free PK and total PK, when applicable) of SIRNA01 and AB01 will be calculated. Parameters may include but are not limited to C max , C last 、T max 、T last , AUC inf , AUC last , %AUC exp ,t 1 / 2 , z 、V z / F and CL / F.

[0753] Immunogenicity data may include, but are not limited to, the presence / absence and titer of anti-drug antibodies (ADA), and neutralization data.

[0754] Participant Groups

[0755] This study will enroll male and female participants aged 18 to 70 years with chronic HBV / HDV co-infection, both non-cirrhotic and cirrhotic, both up to METAVIR-F4 / CPT-A, currently receiving NRTI therapy. For the purposes of this study, "chronic HBV infection" is defined as 2 positive serum HBsAg, HBV DNA, or HBeAg at least 6 months apart based on previous (within the past 12 months) or current laboratory records (any combination of these tests performed 6 months apart is acceptable). Participants will be receiving locally approved NRTI therapy for at least 12 weeks prior to Day 1. Participants will have HBsAg > 0.05 IU / mL at screening; positive HDV antibodies for at least 6 months prior to screening and HDV RNA ≥ 500 IU / mL at screening; serum alanine aminotransferase (ALT) > ULN and < 5× ULN.

[0756] Participants were aged ≥18 years (or the legal age of consent, whichever is greater) to <70 years at screening. Participants had a body mass index (BMI) ≥18 kg / m 2 To ≤40kg / m 2 .

[0757] Additional inclusion criteria included the following:

[0758] ●Female participants must have a negative pregnancy test or confirmed postmenopausal status. Postmenopausal status is defined as the absence of menstruation for 12 months without an alternative medical reason. Women of childbearing potential (WOCBP) must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, must not be breastfeeding, and must be willing to use a highly effective contraceptive method from 14 days prior to study drug administration until 48 weeks after the last dose of SIRNA01 or AB01. Female participants must also agree not to donate eggs and undergo in vitro fertilization from the time of study drug administration until 48 weeks after the last dose of SIRNA01 or AB01.

[0759] ● Male participants with female partners of reproductive potential must agree to meet one of the following contraceptive requirements from the time of study drug administration until 48 weeks after the last dose of SIRNA01 or AB01: vasectomy or documentation of azoospermia, or use of male condoms with their partner using one of the contraceptive options listed for WOCBP contraception. Male participants must also agree not to donate sperm from the time of the first study drug administration until 48 weeks after the last dose of SIRNA01 or AB01.

[0760] ●Able to sign informed consent

[0761] ●12-lead electrocardiogram (ECG) within normal limits; or, as determined by the clinical investigator, no clinically significant abnormalities at screening.

[0762] Agree not to take any medication during the study and for 3 months after the last dose of study medication.

[0763] Donate blood.

[0764] Cohort 1 Specific Inclusion Criteria

[0765] Non-cirrhotic

[0766] ○ Liver biopsy with METAVIR grade F0-F3 within 12 months before screening or liver elastography (eg, )<12 kPa

[0767] ○ Creatinine clearance (CLcr) calculated by the Cockcroft-Gault formula at screening ≥ 30 mL / min

[0768] ○ Platelet count >150,000 cells / mm 3 ( / μL)

[0769] Specific inclusion criteria for cohorts 2a, 2b1, 2b2, 2c, 3, and 4

[0770] Non-cirrhotic

[0771] ○ Liver biopsy with METAVIR grade F0-F3 within 12 months before screening or liver elastography (eg, )<12kPa

[0772] ○ CLcr ≥ 30 mL / min calculated by Cockcroft-Gault formula at screening

[0773] ○ Platelet count >150,000 cells / mm 3 ( / μL)

[0774] CPT-A cirrhosis

[0775] ○ Liver biopsy with METAVIR grade F4 within 12 months before screening or liver elastography (eg, )≥12kPa

[0776] ○ CLcr ≥ 60 mL / min calculated by the Cockcroft-Gault formula at screening

[0777] ○ Platelet count > 90,000 cells / mm 3 ( / μL)

[0778] ○ CPT score of 5 or 6 at screening and study entry

[0779] ●You can use An alternative approach is, for example, 2D shear wave elastography.

[0780] Exclusion criteria included the following :

[0781] ●History of clinically significant liver disease from non-HBV and non-HDV etiologies

[0782] History of clinically significant immune complex disease

[0783] ● History of clinically significant autoimmune disease

[0784] History of HBV-related extrahepatic disease, including but not limited to HBV-related rash, arthritis, or glomerulonephritis

[0785] ● History of allergic reaction, hypersensitivity, or intolerance to the study drug, its metabolites, or excipients

[0786] Anti-HB >10 mIU / mL at screening

[0787] Corrected QT interval (QTc) > 450 milliseconds

[0788] ALT or AST ≥5 × ULN

[0789] Total bilirubin >2.0 mg / dL

[0790] Serum albumin <30 g / L

[0791] Absolute neutrophil count <1000 cells / mm 3 ( / μL)

[0792] International normalized ratio (INR) >1.5

[0793] Hemoglobin <8 g / dL

[0794] History of allergic reactions

[0795] ●History of malignancy diagnosed or treated within 5 years (topically treated squamous or noninvasive basal cell skin cancer permitted; cervical cancer in situ permitted if appropriately treated prior to screening); participants who underwent evaluation for malignancy were ineligible

[0796] History of bone marrow or solid organ transplantation or listed as having undergone a bone marrow or solid organ transplant

[0797] ●A known active infection other than chronic HBV and HDV infection or any clinically significant acute condition such as fever (>38°C) or acute respiratory illness within 7 days prior to Day 1

[0798] ● Co-infection with human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), or hepatitis E virus (HEV); participants who are HCV antibody-positive and HCV RNA-negative are eligible; participants who are HAV or HEV immunoglobulin M antibody (IgM)-positive are not eligible. Participants who are asymptomatic and HAV or HEV immunoglobulin G antibody (IgG)-positive are eligible

[0799] Any clinically significant medical or psychiatric condition that may interfere with study interventions, assessments, or compliance with the protocol, or otherwise render the participant unsuitable for participation in this study; participants with controlled diabetes are eligible

[0800] ●Acute or worsening chronic hepatitis, fluctuating or rapidly worsening liver function, or use of any therapy that has exacerbated liver dysfunction

[0801] ● History or clinical evidence of alcohol or drug abuse within 12 months prior to screening, or a positive drug screen result at screening unless explained by prescription medication; marijuana use is permitted ● Participants need to pierce more than once a month

[0802] ● Participants have refractory encephalopathy or severe central nervous system disease

[0803] ● History of gastric or esophageal varicose bleeding within the past 6 months

[0804] ●Participants had undergone transjugular intrahepatic portosystemic shunt (TIPS)

[0805] Presence of hepatopulmonary or hepatorenal syndrome

[0806] Presence of primary cholestatic liver disease

[0807] Inability or unwillingness to follow dietary recommendations for cirrhosis and liver damage

[0808] Concomitant therapy

[0809] The following concomitant therapies were not permitted during the study:

[0810] ●Treatment with immunomodulators, interferon-alpha (eg, IFN-alpha-2a or IFN-alpha-2b, or pegylated IFN-alpha-2a or alpha-2b), immunosuppressants (eg, disease-modifying antirheumatic drugs), cytotoxic or chemotherapeutic agents, or chronic systemic corticosteroids within six months of screening

[0811] ● Received an HDV active agent (including lonafamib and brevitide) within 90 days or 5 half-lives (if known) before administration of the study drug, whichever is longer, or an HDV active agent is active during the follow-up period of another clinical study involving interventional therapy; participants must also agree not to participate in any other interventional study at any time during participation in this study (including the follow-up period)

[0812] ●Receipt of oligonucleotides with anti-HBV or anti-HDV activity (eg, siRNA, antisense oligonucleotides) within 48 weeks prior to first dose of study drug

[0813] ●Received AB01 or any antibody targeting HBV or HDV within 24 weeks of first study drug administration

[0814] Participants must avoid taking prescription medications other than those prescribed by their prescribing physician for 7 days (14 days if the medication is a potential enzyme inducer) or 5 half-lives (whichever is longer) before the start of the study intervention, as well as over-the-counter medications (including vitamins, recreational drugs, and dietary or herbal supplements) until the end of follow-up unless the medication will not interfere with the study

[0815] Participants with liver impairment who have comorbid conditions requiring medication must have been taking medication without a change in dose for more than 3 months prior to screening

[0816] ● Any of the following systemic medications were prohibited within 14 days prior to study drug administration and throughout the study: chronic systemic steroids (prednisone equivalents > 10 mg / day) or other immunosuppressants (note: corticosteroids were allowed for immune-mediated AEs, and short-term corticosteroids ...

Claims

1. A method of treating hepatitis delta virus (HDV) infection or HDV-related disease in a subject in need thereof, comprising administering to the subject: (a) anti-HBV antibodies; and (b) siRNA targeting HBV mRNA; wherein the subject has a Child-Pugh-Turcotte (CPT) score of 5 or higher before treatment.

2. The method according to claim 1, wherein the HDV-related disease is chronic hepatitis; Acute hepatitis D; Acute fulminant hepatitis D; Chronic hepatitis D; Liver fibrosis; End-stage liver disease; or hepatocellular carcinoma.

3. The method of claim 1, wherein the subject suffers from chronic hepatitis B virus (HBV) / HDV co-infection.

4. The method according to claim 1, wherein the subject has 2 positive serum HBsAg, HBV DNA or HBeAg tests at least 6 months apart before treatment.

5. The method of claim 1, wherein the subject has tested positive for HDV antibodies or HDV RNA prior to treatment.

6. The method of claim 1, wherein the subject has tested positive for HDV antibodies and HDV RNA prior to treatment.

7. The method of claim 1, wherein the subject has tested positive for HDV antibodies for at least 6 months prior to treatment.

8. The method of claim 1, wherein the subject has HDV RNA>500 IU / mL before treatment.

9. The method of claim 1, wherein the subject's HBsAg level before treatment is >0.05 IU / mL.

10. The method of claim 1, wherein the subject has a HBsAg level > 10,000 IU / mL before treatment.

11. The method of claim 1, wherein the subject suffers from chronic hepatitis.

12. The method of claim 1, wherein the subject's alanine aminotransferase (ALT) level before treatment is > upper limit of normal (ULN) and < 5 times ULN.

13. The method of claim 1, wherein the subject has an ALT level > upper limit of normal (ULN) and an aspartate aminotransferase (AST) level > ULN before treatment.

14. The method of claim 1, wherein the subject has an ALT level <5 times ULN and an AST level <5 times ULN before treatment.

15. The method according to claim 1, wherein the subject has a liver biopsy METAVIR grade of F4 or liver elastography within 12 months before treatment. ≥12 kilopascals (kPa).

16. The method according to claim 1, wherein the subject's creatine clearance (CLcr) calculated by the Cockcroft-Gault formula before treatment is ≥ 60 mL / min.

17. The method of claim 1, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 5 or 6 before treatment.

18. The method of claim 1, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 7 or higher before treatment.

19. The method of claim 1, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 7 to 9 before treatment.

20. The method of claim 1, wherein the subject has a Child-Pugh-Turcotte (CPT) score of ≥10 before treatment.

21. The method of claim 1, wherein the subject has not been previously administered anti-HIV antibodies or siRNA targeting HBV mRNA.

22. The method of claim 1, wherein the anti-HBV antibody is a human antibody.

23. The method of claim 1, wherein the antibody is HBC34, or a non-natural variant of HBC34.

24. The method of claim 1, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NO:44, SEQ ID NO:45 or 46, and SEQ ID NO:47, respectively; and (ii) the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NO: 48, SEQ ID NO: 49 or 50, and SEQ ID NO: 52, respectively.

25. The method of claim 24, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

26. The method of claim 24, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 46 and 47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 50 and 52, respectively.

27. The method of claim 1, wherein the anti-HBV antibody comprises: (a) a light chain variable domain (VL) that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:55; and (b) a heavy chain variable domain (VH) that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:

53.

28. The method of claim 1, wherein the anti-HBV antibody comprises: (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:59, and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:

57.

29. The method of claim 1, wherein the siRNA 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, wherein T is replaced by U.

30. The method of claim 1, wherein the antisense strand of the siRNA comprises or consists of the nucleotide sequence 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

31. The method of claim 30, wherein the sense strand of the siRNA comprises or consists of the nucleotide sequence 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

32. The method of claim 31, wherein at least one strand of the siRNA comprises a 3' overhang of at least 1 nucleotide.

33. The method of claim 31, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs in length.

34. The method of claim 31, wherein each strand of the RNAi agent has 15 to 30 nucleotides.

35. The method of claim 31, wherein substantially all of the nucleotides of the sense strand of the siRNA and substantially all of the nucleotides of the antisense strand of the siRNA are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3'-end.

36. The method of claim 35, wherein the ligand is one or more GalNAc derivatives attached via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

37. The method of claim 36, wherein the ligand is 38. The method of claim 37, wherein the siRNA is conjugated to the ligand as shown in the following structure: Wherein X is O or S.

39. The method of claim 38, wherein X is O.

40. The method of claim 31, wherein at least one nucleotide of the siRNA is a modified nucleotide comprising: deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, adenosine-diol nucleic acids, or nucleotides containing 5'-phosphate mimetics.

41. The method of claim 31, wherein the siRNA comprises a phosphate backbone modification, a 2' ribose modification, a 5' triphosphate modification, or a GalNAc conjugation modification.

42. The method of claim 31, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand of the siRNA are modified nucleotides.

43. The method of claim 1, wherein the siRNA comprises: a sense strand having 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand having 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), 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; Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (Agn) is adenosine diol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

44. The method of claim 43, wherein the L96 is conjugated to the sense strand as shown in the following structure: Where X is O.

45. The method of claim 1, further comprising administering a nucleoside reverse transcriptase inhibitor (NRTI) to the subject.

46. ​​The method of claim 1, wherein the subject has been previously administered an NRTI.

47. The method of claim 45, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil (TD), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, dipivoxil, lobcavir, famciclovir, N-acetylcysteine ​​(NAC), PC1323, Theradigm-HBV, thymosin-alpha and ganciclovir, besifovir (ANA-380 / LB-80380) or tenofovir-exaliade (TLX / CMX157).

48. The method of claim 45, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), or tenofovir disoproxil (TD).

49. The method of claim 1, wherein: (a) the anti-HBV antibody comprises or consists of: a light chain amino acid sequence according to SEQ ID NO: 59 and a heavy chain amino acid sequence according to SEQ ID NO: 57; and (b) the siRNA comprises or consists of the following parts: a sense strand comprising or consisting of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand comprising or consisting of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), 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; Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (Agn) is adenosine diol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

50. The method of claim 45, wherein: (a) the anti-HBV antibody comprises or consists of: a light chain amino acid sequence according to SEQ ID NO: 59 and a heavy chain amino acid sequence according to SEQ ID NO: 57; (b) the siRNA comprises or consists of the following parts: a sense strand comprising or consisting of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and an antisense strand comprising or consisting of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), 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; Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (Agn) is adenosine diol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; and (c) The NRTI is tenofovir disoproxil fumarate (TDF) or entecavir.

51. The method of claim 1, wherein the anti-HBV antibody is administered once every 2 weeks, every 4 weeks, or every 8 weeks.

52. The method of claim 1, wherein the siRNA is administered once every 4 weeks or every 8 weeks.

53. The method of claim 45, wherein the NRTI is administered daily.

54. The method of claim 1, wherein the anti-HBV antibody is administered at a dose of 300 mg.

55. The method of claim 1, wherein the siRNA is administered at a dose of 200 mg.

56. The method of claim 45, wherein the NRTI is administered at a dose of 300 mg.

57. The method of claim 45, wherein the NRTI is administered at a dose of 245 mg.

58. The method of claim 1, wherein the subject begins administration of the siRNA and the anti-HBV antibody on the same day.

59. The method of claim 1, wherein the subject is administered the siRNA and the anti-HBV antibody for up to 96 weeks, 96 weeks, at least 96 weeks, or 96 weeks or longer.

60. The method of claim 1, wherein the subject is a human.

61. An anti-HBV antibody; and an siRNA targeting HBV mRNA; for use in the method according to any one of claims 1 to 60.

62. An anti-HBV antibody for use in the method of any one of claims 1 to 60; wherein the subject is also administered siRNA targeting HBV mRNA.

63. Use of an anti-HBV antibody; and a siRNA targeting HBV mRNA; in the manufacture of a medicament for use in the method according to any one of claims 1 to 60.

64. Use of an anti-HBV antibody in the manufacture of a first drug; and use of an siRNA targeting HBV mRNA in the manufacture of a second drug; wherein the first drug and the second drug are to be used in a combination therapy according to any one of claims 1 to 60.

65. An anti-HBV antibody; an siRNA targeting HBV mRNA; and an NRTI; for use in a method according to any one of claims 45 to 60.

66. Use of an anti-HBV antibody; an siRNA targeting HBV mRNA; and a NRTI; in the manufacture of a medicament for use in the method according to any one of claims 45 to 60.

67. Use of anti-HBV antibodies in the manufacture of a first drug; use of siRNA targeting HBV mRNA in the manufacture of a second drug; and use of NRTI in the manufacture of a third drug; wherein the first drug, the second drug and the third drug are to be used in a combination therapy according to any one of claims 45 to 60.

68. A kit comprising: A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient; as well as A pharmaceutical composition comprising siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient.

69. A kit comprising: A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient; A pharmaceutical composition comprising siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient; as well as A pharmaceutical composition comprising a NRTI and a pharmaceutically acceptable excipient.

Images