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

Abstract

The present disclosure provides methods for treating hepatitis D virus (HDV) infection and / or HDV-related diseases using combination therapies, as well as related kits and compositions for use in such methods. The components of the combination therapy can include one or more of an anti-HBV antibody, an siRNA targeting HBV mRNA, and a nucleos(t)ide reverse transcriptase inhibitor (NRTI).

Description

Technical Field

[0001] Reference to the Electronic Sequence Listing The content of the electronic sequence listing (444WO_SeqListing.xmL; size: 70.5 KB; creation date: May 5, 2023) is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] Hepatitis D, also known as "delta hepatitis", is a viral infection caused by the hepatitis D virus (HDV). HDV is a defective RNA antigen that does not encode its own envelope protein and depends on the expression of the hepatitis B virus (HBV) surface antigen (HBsAg) to complete its life cycle and produce infectious HDV virions. Therefore, HDV needs to coinfect or superinfect with HBV.

[0003] 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 modeling study, Lancet Gastroenterol Hepatol. June 3, 2018(6): 383-403) and are at risk of serious sequelae including cirrhosis, liver failure, and hepatocellular carcinoma (HCC). The global prevalence of HDV has been 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. September 2020, 73(3): 523-532) to 60-72 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. April 27, 2020, 221(10):1677-1687). HDV infection is the most aggressive form of hepatitis D due to the rapid progression to liver failure, cirrhosis, HCC, and death observed in people with chronic HBV / HDV coinfection (Lee AU and Lee C, Viral Review: Challenges for the Resource-Poor Setting, Viruses. September 23, 2021, 13(10):1912; Stockdale et al., 2020, supra).Among people with chronic HDV infection, 85–95% may develop cirrhosis and liver failure within 10 years of infection, and some (about 15%) develop these complications as early as 1–2 years after infection (Kamili S et al., Delta hepatitis: Toward improved diagnostics, Hepatology, December 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. July 1, 2015, 5(7), a021576; WHO, Hepatitis B, https: / / www.who.int / news-room / factsheets / detail / hepatitis-b, published July 2021, accessed April 10, 2022). In particular, about 50% of patients with HDV infection are cirrhotic 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. May 1987, 155(5): 931–5).

[0004] 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) and has four open reading frames encoding seven proteins: HBcAg (HBV core antigen, viral capsid protein), HBeAg (hepatitis B e antigen), HBV Pol / RT (polymerase, reverse transcriptase), preS1 / preS2 / HBsAg (large, medium, and small surface envelope glycoproteins), and HBx (HBVx antigen, a transcriptional regulatory factor required for the initiation of 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).

[0005] In hepatocytes, the rcDNA, which is the form of HBV nucleic acid introduced by the infecting virion, is converted into covalently closed circular DNA (cccDNA), which persists in the host cell nucleus as an episomal chromatinized structure (Allweiss L et al., The Role of cccDNA in HBV Maintenance, Viruses 2017, 9:156). The cccDNA functions as the transcription template for all viral transcripts (Lucifora J et al., Attacking hepatitis B virus cccDNA - The holy grail to hepatitis B cure, Journal of Hepatology 2016, 64(1): S41 - S48). The pregenomic RNA (pgRNA) transcript is reverse transcribed into new rcDNA for new virions, which is secreted without causing cytotoxicity. In addition to infectious virions, infected hepatocytes secrete large amounts of genome - free subviral particles that can exceed the number of secreted virions by more than 10,000 - fold (Seeger et al., 2015, supra). Random integration of the virus into the host genome can also occur, which is a mechanism contributing to hepatocyte transformation (Levrero M et al., Mechanisms of HBV - induced hepatocellular carcinoma, Journal of Hepatology 2016, 64(1): S84 - S101). HBV persists in hepatocytes in the form of cccDNA and integrated DNA (intDNA).

[0006] Hepatitis B infection is characterized by serological viral markers and antibodies. In acute resolving infection, the virus is cleared by effective innate and adaptive immune responses, including the induction of cytotoxic T cells that cause the death of infected hepatocytes and B cells that 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 dysfunction of T and B cells mediated by multiple regulatory mechanisms, including the presentation of viral epitopes on hepatocytes and the secretion of subviral particles (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 thought to be an important step in the host's inability to eliminate the infection.

[0007] Treatment options for HDV infection are limited to peginterferon alpha (PEG-IFNα) and bulevirtide. PEG-IFNα results in sustained virological response (SVR (clearance of serum HDV maintained 6 months after treatment discontinuation)) in only about 25 - 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, 201 l(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 administering PEG-IFNa to patients with chronic HDV infection for at least 48 weeks (European Association for the Study of the Liver (EASL), EASL 2017 Clinical Practice Guidelines on the management of Hepatitis B virus infection. J. Hepatol. August 2017, 67(2): 370-398; Sarin SK et al., Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update, Hepatol Int. January 2016, 10(1):1-98; Terrault NA et al., Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance, Hepatology April 2018, 67(4):1560-1599).In addition to the low SVR rate, the major adverse reactions of PEG-IFNα therapy have been well documented, and PEG-IFNα is contraindicated in patients with autoimmune diseases, patients with major psychiatric syndromes, and patients with hepatitis D in Child-Pugh-Turcotte (CPT)-B or CPT-C stage (Rizzetto M, Cirrhosis Virus: Introduction and Epidemiology, Cold Spring Harb Perspect Med. July 1, 2015, 5(7): a021576; Sleijfer S et al., Side effects of interferonine alpha therapy, Pharm World Sci. December 2005, 27(6): 423-31). As a further limitation of its usefulness, a decrease in the efficacy of PEG-IFNa has been observed when treating cirrhotic patients with chronic HDV (Gunsar F et al., Two-year interferon therapy with or without ribavirin in chronic delta hepatitis, Antivir Ther. 2005, 10(6): 721-6).

[0008] The limitations regarding the use of PEG-IFNα in cirrhotic HDV patients are of particular interest because 50% of HDV-infected patients are cirrhotic at the time of diagnosis (Fattovich et al., 1987, supra). These limitations and the insufficient efficacy of current agents highlight the unmet need for patients with chronic HDV infection at all stages of the disease. Brevibulstatin (BLV), an entry inhibitor targeting the taurocholate sodium cotransporting polypeptide (NTCP) receptor, has conditional approval in the EU for the treatment of chronic HDV. Interim results from the Phase 3 MYR301 trial indicate that 36.7% of participants achieved a combination of virological and biochemical endpoints after 24 weeks of a subcutaneous (SC) dose of 2 mg of BLV per day. Nucleoside reverse transcriptase inhibitor (NRTI) therapy can suppress HBV replication but does not directly affect HBsAg production, HDV replication in the liver, or HDV viremia.

Summary of the Invention

[0009] In some aspects, the present disclosure provides a method of treating hepatitis D virus (HDV) infection or an HDV-related disease in a subject in need thereof, the method comprising administering to the subject (a) an anti-HBV antibody and (b) an siRNA targeting HBV mRNA. In some embodiments, the subject is cirrhotic, e.g., having METAVIR F4 or a liver elastography (Fibroscan®) of 12 kilopascals (kPa) or more within 12 months prior to treatment; a creatinine clearance (CLcr) of 60 mL / min or more calculated by the Cockcroft-Gault formula prior to treatment; and a liver biopsy with a Child-Pugh-Turcotte (CPT) score of 5 or more prior to treatment. In some aspects, compositions for use in treatment, compositions for use in the manufacture of a medicament, and kits are provided.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] The present disclosure provides methods and compositions for use in the treatment of hepatitis D virus (HDV) infection or HDV-related diseases, wherein one or more of an anti-HBV antibody and an anti-HBV siRNA are administered to a subject, as well as related kits.

[0012] I. Terms The following sections provide a detailed description of combination therapies for treating HDV infection or HDV-related diseases, and kits related to the combination therapies. Before describing the present disclosure in more detail, it may be helpful to provide definitions of certain terms used herein. Additional definitions are set forth throughout the present disclosure.

[0013] As used herein, the term “about” means +20% of the indicated range, value, or structure, unless otherwise indicated.

[0014] The term “comprise” (and similar terms such as “comprising of” and “comprised of”) means the presence of the recited features, integers, steps, or components recited in the claims, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. The term “consisting essentially of” limits the claims to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0015] It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the recited components. The use of an alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives, and may be used synonymously with “and / or”. As used herein, the terms “include” and “have” are used synonymously, and these terms and their variations are intended to be interpreted in a non-limiting or open-ended manner.

[0016] The term "substantially" does not exclude "completely". For example, a composition "substantially free of" Y may be completely free of Y. If necessary, the term "substantially" may be omitted from the definitions provided herein.

[0017] The term "disease", as used herein, is intended to be generally synonymous with the terms "disorder" and "condition" (as in "medical condition") and is used interchangeably, all reflecting an abnormal condition of one or more of the body or a part thereof of a human or animal that impairs normal function, typically manifested by characteristic signs and symptoms, and that reduces the lifespan or quality of life of a human or animal.

[0018] As used herein, the terms "peptide," "polypeptide," and "protein," and variations of these terms, each refer to a molecule, particularly a peptide, oligopeptide, polypeptide, or protein (including fusion proteins), which contain at least two amino acids linked to each other by normal peptide bonds or modified peptide bonds (e.g., in the case of isosteric peptides). For example, a peptide, polypeptide, or protein can be composed of amino acids selected from the 20 amino acids defined by the genetic code, linked to each other by normal peptide bonds ("classical" polypeptides). Peptides, polypeptides, or proteins can be composed of L-amino acids and / or D-amino acids. In particular, the terms "peptide," "polypeptide," and "protein" also include "peptidomimetics," which are defined as peptide analogs containing non-peptidic structural elements that can mimic or antagonize the biological action(s) of the native parent peptide. Peptidomimetics lack classical peptide properties such as enzymatically cleavable peptide bonds. In particular, a peptide, polypeptide, or protein may, in addition to these amino acids, contain amino acids other than the 20 amino acids defined by the genetic code, or may be composed of amino acids other than the 20 amino acids defined by the genetic code. In particular, a peptide, polypeptide, or protein in the context of the present disclosure can equally be composed of amino acids modified by natural processes such as post-translational maturation processes well known to those skilled in the art, or by chemical processes. Such modifications are fully detailed in the literature. These modifications can occur anywhere in the polypeptide: in the peptide backbone, in the amino acid chain, or even at the carboxy or amino terminus. In particular, a peptide or polypeptide may be branched after ubiquitination, or may be cyclic, with or without branching. This type of modification can be the result of natural or synthetic post-translational processes well known to those skilled in the art. The terms "peptide," "polypeptide," or "protein" in the context of the present disclosure specifically also include modified peptides, polypeptides, and proteins.For example, peptide, polypeptide, or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including PEGylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, senoylation, sulfation, amino acid addition such as arginylation, or ubiquitination. Such modifications are fully detailed in the literature (Proteins Structure and Molecular Properties, Part 2, T.E. Creighton, New York (1993); Post-translational Covalent Modifications of Proteins, B.C. Johnson, ed., Academic Press, New York (1983); Seifter et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 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.

[0019] As used herein, "(poly)peptide" includes a single chain of amino acid monomers linked by peptide bonds as described above. "Protein", as used herein, when referring to 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 described above. In certain embodiments, the proteins according to the present disclosure include 1, 2, 3, or 4 polypeptides.

[0020] The term "recombinant", as used herein (e.g., recombinant antibody, recombinant protein, recombinant nucleic acid, etc.), refers to any molecule (antibody, protein, nucleic acid, siRNA, etc.) that is prepared, expressed, produced, or isolated by recombinant means and 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 or double-stranded. In certain embodiments, the nucleic acid molecule is double-stranded RNA.

[0021] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such names include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny having the same function or biological activity as that screened in the original transformed cell are included. Where different names are intended, it will be apparent from the context.

[0022] As used herein, the term "array variant" refers to any array having one or more modifications as compared to a reference array, where the reference array is any of the arrays listed in the sequence listing, i.e., SEQ ID NO: 1 to SEQ ID NO: 61. Thus, the term "array variant" includes nucleotide sequence variants and amino acid sequence variants. For array variants in the context of nucleotide sequences, the reference array is also a nucleotide sequence, while for array variants in the context of amino acid sequences, the reference array is also an amino acid sequence. An "array variant" as used herein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the reference array. Sequence identity is usually calculated with respect to the full length of the reference array (i.e., the sequences listed in this application), unless otherwise specified. The percentage of identity, when referred to herein, can be determined, for example, using BLAST with the default parameters defined by NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap open penalty = 11 and gap extension penalty = 1]. A "sequence variant" in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more nucleotides in the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one-letter notation (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 respective reference amino acid sequence, i.e., a change in an amino acid "sequence variant". In certain embodiments, a nucleotide sequence variant is a variant that does not result in an amino acid sequence variant (i.e., a silent mutation).However, nucleotide sequence variants that result in "non-silent" mutations, particularly nucleotide sequence variants that result in amino acid sequences that are 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, are also within the scope. A "sequence variant" in the context of an amino acid sequence has an altered sequence in which one or more amino acids are deleted, substituted, or inserted, as compared to the reference amino acid sequence. As a result of the modification, such sequence variants have amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having 10 or fewer modifications, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids of the reference sequence is "at least 90% identical" to the reference sequence.

[0023] It is possible to have non-conservative amino acid substitutions, although in certain embodiments the substitutions are conservative amino acid substitutions, and the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the reference sequence. As examples, conservative amino acid substitutions include substitution of one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, with another, substitution of one hydroxyl-containing amino acid, such as serine and threonine, with another, substitution of one acidic residue, such as glutamic acid or aspartic acid, with another, substitution of one amide-containing residue, such as asparagine and glutamine, with another, substitution of one aromatic residue, such as phenylalanine and tyrosine, with another, substitution of one basic residue, such as lysine, arginine, and histidine, with another, and substitution of one small amino acid, such as alanine, serine, threonine, methionine, and glycine, with another.

[0024] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of lengths ranging from 1 residue to polypeptides containing 100 or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusions of amino acid sequences to the N- or C-terminus of a reporter molecule or an enzyme.

[0025] Unless otherwise specified, changes in sequence variants do not abrogate the functionality of the respective reference sequences, e.g., in the case of the present invention, the functionality of anti-HBV antibodies or siRNA sequences that sufficiently neutralize HBV infection or reduce HBV protein expression, respectively. Guidance on which nucleotide and amino acid residues can be substituted, inserted, or deleted without impairing such functionality can be found by using computer programs well known in the art.

[0026] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide, or protein refers to the origin of the nucleic acid, peptide, polypeptide, or protein. In some embodiments, a nucleic acid sequence or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to the sequence from which it is derived or a portion thereof, and "essentially identical" includes sequence variants as defined above. In certain embodiments, a nucleic acid sequence or amino acid sequence derived from a particular peptide or protein is derived from the corresponding domain in the particular peptide or protein. Here, "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). Thus, the "corresponding" portions of peptides, proteins, and nucleic acids are identifiable to those of ordinary skill in the art. Similarly, sequences "derived from" other sequences are generally identifiable by those of ordinary skill in the art as having their origin within the sequence.

[0027] In some embodiments, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein from which it is derived. However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide, or protein from which it is derived, and in particular, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a functional sequence variant as described above of the starting nucleic acid, peptide, polypeptide, or protein from which it is derived. For example, in a peptide / protein, one or more amino acid residues may be substituted with other amino acid residues, or insertions or deletions of one or more amino acid residues may occur.

[0028] As used herein, the term "mutation" refers to a change in a nucleic acid sequence and / or an amino acid sequence as compared to a reference sequence, e.g., a corresponding genomic sequence. A mutation can be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation as compared to a genomic sequence, an induced mutation induced by, e.g., an enzyme, a chemical, or radiation, or a mutation obtained by site-directed mutagenesis (a molecular biological method for creating specific and intentional changes in a nucleic acid sequence and / or an amino acid sequence). Thus, the term "mutation" or "mutating" is understood to include, for example, physically creating a mutation in a nucleic acid sequence or an amino acid sequence. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, the mutation can be introduced into the nucleotide sequence encoding the amino acid sequence in order to express a (recombinant) mutant polypeptide. A mutation can be achieved, for example, by modifying the codon of a nucleic acid molecule encoding one amino acid to result in a codon encoding a different amino acid, e.g., by site-directed mutagenesis, or by synthesizing a sequence variant, e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and designing the synthesis of a nucleic acid molecule containing a nucleotide sequence encoding a variant of the polypeptide without mutating one or more nucleotides of the nucleic acid molecule.

[0029] As used herein, the term "coding sequence" is intended to refer to a polynucleotide molecule that encodes the amino acid sequence of a protein product. The boundaries of a coding sequence are generally determined by an open reading frame that usually begins with an ATG start codon.

[0030] As used herein, the term "expression" refers to any process involved in the production of a polypeptide, including transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0031] As used herein, the term "vaccine" is understood to be a prophylactic or therapeutic material that provides at least one antigen or immunogen, typically including a viral vector vaccine containing a nucleic acid encoding an antigen(s) or immunogen(s). The antigen or immunogen can be derived from any material suitable for vaccination. For example, the antigen or immunogen can be derived from a pathogen, such as a bacterium or viral particle, or a tumor or cancerous tissue. The antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response. In particular, an "antigen" or "immunogen" is typically a substance that can be recognized by the immune system (e.g., the adaptive immune system) and can induce an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen can be or include a peptide or protein that can be presented to T cells by MHC.

[0032] As used herein, the term "hepatitis B virus," which is used interchangeably with the term "HBV," refers to a well-known non-cytopathic liver tropic DNA virus belonging to the Hepadnaviridae family. The HBV genome is a partially double-stranded circular DNA having four overlapping reading frames (which may be referred to herein as "genes," "open reading frames," or "transcripts"): C, X, P, and S. The core protein is encoded by gene C (HBcAg). The hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the precore (preC) protein. DNA polymerase is encoded by gene P. Gene S is the gene encoding the surface antigen (HBsAg). The HBsAg gene is a single long open reading frame containing three in-frame "start" (ATG) codons, resulting in three different sizes of polypeptides called the large, middle, and small S antigens, pre-S1+pre-S2+S, pre-S2+S, or S. The surface antigen, in addition to decorating the envelope of HBV, is also part of subviral particles produced in large excess compared to virion particles and plays a role in immune tolerance and sequestration of anti-HBsAg antibodies, thereby allowing infectious particles to escape immune detection. The protein encoded by gene X plays a role in transcriptional transactivation and replication and is associated with the development of liver cancer.

[0033] Nine genotypes of HBV, named A - I, have been determined, and an additional genotype J has been proposed, each having a distinct 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 of the genotypes (A - J) of HBV. The complete coding sequence of the reference sequence of the HBV genome can be found, for example, in GenBank accession numbers GI:21326584 and GI:3582357. The amino acid sequences of the C, X, P, and S proteins can be found, for example, in NCBI 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). Further examples of HBV messenger RNA (mRNA) sequences are available using publicly available databases such as GenBank, UniProt, and OMIM. The International Repository for Hepatitis B Virus Strain Data can be accessed at http: / / www.hpa - bioinformatics.org.uk / HepSEQ / main.php. The term "HBV" as used herein also refers to naturally occurring DNA sequence variations of the HBV genome, namely genotypes A - J and their variants.

[0034] In some embodiments, the present disclosure provides a combination therapy for treating HBV that includes an anti-HBV siRNA. siRNA mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, thereby resulting in the inhibition of gene expression. This process is often referred to as "RNA interference" (RNAi). Without wishing to be bound by a particular theory, long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al., 2001, 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19- to 23-base pair siRNAs with characteristic 2-base 3' overhangs (Bernstein et al., Nature 2001, 409:363). The siRNA is then incorporated into RISC, where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen et al., Cell 2001, 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., Genes Dev. 2001, 15:188).

[0035] Terms such as "silence", "inhibit the expression of", "downregulate the expression of", "suppress the expression of", as long as they refer to the HBV gene, herein refer to at least a partial decrease in the expression of the HBV gene, as indicated by a decrease in the amount of HBV mRNA that can be isolated or detected from a first cell or cell population that has been transcribed so that the expression of the HBV gene is inhibited and treated with an inhibitor of HBV gene expression, compared to a second cell or cell population (control cells) that is substantially identical to the first cell or cell population but has not been so treated or has been treated. The degree of inhibition can be measured, for example, as the difference between the degree of mRNA expression in the treated cells minus the degree of mRNA expression in the control cells. Alternatively, the degree of inhibition can be given in terms of a parameter that is functionally related to HBV gene expression, such as the amount of protein encoded by the HBV gene, or a decrease in the number of cells exhibiting a particular phenotype, such as the HBV infection phenotype. In principle, HBV gene silencing can be determined by any appropriate assay in any cell that expresses the HBV gene, such as an HBV-infected cell, or a cell that has been modified to express the HBV gene.

[0036] The level of HBV RNA expressed by a cell or cell population, or the level of circulating HBV RNA, can be determined using any method known in the art for assessing mRNA expression, such as the rtPCR methods provided in Example 2 of International Application Publication No. WO 2016 / 077321A1 and U.S. Patent Application Publication No. US2017 / 0349900A1, which are incorporated herein by reference. In some embodiments, the expression level of an HBV gene in a sample (e.g., total HBV RNA, HBV transcript, e.g., HBV 3.5 kb transcript) is determined by detecting the transcribed polynucleotide or a portion thereof, e.g., the RNA of the HBV gene. RNA can be extracted from cells by RNA extraction techniques including, for example, acid phenol / guanidinium isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen®), or PAXgene (PreAnalytix, Switzerland). Exemplary assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton D A 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 No. WO 2012 / 177906A1 and U.S. Patent Application Publication No. US2014 / 0275211A1, which are incorporated herein by reference.

[0037] As used herein, the term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a HBV gene, including the mRNA that is the product of RNA processing of the primary transcript. The target portion of the sequence is at least long enough to function as a substrate for RNAi-directed cleavage at or near that portion. For example, the target sequence is generally 9 to 36 nucleotides in length, such as 15 to 30 nucleotides in length, including all sub-ranges therebetween. By way of non-limiting example, the target sequence can be 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

[0038] As used herein, the term "strand containing a sequence" refers to an oligonucleotide that includes a strand of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0039] As used herein and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence with respect to a second nucleotide sequence, means that an oligonucleotide or polynucleotide containing the first nucleotide sequence is capable of hybridizing to an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions to form a double-stranded structure, as understood by those skilled in the art. Such conditions can be, for example, stringent conditions, where stringent conditions can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, and incubation at 50 °C or 70 °C for 12 - 16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered within an organism, can be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0040] The complementary sequences within the siRNAs described herein include base pairing of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence, over the full length of one or both nucleotide sequences. Such sequences can be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences can be fully complementary, or they can form one or more, but generally 5, 4, 3, or 2 or fewer mismatched base pairs upon hybridization for duplexes up to 30 base pairs while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via the RISC pathway. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches with respect to determination of complementarity. For example, an siRNA comprising one oligonucleotide of length 21 nucleotides and another oligonucleotide of length 23 nucleotides, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, can be referred to as "fully complementary" for the purposes described herein.

[0041] As used herein, "complementary" sequences can also include base pairs formed from non-Watson-Crick base pairs and / or non-natural nucleotides and modified nucleotides, or can consist entirely of non-Watson-Crick base pairs and / or non-natural nucleotides and modified nucleotides, provided that the above requirements regarding the ability to hybridize are met. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen-type base pairs.

[0042] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" can be used with respect 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 understood from the context of their use.

[0043] 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 an adjacent portion of the mRNA of interest (e.g., the mRNA encoding an HBV protein). For example, a polynucleotide is complementary to at least a portion of an HBV mRNA if its sequence is substantially complementary to an uninterrupted portion of the HBV mRNA.

[0044] As used herein, the term "siRNA" refers to an RNA interference molecule that includes an RNA molecule or a complex of molecules having a hybridized double-stranded region that includes two anti-parallel and substantially complementary nucleic acid strands that are said to have "sense" and "antisense" orientations with respect to a target RNA. The double-stranded region can be of any length that permits specific degradation of a desired target RNA via the RISC pathway, but is typically in the range of 9 to 36 base pairs in length, for example, in the range of 15 to 30 base pairs in length. Considering double-strands of 9 to 36 base pairs, the double-strand can be of any length within this range, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36, and any partial range length between these, 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 base pairs, 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 intracellularly by processing with Dicer and similar enzymes are generally in the range of 19 to 22 base pairs in length.

[0045] One strand of the double-stranded region of the siRNA contains a sequence that is substantially complementary to the region of the target RNA. The two strands forming the double-stranded structure can be derived from a single RNA molecule having at least one self-complementary region or can be formed from two or more separate RNA molecules. When the double-stranded region is formed from two strands of a single molecule, the molecule can have a double-stranded region separated by a single strand of nucleotides (referred to herein as a "hairpin loop") between the 3' end of one strand forming the double-stranded structure and the 5' end of each other strand. The hairpin loop can contain at least one unpaired nucleotide. In some embodiments, the hairpin loop can contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. When the two substantially complementary strands of the siRNA are constituted by separate RNA molecules, these molecules need not be covalently linked but can be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is called a "linker".

[0046] The siRNAs described herein can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0047] The term "antisense strand" or "guide strand" refers to the strand of the siRNA that contains a region that is substantially complementary to the target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., the target sequence as defined herein. If the complementary region is not completely complementary to the target sequence, mismatches can be present in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are within 5, 4, 3, or 2 nucleotides of the terminal regions, e.g., the 5' and / or 3' termini.

[0048] As used herein, the terms "sense strand" or "passenger strand" refer to the strand of siRNA that includes a region that is substantially complementary to the region of the antisense strand as defined herein.

[0049] The term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules that are naturally expressed or found, but also analogs and derivatives of RNA that include one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, a "ribonucleoside" contains a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside having one, two, or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleotide" can be considered equivalent when used herein. RNA can be modified, for example, in the nucleobase structure or the ribose-phosphate backbone structure, as described in more detail below. However, siRNA molecules containing ribonucleoside analogs or derivatives retain the ability to form double strands. By way of non-limiting example, RNA molecules can also include at least one modified ribonucleoside including, but not limited to, 2'-O-methyl modified nucleosides, nucleosides containing 5'-phosphorothioate groups, terminal nucleosides linked to cholesteryl derivatives or didodecylamid groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino modified nucleosides, 2'-alkyl modified nucleosides, morpholino nucleosides, phosphoramidates, or non-natural bases containing nucleosides, or any combination thereof. In another example, an RNA molecule can include at least two modified ribonucleosides, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, or more, up to the full length of the siRNA molecule. The modifications need not be the same for each of such multiple modified ribonucleosides in the RNA molecule. In some embodiments, the modified ribonucleosides include deoxyribonucleosides. For example, an siRNA can include one or more deoxynucleosides, such as deoxynucleotide overhang(s), or one or more deoxynucleosides within the double-stranded portion of the siRNA.However, as used herein, the term "siRNA" does not include complete DNA molecules.

[0050] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an siRNA. For example, when the 3' end of one strand of an siRNA extends beyond the 5' end of the other strand, or vice versa, a nucleotide overhang is present. An siRNA can include an overhang of at least one nucleotide. Alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang can include, or consist of, nucleotide / nucleoside analogs including deoxynucleotides / nucleosides. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotide(s) of the overhang can be present at the 5' end, 3' end, or both ends of either the antisense or sense strand of the siRNA.

[0051] As used herein in connection with siRNA, the term "blunt" or "blunt-ended" means that there are no unpaired nucleotides or nucleotide analogs at a given end of the siRNA, i.e., there is no nucleotide overhang. One or both ends of the siRNA can be blunt. When both ends of the siRNA are blunt, the siRNA is said to be "blunt-ended." A "blunt-ended" siRNA is an siRNA in which both ends are blunt, i.e., it has no nucleotide overhangs at either end of the molecule. Often, such molecules are double-stranded over their entire length.

[0052] In some embodiments, the present disclosure provides a combination therapy for treating HBV that includes an anti-HBV antibody. In certain embodiments, the anti-HBV antibody or an antigen-binding fragment thereof binds to the antigen loop region of HBsAg and neutralizes infection by hepatitis B virus. In some embodiments, the anti-HBV antibody or an antigen-binding fragment thereof binds to the antigen loop region of HBsAg and neutralizes infection by hepatitis D virus.

[0053] As used herein, the term "antibody" includes various forms of antibodies including, but not limited to, whole antibodies, antibody fragments, antigen-binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered antibodies (variant or mutant antibodies), so 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 certain 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 fragment of an antibody of a combination therapy that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single-chain antibodies, Fab, Fab’, F(ab’)2, Fv, or scFv. Further, the term "antibody" as used herein includes both the antibody and its antigen-binding fragments.

[0054] As used herein, a "neutralizing antibody" is an antibody that can neutralize, i.e., prevent, inhibit, reduce, interfere with, or block, the ability of a pathogen to initiate and / or perpetuate infection in a host. The terms "neutralizing antibody" and "antibody(ies) that neutralize" are used interchangeably herein. These antibodies can be used alone or in combination as prophylactic or therapeutic agents upon appropriate formulation, in connection with active vaccination, as diagnostic tools, or as production tools as described herein.

[0055] Human antibodies are well known in the 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 capable of producing a complete repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production upon immunization. Transfer of the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits A et al., Proc. Natl. Acad. Sci. USA 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. are also available for the preparation of 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 immortalization as described by Traggiai E et al. (Nat Med. 2004, 10(8):871-5). The term "human antibody" as used herein also includes antibodies that have been modified, e.g., in the variable regions, to produce the characteristics described herein.

[0056] The antibody for combination therapy can be of any isotype (e.g., IgA, IgG, IgM, i.e., κ, γ, or β heavy chain), but in certain embodiments, the antibody is IgG. Within the IgG isotype, the antibody can be of the IgG1, IgG2, IgG3, or IgG4 subclass. In certain embodiments, the antibody is IgG1. The antibody for combination therapy can have a K or X light chain. The IgG-type HBsAg-specific antibody can also advantageously block the release of HBV and HBsAg from infected cells based on the antigen-independent uptake of IgG into hepatocytes via the FcRn-IgG receptor. Thus, the IgG-type HBsAg-specific antibody can bind intracellularly and thereby block the release of HBV virions and HBsAg.

[0057] As used herein, "variable region" (the variable region of the light chain (VL), the variable region of the heavy chain (V HThe term "()" includes complementarity-determining regions ("CDRs") and framework regions ("FRs"), and refers to the portions of the antibody light chain (LC) or heavy chain (HC) that are directly involved in binding of the antibody to an antigen (typically, about 105-120 amino-terminal amino acids of the mature antibody heavy or light chain). The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-adjacent sequences of amino acids within the variable region of an antibody that confer antigen specificity and / or binding affinity. Generally, each variable region of an immunoglobulin binding protein has three CDRs. For example, with respect to an antibody, the VH and VL regions generally include six CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3). Immunoglobulin sequences can be aligned to 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 will be understood that in certain embodiments, the antibodies or antigen-binding fragments of the present disclosure can include all or a portion of the heavy chain (HC), light chain (LC), or both. For example, a full-length intact IgG antibody monomer typically includes V H , CH1, CH2, CH3, V L , and CL.

[0058] In certain embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy according to the present disclosure is a purified antibody, single-chain antibody, Fab, Fab’, F(ab’)2, Fv, or scFv. Thus, the antibody of the combination therapy can be a human antibody, monoclonal antibody, human monoclonal antibody, recombinant antibody, and / or purified antibody. The present disclosure also provides fragments of the antibody, particularly fragments that retain the antigen-binding activity of the antibody. Such fragments include, but are not limited to, single-chain antibodies, Fab, Fab’, F(ab’)2, Fv, or scFv. In some places, the present disclosure may explicitly refer to antigen-binding fragment(s) of the antibody, antibody fragment(s), variant(s), and / or derivative(s), but as used herein, the term “antibody” or “antibody of the combination therapy” includes all categories of antibodies, namely, antigen-binding fragment(s) of the antibody, antibody fragment(s), variant(s), and derivative(s).

[0059] Fragments of the antibody can be obtained from the antibody by methods including digestion with enzymes such as pepsin or papain and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibody can be obtained by cloning and expressing a portion of the heavy or light chain sequence. The present disclosure also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of the antibodies of the present disclosure. For example, the present disclosure includes scFv containing CDRs derived from the 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, such as single-chain Fv in which the heavy and light chain variable domains are linked by a peptide linker.

[0060] The antibody fragments of the present disclosure can confer monovalent or multivalent interactions and can be included in various structures as described above. For example, scFv molecules can be synthesized to produce trivalent "triabodies" or tetravalent "tetrabodies". The scFv molecule may include a domain of the Fc region that results in a bivalent minibody. Further, the sequence of the antibody / antibody fragment can be a component of a multispecific molecule where the sequence targets an epitope described herein and other regions of the multispecific molecule bind to other targets. Exemplary multispecific molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, Nature Biotechnology 2005, 9:1126-36).

[0061] Antibodies according to the present disclosure can be provided in a purified form. Typically, the antibody is present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight), usually less than 60%, more usually less than 50% of the composition is composed of other polypeptides.

[0062] The antibodies and antigen-binding fragments of the present disclosure may, in embodiments, be multispecific (e.g., bispecific, trispecific, quadrispecific, etc.) and may be provided in any multispecific format as disclosed herein. In certain embodiments, the antibody or antigen-binding fragment of the present disclosure is a multispecific antibody such as a bispecific or trispecific antibody. Formats of bispecific antibodies are disclosed, for example, in Spiess et al. (Mol. Immunol. 2015, 67(2): 95), and Brinkmann and Kontermann (mAbs 2017, 9(2):182-212), and these bispecific formats and methods for making them are incorporated herein by reference, for example, bispecific T cell engager (BiTE), DART, Knobs-Into-Holes (KIH) assembly, scFv-CH3-KIH assembly, KIH common light chain antibody, TandAb, triple body, TriBi minibody, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2-scFv2, tetravalent HCAb, intrabody, CrossMab, dual action Fab (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, charge pair, Fab arm exchange, SEEDbodies, Triomabs, LUZ-Y assembly, Fcabs, Kk-bodies, orthogonal Fabs, DVD-IgGs, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (four-in-one). Bispecific or multispecific antibodies can comprise a HBVand / or HDV-specific binding domain of the present disclosure in combination with another such binding domain of the present disclosure or in combination with a different binding domain that specifically binds to HBV and / or HDV (e.g., at the same or different epitopes).

[0063] II. siRNA Targeting HBV In some embodiments, the present disclosure provides a treatment method comprising administering an siRNA targeting HBV mRNA, as well as related compositions and kits. Since HDV is a defective satellite virus that depends on HBsAg for the completion of its life cycle, siRNAs that reduce HBsAg production interfere with the production of infectious HDV virions. In in vitro experiments, treatment of primary human hepatocytes co-infected with HBV / HDV with an siRNA targeting HBV mRNA (SIRNA01) resulted in a dose-dependent decrease in the secreted infectious HDV virions. In patients co-infected with HBV / HDV, the HBsAg level correlates with the HDV RNA level, indicating that reducing HBsAg in serum can lead to a decrease 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). Furthermore, in preclinical models, reducing intrahepatic HBsAg with an siRNA agent decreased HDV viremia (Ye X et al., Hepatitis B Virus Therapeutic Agent ARB1740 Has Inhibitory Effect on Hepatitis Delta Virus in a New Dually-Infected Humanized Mouse Model, ACS Infect Dis. May 10, 2019 5(5): 738-749) and restricted virus spread to uninfected hepatocytes.

[0064] In some embodiments described herein, the siRNA targeting HBV mRNA is SIRNA01. SIRNA01 is a synthetic chemically modified siRNA targeting HBV RNA that has a covalently linked trisubstituted N-acetyl-galactosamine (GalNAc) ligand that enables specific uptake by hepatocytes. SIRNA01 targets mRNA encoded by a region of the HBV genome that is common to all HBV viral transcripts and is pharmacologically active against HBV genotypes A - J. In preclinical models, SIRNA01 has been shown to inhibit viral replication, translation, and the secretion of HBsAg, and may provide or contribute to a functional cure of chronic HBV infection. The siRNA can have multiple antiviral effects, including degradation of pgRNA, thus inhibiting viral replication, and degradation of all viral mRNA transcripts, thereby preventing the expression of viral proteins. This can lead to the restoration of a functional immune response against HBV, either alone or in combination with other therapies. The ability of SIRNA01 to reduce HBsAg-containing non-infectious subviral particles also distinguishes it from currently available treatments.

[0065] SIRNA01 targets and inhibits the expression of mRNA encoded by the HBV genome described in 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 that contains the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO:2) corresponding to nucleotides 1579 - 1597 of SEQ ID NO:1. Since transcription of the HBV genome results in polycistronic overlapping RNAs, SIRNA01 results in significant inhibition of the expression of most or all HBV transcripts. Exemplary methods for synthesizing SIRNA01 and experimental data demonstrating the silencing of HBV gene expression are described in International Application Publication No. WO 2020 / 036862A1, which are incorporated herein by reference.

[0066] SIRNA01 has a sense strand containing 5’-GUGUGCACUUCGCUUCACA-3’ (SEQ ID NO: 3) and an antisense strand containing 5’-UGUGAAGCGAAGUGCACACUU-3’ (SEQ ID NO: 4), and the nucleotides have 2’-fluoro (2’F) and 2’-O-methoxy (2’OMe) ribose sugar modifications, phosphorothioate backbone modifications, glycol nucleic acid (GNA) modifications, and conjugation to a tris-branched N-acetyl-galactosamine (GalNAc) ligand at the 3’ end of the sense strand to facilitate delivery to hepatocytes via the asialoglycoprotein receptor (ASGPR). Including the modifications, the sense strand of SIRNA01 contains 5’-gsusguGfcAfCfUfucgcuucacaL96-3’ (SEQ ID NO: 5), the antisense strand contains 5’-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3’ (SEQ ID NO: 6), and the modifications are abbreviated as shown in Table 1.

[0067]

Table 1

[0068] 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 sequence variants of SIRNA01. In certain embodiments, the portion of the HBV transcript(s) targeted by the sequence variant of SIRNA01 overlaps with the portion of the HBV transcript(s) targeted by SIRNA01.

[0069] 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, respectively, by 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides, 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, respectively, by 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides.

[0070] In some embodiments, shorter double-strands are used that have a sequence with only a few nucleotides subtracted from one or both ends from one of the sequences of SEQ ID NO: 4 or SEQ ID NO: 6. Thus, siRNAs are contemplated herein that have a subsequence of 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 whose ability to inhibit HBV gene expression differs from that of the siRNA containing the full sequence by 5, 10, 15, 20, 25, or 30% or less inhibition. In some embodiments, siRNAs are provided that have blunt ends at one or both ends, formed by removing nucleotides from one or both ends of SIRNAO1.

[0071] 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 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides, respectively, or (2) the antisense strand comprises SEQ ID NO: 8, or a sequence that differs from SEQ ID NO: 8 by 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides, respectively.

[0072] In some embodiments, shorter double-strands having sequences with only a few nucleotides subtracted from one or both ends of the sequence of SEQ ID NO: 8 are used. Thus, siRNAs having a subsequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from SEQ ID NO: 8 and having an ability to inhibit the expression of the HBV gene that differs by 5, 10, 15, 20, 25, or 30% or less inhibition from the siRNA containing the full sequence are contemplated herein. In some embodiments, siRNAs having blunt ends at one or both ends, formed by removing nucleotides from one or both ends of SEQ ID NO: 8, are provided.

[0073] In some embodiments, the siRNAs described herein may contain one or more mismatches to the target sequence. In some embodiments, the siRNAs described herein contain three or fewer mismatches. In some embodiments, when the antisense strand of the siRNA contains a mismatch to the target sequence, the region of the mismatch is not located in the center of the complementary region. In certain embodiments, when the antisense strand contains a mismatch to the target sequence, the mismatch is restricted within the last 5 nucleotides from either the 5' or 3' end of the complementary region. For example, for a 23-nucleotide siRNA strand complementary to a region of the HBV gene, the RNA strand may not contain a mismatch within the central 13 nucleotides. Using the methods described herein or methods known in the art, it can be determined whether an siRNA containing a mismatch to the target sequence is effective in inhibiting the expression of the HBV gene.

[0074] In some embodiments, the siRNAs used in the methods, compositions, and kits described herein comprise two oligonucleotides, where one oligonucleotide is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described herein and as known in the art, the complementary sequences of the siRNA can also be included as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0075] In some embodiments, single-stranded antisense RNA molecules comprising the antisense strand of the siRNAs described herein are used in the methods, compositions, and kits described herein. The antisense RNA molecule can have 15 to 30 nucleotides complementary to the target.

[0076] In some embodiments, single-stranded antisense RNA molecules comprising the antisense strand of SIRNA01 or a sequence variant thereof are used in the methods, compositions, and kits described herein. The antisense RNA molecule can have 15 to 30 nucleotides complementary to the target. For example, the antisense RNA molecule can have a sequence of at least 15, 16, 17, 18, 19, 20, 21, or more consecutive nucleotides from SEQ ID NO: 4 or SEQ ID NO: 6.

[0077]

[0078] In some embodiments, the siRNA comprises a sense strand and an antisense strand, the sense strand comprises SEQ ID NO: 5, the antisense strand comprises SEQ ID NO: 6, and further comprises additional nucleotides, modifications, or conjugates described herein. For example, in some embodiments, the siRNA can comprise additional modifications in addition to those shown in SEQ ID NOs: 5 and 6. Such modifications can 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, and examples of such modifications are described in more detail below.

[0078] In some embodiments, substantially all or all of the nucleotides of the sense strand of the siRNA, and substantially all or all of the nucleotides of the antisense strand, are modified nucleotides. The nucleotides can be modified as described below.

[0079] a. Modified siRNA Examples of the modifications disclosed herein include, for example, (a) sugar modifications (e.g., at the 2'- or 4'-position) or substitution of the sugar; (b) backbone modifications including modification or substitution of the phosphodiester bond; (c) base modifications, e.g., substitution of a base with a stabilized base, a destabilized base, or a base that pairs with an expanded repertoire of partners, removal of a base (abasic nucleotide), or a conjugated base; and (d) terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, inverse ligation, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverse ligation, etc.). Some specific examples of modifications that can be incorporated into the siRNAs of the present application are shown in Table 1.

[0080] The modification includes a substituted sugar moiety. The siRNAs discussed herein can include at the 2'-position one of the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. In some other embodiments, the siRNA has at the 2'-position a C1-C 10Lower alkyl, substituted lower alkyl, aralkyl, aralkyl, O-aralkyl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloaryl, 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 one of other substituents having similar properties. In some embodiments, the modification is 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (see Martin et al., Helv. Chim. Acta 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group also known as 2'-DMA0E, and 2'-dimethylaminoethoxyethoxy (in the art 2 * -O-dimethylaminoethylethyl or 2 * -DMAEOE, also known), 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 be made at other positions on the RNA of siRNA, particularly at the 3' terminal nucleotide or at the 3' position of the sugar in a 2'-5' linked siRNA, and at the 5' position of the 5' terminal nucleotide. The modification can also include sugar mimetics such as cyclobutyl moieties instead of the pentofuranosyl sugar.

[0081] Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is incorporated herein by reference for the teachings related to methods of preparing such modifications.

[0082] Modified RNA backbones include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinate, phosphoramidate (including 3'-aminophosphoramidate and aminoalkyl phosphoramidate), thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and boranophosphoric acid having a normal 3'-5' linkage, 2'-5' linkage analogs thereof, and boranophosphoric acid having an inverse polarity in which adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0083] Representative U.S. patents that teach the preparation of such phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, 6,239,265, 6,277,603, 6,326,199, 6,346,614, 6,444,423, 6,531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Reissue Patent No. 39464, each of which is hereby incorporated by reference herein for its teachings regarding methods of preparing such modifications.

[0084] Examples of RNAs having a modified backbone include, inter alia, those having no phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs having no phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. The modified RNA backbone containing no phosphorus atom therein is formed by a short-chain alkyl or cycloalkyl internucleoside linkage, a mixed heteroatom and alkyl or cycloalkyl internucleoside linkage, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those having a morpholino linkage (partially formed from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioformylacetyl backbones; methyleneformylacetyl and thioformylacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component moieties.

[0085] Representative U.S. patents teaching the preparation of such oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, each of which is incorporated herein by reference for its teachings regarding methods of preparing such modifications.

[0086] In some embodiments, both the sugar of the nucleotide unit and the internucleoside linkage, i.e., the backbone, are replaced with novel groups. The base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, 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 with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the azanitrogen atoms of the amide portions of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference for its teachings regarding such preparation methods. Further teachings of PNA compounds can be found, for example, in Nielsen et al. (Science 1991, 254:1497-1500).

[0087] Some embodiments characterized in the techniques described herein include RNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene(methylimino) or MMI backbone], -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- of U.S. Patent No. 5,489,677 [the native phosphodiester backbone is represented as -O-P-O-CH2-] and the amide backbone of U.S. Patent No. 5,602,240. In some embodiments, the RNAs taken up herein have the morpholino backbone structure of U.S. Patent No. 5,034,506.

[0088] The modifications of siRNAs disclosed herein can also include nucleobase (often simply referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 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 U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine (edited by Herdewijn P, Wiley-VCH, 2008); those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering (pages 858 - 859, edited by Kroschwitz JL, John Wiley & Sons, 1990); those disclosed in Englisch et al. (Angewandte Chemie, International Edition 30, 613, 1991); and those disclosed in Sanghvi YS (Chapter 15, dsRNA Research and Applications, pages 289 - 302, edited by Crooke ST and Lebleu B, CRC Press, 1993). Some of these nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds characterized in the techniques described herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 - 1.2 °C (Sanghvi YS et al., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, pp. 276 - 278, 1993), and is an exemplary base substitution, more specifically in combination with 2'-O-methoxyethyl sugar modification.

[0089] Representative U.S. patents that teach the preparation of the specific modified nucleobases as well as other modified nucleobases include, but are not limited to, U.S. Patent No. 3,687,808, U.S. Patent No. 4,845,205, U.S. Patent No. 5,130,30, U.S. Patent No. 5,134,066, U.S. Patent No. 5,175,273, U.S. Patent No. 5,367,066, U.S. Patent No. 5,432,272, U.S. Patent No. 5,457,187, U.S. Patent No. 5,459,255, U.S. Patent No. 5,484,908, U.S. Patent No. 5,502,177, U.S. Patent No. 5,525,711, U.S. Patent No. 5,552,540, U.S. Patent No. 5,587,469, U.S. Patent No. 5,594,121, U.S. Patent No. 5,596,091, U.S. Patent No. 5,614,617, U.S. Patent No. 5,681,941, U.S. Patent No. 5,750,692, U.S. Patent No. 6,015,886, U.S. Patent No. 6,147,200, U.S. Patent No. 6,166,197, U.S. Patent No. 6,222,025, U.S. Patent No. 6,235,887, U.S. Patent No. 6,380,368, U.S. Patent No. 6,528,640, U.S. Patent No. 6,639,062, U.S. Patent No. 6,617,438, U.S. Patent No. 7,045,610, U.S. Patent No. 7,427,672, and U.S. Patent No. 7,495,088, each of which is incorporated herein by reference for the teachings related to methods of preparing such modifications.

[0090] siRNA can also be modified to include one or more glycol nucleic acids such as adenosine-glycol nucleic acid (GNA). The 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 the teachings related to methods of preparing GNA modifications.

[0091] The RNA of siRNA can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides having a modified ribose moiety, where the ribose moiety includes an extra bridge connecting the 2’ and 4’ carbons. This structure effectively “locks” the ribose into a 3’-end conformational structure. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability 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).

[0092] Exemplary U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, 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 the teachings related to methods of preparing such modifications.

[0093] In some embodiments, the siRNA comprises a modification that chemically links to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the siRNA.Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (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 such as beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660: 306-9; Manoharan et al., Biorg. Med. Chem. Let. 1993, 3: 2765-70), thiocolesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20: 533-38), aliphatic chains such as dodecanediol or undecyl residues (Sezon-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 such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al. 1995, 36: 3651-54; Shea et al., Nucl. Acids Res. 1990, 18: 3777-83), polyamine 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), palmitoyl moieties (Mishra et al. biochim. Biophys. Acta 1995, 1264: 229-37), or octadecylamine or hexylaminocarbonyloxy cholesterol moieties (Crooke et al. Exp. Ther. 1996, 277: 923-37).

[0094] In some embodiments, the ligand alters the distribution, targeting, or lifespan of the siRNA into which it is incorporated. In some embodiments, the ligand provides, for example, an enhanced affinity for a selected target, such as a molecule, cell, or cell type, compartment, such as a cellular or organ compartment, tissue, organ, or region of the body, compared to species in which such ligand is absent. In such embodiments, the ligand is not involved in double-stranded base pairing in the double-stranded nucleic acid.

[0095] 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 or synthetic molecule, such as a synthetic polymer, such as a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salts of polyamines, and α-helix peptides.

[0096] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as an antibody that binds to a specific cell type, such as a lectin, glycoprotein, lipid or protein, such as a hepatocyte. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (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, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl) lithocholic acid, 03-(oleoyl) cholic acid, dimethoxytrityl, or phenoxazine), peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption promoter (e.g., aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ complex of tetraaza macrocyclic molecule), dinitrophenyl, HRP, and AP.

[0097] A ligand can be a molecule having specific affinity for a protein, such as a glycoprotein, or a peptide, such as a co-ligand, or an antibody, such as an antibody that binds to a specific cell type such as a hepatocyte. Ligands can also include hormones and hormone receptors. They can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, and polyvalent fucose. A ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0098] A ligand can be a substance, such as a drug, that can increase the uptake of siRNA into cells, for example, by disrupting the cytoskeleton of the cells, such as by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cells. Drugs can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0099] In some embodiments, the ligand is a moiety that is taken up by a target cell, such as a hepatocyte, such as a vitamin. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include vitamins B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells such as hepatocytes. HSA and low density lipoprotein (LDL) are also included.

[0100] In some embodiments, the ligand bound to the siRNA described herein acts as a pharmacokinetic (PK) modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like.

[0101] Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing some phosphorothioate linkages are also known to bind to serum proteins, and thus short oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, are also suitable for the techniques described herein as ligands (e.g., as PK modulator ligands). In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulator ligands in the embodiments described herein.

[0102] (i) Lipid conjugates. In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Lipid or lipid-based ligands can be used to (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA. Such lipid or lipid-based molecules can bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including hepatocytes of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, neproxine or aspirin can be used.

[0103] Lipid-based ligands can be used to inhibit, e.g., control the binding of the conjugate to the target tissue. For example, lipids or lipid-based ligands that bind strongly to HSA are less likely to be targeted to the kidney and thus less likely to be eliminated from the body. Lipids or lipid-based ligands that do not bind too strongly to HSA can be used to target the conjugate to the kidney.

[0104] In some embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity such that the conjugate is distributed to non-renal tissues. In certain embodiments, the HSA-ligand binding is reversible.

[0105] In some embodiments, the lipid-based ligand binds weakly or not at all to HSA such that the conjugate is distributed to the kidney. Other moieties that target kidney cells can also be used instead of or in addition to the lipid-based ligand.

[0106] (ii) Cell-penetrating peptides and agents. In another aspect, the ligand is a cell-penetrating agent such as a helical cell-penetrating agent. In some embodiments, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennapedia. If the agent is a peptide, it can be modified including the use of peptidomimetics, invertomers, non-peptide or pseudo-peptide bonds, and D-amino acids. In some embodiments, the helical agent is an α-helical agent. In certain embodiments, the helical agent has a lipophilic phase and a lipophobic phase.

[0107] "Cell-penetrating peptides" can penetrate cells, such as microbial cells like bacterial or fungal cells, or mammalian cells like human cells. Microbial cell-penetrating peptides can be, for example, α-helix linear peptides (such as LL-37 or seropin PI), disulfide bond-containing peptides (such as α-defensin, β-defensin, or bactericidin), or peptides containing only one or two dominant amino acids (such as PR-39 or indolicidin).

[0108] The ligand can be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to a natural peptide. The binding of peptides and peptidomimetics to siRNA can affect the pharmacokinetic distribution of RNAi, for example, by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0109] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety 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., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry large polar molecules including peptides, oligonucleotides, and proteins across the cell membrane. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWK (SEQ ID NO: 12)) have been found to function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence such as a peptide identified from a phage display library or a one-bead one-compound (OBOC) combinatorial library (Lam et al., Nature 1991, 354:82-84).

[0110] The cell-penetrating peptide can also include a nuclear localization signal (NLS). For example, the cell-penetrating peptide can be a bipartite amphiphilic peptide such as MPG derived from the fusion peptide domain of HIV1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 1993, 31:2717-24).

[0111] (iii) Carbohydrate conjugate. In some embodiments, the siRNA oligonucleotides described herein further comprise a carbohydrate conjugate. Carbohydrate conjugates can be advantageous for in vivo delivery of nucleic acids and for compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound composed of one or more monosaccharide units having at least 6 carbon atoms (which is a carbohydrate compound itself composed of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched, or cyclic) having a sulfur atom; or as part of it, refers to a compound having a carbohydrate moiety composed of one or more monosaccharide units each having at least 6 carbon atoms (which can be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. 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 with C5 or more (in some embodiments, C5 - C8), and disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (in some embodiments, C5 - C8).

[0112] In some embodiments, the carbohydrate conjugate is

[0113]

Chemical formula

[0114]

Chemical formula

[0115]

Chemical formula

[0116]

Chemical formula

[0117] [Chemical formula] is selected from the group consisting of.

[0118] Another representative carbohydrate conjugate for use in the embodiments described herein is

[0119] [Chemical formula]

[0120] (Formula XXII), where when one of X or Y is an oligonucleotide, the other is hydrogen.

[0121] In some embodiments, the carbohydrate conjugate further comprises another ligand such as, but not limited to, a PK modulator, an endosome-degradable ligand, or a cell-penetrating peptide.

[0122] (iv) Linker. In some embodiments, the conjugates described herein can be attached to the siRNA oligonucleotide using various linkers that can be cleavable or non-cleavable.

[0123] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound. A linker typically is a direct bond or an atom such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or, without limitation, 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, including chains of atoms in which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle. In the formula, R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In certain embodiments, the linker is from 1 to 24 atoms, from 4 to 24 atoms, from 6 to 18 atoms, from 8 to 18 atoms, or from 8 to 16 atoms.

[0124] The cleavable linker group is sufficiently stable extracellularly but is cleaved upon entry into the target cell, releasing the two moieties that the linker holds together. In certain embodiments, the cleavable linker group is cleaved at least 10-fold or at least 100-fold faster in the target cell, or under a first reference condition (e.g., selected to mimic or represent intracellular conditions), or under a second reference condition (e.g., selected to mimic or represent conditions found in blood or serum), than in the subject's blood.

[0125] Cleavable linker groups are susceptible to the influence of cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities intracellularly than in serum or blood. Examples of such degrading agents include redox agents that are selected for or lack substrate specificity for a particular substrate, such as oxidases or reductases or reducing agents such as mercaptans that are present intracellularly and can degrade redox-cleavable linker groups by reduction; esterases; endosomes or agents that can create an acidic environment, such as those that result in a pH of 5 or less; general acids, peptidases (which may be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linker groups by acting as phosphatases. Cleavable linker groups, such as disulfide bonds, can be pH-sensitive. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, in the range of about 7.1 to 7.3. Endosomes have a more 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 have cleavable linker groups that are cleaved at a particular pH, thereby releasing a ligand intracellularly or a cationic lipid within a desired compartment of the cell.

[0126] The linker can include a cleavable linking group that is cleavable 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 linked to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and thus the linker is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0127] Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as hepatocytes and synoviocytes.

[0128] In general, the suitability of a cleavable linking group candidate can be evaluated by testing the ability of the degrading agent (or condition) that cleaves the linking group candidate. It may also be desirable to test the ability of the cleavable linking group candidate to resist cleavage when in contact with blood or other non-target tissues. Thus, the relative sensitivity to cleavage between a first condition and a second condition can be determined, where the first condition is selected to indicate cleavage in the target cell and the second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. The evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to perform an initial evaluation under cell-free or culture conditions and confirm with further evaluation in whole animals. In certain embodiments, a useful candidate compound is cleaved at least 2-fold, at least 4-fold, at least 10-fold, or at least 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0129] One class of cleavable linking groups are redox-cleavable linking groups that are cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-S-S-). To determine whether a cleavable linking group candidate is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular RNAi moiety and a particular targeting agent, the methods described herein can be examined. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. A candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In some embodiments, a candidate compound is cleaved up to 10% in blood. In certain embodiments, a useful candidate compound is degraded at least 2-fold, at least 4-fold, at least 10-fold, or at least 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of a candidate compound can be determined using standard enzyme kinetics assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0130] Phosphate-based cleavable linkers are cleaved by agents that decompose or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linkers are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -OP-(O)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(ORk)-O-, -OP(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-. In certain embodiments, the phosphate-based linker is selected from -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -OP(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-O-, -O-P(O)(H)-O-, -OP(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S- and -O-P(S)(H)-S-. In certain embodiments, the phosphate linker is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to the methods described above.

[0131] An 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 having a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by an agent such as an enzyme that can act as a general acid. In cells, certain 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, hydrazones, esters, and esters of amino acids. The acid-cleavable group can generally have the formula -C=N-, C(O)O, or -OC(O). In some embodiments, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0132] Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkynylene groups. The ester-cleavable linking group generally has the formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0133] Peptide-based cleavable linkers are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linkers are peptide bonds formed between amino acids and include oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not contain an amide group. The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids that gives rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that give rise to peptides and proteins and do not include the entire amide functional group. Peptide-based cleavable linkers generally have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to the above method.

[0134] Representative carbohydrate conjugates having a linker include

[0135]

Chemical formula

[0136]

Chemical formula

[0137] In certain embodiments of the compositions and methods described herein, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives linked via a divalent or trivalent branched linker. For example, in some embodiments, the siRNA is conjugated to a GalNAc ligand as shown in the following schematic diagram,

[0138] [Chemical formula] X is O or S. In some of these embodiments, X is O.

[0139] In some embodiments, the combination therapy comprises siRNA conjugated to a divalent or trivalent branched linker selected from the group of structures shown in formulas (XXXI)-(XXXIV):

[0140] [Chemical formula] wherein q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent from 0 to 20 upon each occurrence, and the repeating units may be the same or different, p p 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , and T 5C each independently represent, upon each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O, Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , and Q 5C each independently represent, upon each occurrence, absent, alkylene, or substituted alkylene, and one or more methylenes are O, S, S(O), SO2, N(R N)、C(R’=C(R’’),

[0141]

Chem.

[0142]

Chem.

[0143]

Chem.

[0144] Examples of suitable divalent and trivalent branched linker groups that bind to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas I, VI, X, IX, and XII.

[0145] Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; and 7,037,646, each of which is incorporated herein by reference for the teachings related to such preparation methods.

[0146] In certain instances, the RNA of the siRNA can be modified by non-ligand groups. A number of non-ligand molecules have been conjugated to siRNAs to enhance their activity, cellular distribution, or cellular uptake, and procedures for doing such conjugation are available in the scientific literature.Such non-ligand moieties include 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, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. New York Acad. Sci. 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3:2765), thiocolesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20:533), aliphatic chains, e.g., dodecanediol 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 triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (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), palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al. Exp. Ther. 1996, 277:923).

[0147] Typical conjugation protocols involve the synthesis of RNA having an amino linker at one or more positions of the array. The amino group is then reacted with the molecule to be conjugated using a suitable coupling or activating reagent. The conjugation reaction can be carried out using RNA still attached to a solid support or in solution phase after cleavage of the RNA. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0148] b. Pharmaceutical Compositions and Delivery of siRNA In some embodiments, pharmaceutical compositions are provided that contain the siRNAs described herein and a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions containing siRNAs can be used to treat HBV infection. Such pharmaceutical compositions are typically formulated based on the mode of delivery. For example, the composition can be formulated for systemic administration via parenteral delivery, such as subcutaneous (SC) delivery.

[0149] 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 (e.g., nucleic acids) to an animal. Excipients can be liquid or solid and are selected considering the planned mode of administration to provide the desired bulk, viscosity, 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 binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropylmethylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate), but are not limited thereto.

[0150] Pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with nucleic acids can also be used to formulate siRNA compositions. Suitable pharmaceutically acceptable carriers for formulations used in non-parenteral delivery include water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, but are not limited thereto.

[0151] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of nucleic acids in liquid or solid oil-based vehicles. The solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with nucleic acids can be used for parenteral administration.

[0152] In some embodiments, administration of the pharmaceutical compositions and formulations described herein can be topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer), intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subcutaneous administration (e.g., via an implanted device); or intracranial administration (e.g., by parenchymal, intrathecal, or intraventricular administration).

[0153] In some embodiments, the pharmaceutical composition comprises a sterile solution of siRNA (e.g., SIRNA01) formulated in water for subcutaneous injection. In some embodiments, the pharmaceutical composition comprises a sterile solution of SIRNA01 formulated in water for subcutaneous injection at a free acid concentration of 200 mg / mL.

[0154] In some embodiments, the pharmaceutical composition containing the siRNA described herein is administered in a dose sufficient to inhibit the expression of the HBV gene. In some embodiments, the dose of siRNA ranges from 0.001 to 200.0 milligrams per kilogram of the recipient's body weight per day, or from 1 to 50 milligrams per kilogram of body weight per day. For example, the siRNA can be administered at 0.01 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg per single dose. The pharmaceutical composition can be administered once a day, or can be administered as two, three, or more sub-doses at appropriate intervals throughout the day, or can also be administered using continuous infusion or delivery by a controlled release formulation. In that case, the siRNA contained in each sub-dose must correspondingly be smaller in order to achieve the total daily dosage. The dosage unit can also be formulated for delivery over several days, for example, using a conventional sustained release formulation that provides for the sustained release of the siRNA over several days. Sustained release formulations are well known in the art and are particularly useful for the delivery of a drug at a specific site such that they can be used in conjunction with the drugs of the technology described herein. In such embodiments, the dosage unit contains a corresponding plurality of daily doses.

[0155] In some embodiments, the pharmaceutical composition containing siRNA targeting the HBV mRNA described herein (e.g., 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.

[0156] In some embodiments, a pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, or 900 mg. In some embodiments, a pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 20 mg to 900 mg. In some embodiments, a pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 100 mg to 300 mg.

[0157] In some embodiments, a pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg.

[0158] In some embodiments, a pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 200 mg.

[0159] III. Anti-HBV Antibodies The present disclosure also provides anti-HBV antibodies for use in combination therapies for treating HDV or HDV-related diseases. Antibodies that bind to the antigenic loops of HBsAg on the surface of HDV virions can act as entry inhibitors by blocking the interaction between HBsAg and its receptor NTCP. Inhibition of viral entry prevents new rounds of HDV infection in the liver and ultimately reduces HDV viremia. Antibodies can also promote HDV clearance by opsonizing virions. Antibodies can also have indirect antiviral activity against HDV by stimulating an immune response against HBsAg present in co-infected hepatocytes that produce new HDV virions.

[0160] a. An antibody that binds to an HBV protein In some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy binds to the antigen loop region of HBsAg. The envelope of hepatitis B virus contains the following three "HBV envelope proteins" (also known as "hepatitis B surface antigen"): S protein (representing "small", also called S-HBsAg), M protein (representing "medium", also called M-HBsAg), and L protein (representing "large", also called L-HBsAg). S-HBsAg, M-HBsAg, and L-HBsAg share the same C-terminus (also called the "S domain", 226 amino acids), which corresponds to the S protein (S-HBsAg) and is involved in virus assembly and infectivity. S-HBsAg, M-HBsAg, and L-HBsAg are synthesized in the endoplasmic reticulum (ER), assembled, and secreted as particles via the Golgi apparatus. 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. Both transmembrane domains TM1 and TM2 are required for co-translational protein integration into the ER membrane, and transmembrane domains TM3 and TM4 are located in the C-terminal third of the S domain. The "antigen loop region" of HBsAg is located between the predicted TM3 transmembrane domain and TM4 transmembrane domain of the S domain of HBsAg, whereby the antigen loop region contains amino acids 101-172 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 antigen loop region of the HBV envelope protein. In particular, the residues between 119 and 125 of HBsAg contain a CXXC motif, which has been demonstrated to be the most important sequence required for HBV infectivity (Jaoude GA and Sureau C, Journal of Virology 2005, 79:10460-6).

[0161] As used herein, the S domain of HBsAg refers to the amino acid sequence set forth in SEQ ID NO: 13 (shown below), or a natural or artificial sequence variant thereof.

[0162] [Table 2] (SEQ ID NO: 13; amino acids 101 - 172 are underlined).

[0163] For example, the expression "amino acids 101 - 172 of the S domain" refers to the amino acid residues from positions 101 - 172 of the polypeptide set forth in SEQ ID NO: 13. However, one of ordinary skill in the art would understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions of different genotypes of HBsAg or different HBsAg mutants as described herein) can occur naturally in the amino acid sequence of the S domain of HBsAg or can be introduced artificially into the amino acid sequence of the S domain of HBsAg without affecting its biological properties. Thus, the term "S domain of HBsAg" includes all such polypeptides, including, for example, the polypeptide set forth in SEQ ID NO: 13 and its natural or artificial mutants. In addition, when sequence fragments of the S domain of HBsAg are described herein (e.g., amino acids 101 - 172 or amino acids 120 - 130 of the S domain of HBsAg), they include not only the corresponding sequence fragments of SEQ ID NO: 13 but also the corresponding sequence fragments of its natural or artificial mutants. For example, the expression "amino acid residues from positions 101 - 172 of the S domain of HBsAg" includes the amino acid residues from positions 101 - 172 of SEQ ID NO: 13 and the corresponding fragments of its variants (natural or artificial mutants).

[0164] As used herein, the expression "corresponding array fragment" or "corresponding fragment" refers to a fragment located at an equal position of an array when the array is subjected to an optimized alignment, i.e., when the array is aligned to obtain the highest percentage of identity. The M protein (M-HBsAg) corresponds to the S protein extended by an N-terminal domain of 55 amino acids called "pre-S2". The L protein (L-HBsAg) corresponds to the M protein extended by an N-terminal domain of 108 amino acids called "pre-S1" (genotype D). The pre-S1 and pre-S2 domains of the L protein are present on the inner surface (cytoplasmic side of the ER) of the viral particle and play an important role in virus assembly, or are present on the outer surface (lumen side of the ER) and are available for interaction with target cells and are necessary for virus infection. Furthermore, the HBV surface protein (HBsAg) is not only incorporated into the virion envelope but also spontaneously buds from the ER-Golgi intermediate compartment membrane to form empty "subviral particles" (SVPs) that are released from the cell by secretion.

[0165] Since 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 "antigen loop region". Therefore, an antibody or an antigen-binding fragment thereof that binds to the antigen loop region of HBsAg binds to all three HBV envelope proteins: S-HBsAg, M-HBsAg, and L-HBsAg.

[0166] Furthermore, in some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy neutralizes infection by hepatitis B virus. 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 infection by hepatitis D virus. In other words, the antibody or antibody-binding fragment thereof can reduce the viral infectivity of hepatitis D virus (see below for further description of hepatitis B virus as an absolute satellite of hepatitis D virus).

[0167] To test and quantify viral infectivity (or "neutralization") in the laboratory, those skilled in the art are aware of various standard "neutralization assays". For a neutralization assay, an animal virus is typically grown in cells and / or cell lines. In the context of the present disclosure, for a neutralization assay, cultured cells can be incubated with a fixed amount of HBV in the presence (or absence) of the antibody being tested. As a readout, the levels 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 evaluated. In one embodiment of the HBV neutralization assay, cultured cells, such as HepaRG cells, particularly differentiated HepaRG cells, are incubated with a fixed amount of HBV in the presence or absence of the antibody being tested, for example, at 37°C for 16 hours. The incubation can be carried out in a medium (e.g., supplemented with 4% PEG8000). After incubation, the cells can be washed and further cultured. To measure viral infectivity, for example, between 7 and 11 days post-infection, the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant can be determined by enzyme-linked immunosorbent assay (ELISA). Additionally, HBeAg staining can be evaluated in an immunofluorescence assay.

[0168] In some embodiments, the antibodies and antigen-binding fragments have high neutralizing potency. The concentration of the antibodies of the present disclosure required for 50% neutralization of hepatitis B virus (HBV) is, for example, about 10 pg / mL or less. In certain embodiments, the concentration of the antibodies of the present disclosure required for 50% neutralization of HBV is about 5 pg / mL, about 1 pg / mL, or about 750 ng / mL. In certain embodiments, the concentration of the antibodies of the present disclosure required for 50% neutralization of HBV is 500 ng / mL or less, for example, 450, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, or about 50 ng / mL or less. This means that only low concentrations of antibodies are required for 50% neutralization of HBV. Specificity and potency can be measured using standard assays known to those of skill in the art.

[0169] In some embodiments, the anti-HBV antibodies as components of combination therapies are useful in the prevention and / or treatment of hepatitis B or hepatitis B-related diseases.

[0170] In some embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure promote the clearance of HBsAg and HBV. In particular, the antibodies or antigen-binding fragments thereof according to the present disclosure can promote the clearance of both HBV and subviral particles of hepatitis B virus (SVP). The clearance of HBsAg or subviral particles can be evaluated, for example, by measuring the level of HBsAg in a blood sample from a hepatitis B patient. Similarly, the clearance of HBV can be evaluated, for example, by measuring the level of HBV in a blood sample from a hepatitis B patient.

[0171] In the sera of patients infected with HBV, in addition to infectious particles (HBV), there are typically present in excess (typically 1,000 - 100,000 - fold) empty sub - viral particles (SVP) composed solely of HBV envelope protein (HBsAg) in the form of relatively small spheres and filaments of variable length. Sub - viral particles have been shown to strongly enhance intracellular virus replication and HBV gene expression (Bruns M et al., J Virol 1998, 72(2):1462 - 8). Since infectivity depends on the number of not only the virus but also SVP, this is also important regarding the infectivity of sera containing HBV (Bruns et al., 1998, supra).

[0172] Furthermore, excess sub - viral particles can act as decoys by absorbing neutralizing antibodies and thus can delay the clearance of infection. Thus, typically, the achievement of hepatitis B surface antigen (HBsAg) loss is considered the ideal endpoint of treatment and the closest outcome to a cure for chronic hepatitis B (CHB). Thus, in some embodiments, the antibodies or antigen - binding fragments thereof according to the present disclosure that promote the clearance of HBsAg, particularly the clearance of hepatitis B virus and HBV sub - viral particles, enable an improved treatment of hepatitis B, particularly in the context of chronic hepatitis B. Thereby, the antibodies or antigen - binding fragments thereof according to the present disclosure can strongly neutralize HBV because fewer antibodies are absorbed by SVP acting as decoys. Further, in certain embodiments, the antibodies or antigen - binding fragments thereof according to the present disclosure promote the clearance of HBV sub - viral particles and reduce the infectivity of HBV in sera.

[0173] HBV is distinguished into many genotypes by its genomic sequence. To date, eight well - known genotypes (A - H) of the HBV genome have been defined.

[0174] Furthermore, two new genotypes I and J have also been identified (Sunbul M, World J Gastroenterol 2014, 20(18): 5427-34). Genotypes are known to affect the progression of the disease, and differences between genotypes in response to antiviral treatment have been determined. For example, genotype A has a tendency to become chronic, but viral mutations are frequently encountered in genotype C. Both chronicity and mutation frequency are common in genotype D. Furthermore, the genotypes of HBV are differentially distributed worldwide (Sunbul, 2014, supra). In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to at least 6, at least 8, or all 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. Examples of different genotypes of HBsAg include GenBank accession number J02203 (HBV-D, ayw3), GenBank accession number FJ899792.1 (HBV-D, adw2), GenBank accession number AM282986 (HBV-A), GenBank accession number D23678 (HBV-B1 Japan), GenBank accession number AB 117758 (HBV-C1 Cambodia), GenBank accession number AB205192 (HBV-E Ghana), GenBank accession number X69798 (HBV-F4 Brazil), GenBank accession number AF 160501 (HBV-G United States), GenBank accession number AY090454 (HBV-H Nicaragua), GenBank accession number AF241409 (HBV-I Vietnam), and GenBank accession number AB486012 (HBV-J Borneo). Table 2 shows the amino acid sequences of the antigen loop regions of the S domain of HBsAg of different genotypes (SEQ ID NOs: 14-42).

[0175] [Table 3-1]

[0176]

Table 3-2

[0177] In certain embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the HBsAg mutants having mutations in the antigen loop region: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. These mutants are naturally occurring mutants based on the S domain of HBsAg genotype D (SEQ ID NO: 43), Genbank accession number FJ899792 (the mutated amino acid residue(s) are indicated in the name).

[0178]

Table 4

[0179] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to at least 12, at least 15, or all 18 of the infectious HBsAg mutants having mutations in the antigen loop region: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A.

[0180] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to an epitope comprising at least one, at least two, at least three, or at least four amino acids in the antigen loop region of HBsAg, and the at least two, at least three, or at least four amino acids are selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. It should be noted that the amino acid positions (e.g., 115-133, 120-133, 120-130) refer to the S domain of HBsAg as described above, which is present in all three HBV envelope proteins S-HBsAg, M-HBsAg, and L-HBsAg.

[0181] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to an epitope within the antigen loop region of HBsAg, whereby the epitope is formed by one or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg.

[0182] As used herein in the context of an epitope, the term "formed by" means that the epitope to which an antibody or antigen-binding fragment thereof of the present disclosure binds can be linear (continuous) or conformational (discontinuous). A linear or continuous epitope is an epitope recognized by an antibody by its linear sequence or primary structure of amino acids. In contrast, a conformational epitope has a specific three-dimensional shape and protein structure. Thus, when the epitope is a linear epitope and contains two or more amino acids located at positions selected from amino acids 115 to 133, or amino acids 120 to 133 of the S domain of HBsAg, the amino acids included in the epitope can be located at adjacent positions in the primary structure (i.e., contiguous amino acids in the amino acid sequence). In the case of a conformational epitope (3D structure), in contrast, the amino acid sequence typically forms a 3D structure as an epitope, and thus the amino acids forming the epitope (or the amino acids "included" in the epitope) may or may not be located at adjacent positions in the primary structure (i.e., may or may not be contiguous amino acids in the amino acid sequence). In certain embodiments, the epitope to which an antibody or antigen-binding fragment thereof of the present disclosure binds is formed only by the amino acid(s) 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, (additional) amino acids located outside positions 115 to 133, 120 to 133, or 120 to 130 are not required to form the epitope to which an antibody or antigen-binding fragment thereof of the present disclosure binds.

[0183] In certain embodiments, the epitope within the antigen loop region of HBsAg to which the antibody or antigen-binding fragment thereof of the present disclosure binds is formed by two or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. In certain embodiments, the epitope in the antigen loop region of HBsAg to which the antibody or antigen-binding fragment thereof of the present disclosure binds is formed by three or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, and amino acid positions 120-130 of the S domain of HBsAg. In some embodiments, the epitope within the antigen loop region of HBsAg to which the antibody or antigen-binding fragment thereof of the present disclosure binds is formed by four or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. Thus, the antibody or antigen-binding fragment thereof according to the present disclosure can bind to at least one, at least two, at least three, or at least four amino acids in the antigen loop region of HBsAg selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. In certain embodiments, 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 antigen loop region of HBsAg, and the at least two, at least three, or at least four amino acids are selected from amino acids 120-133 or amino acids 120-130 of the S domain of HBsAg, and the at least two, at least three, or at least four amino acids are located at adjacent positions (i.e., are contiguous amino acids in the amino acid sequence / primary structure).

[0184] In certain embodiments, the epitope to which the antibody or antigen-binding fragment thereof according to the present disclosure binds is a conformational epitope. Thus, the antibody or antigen-binding fragment thereof according to the present disclosure can bind to an epitope comprising at least two, at least three, or at least four amino acids in the antigen loop region of HBsAg, and the at least two, at least three, or at least four amino acids are selected from amino acids 120-133 or amino acids 120-130 in the S domain of HBsAg, and the at least two, at least three, or at least four amino acids are not located at adjacent positions (in the primary structure).

[0185] In certain specific embodiments, the antibody of the present disclosure is a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating immune effector cells (e.g., by targeting a T cell surface protein, such as the extracellular portion of the CD3 protein). The second specificity can, for example, cause a cytotoxic effect or a vaccine effect.

[0186] b. Fc portion In some embodiments, the binding protein (e.g., an antibody or antigen-binding fragment thereof) comprises an Fc portion. In certain embodiments, the Fc portion can be of human origin, for example, derived from human IgG1, IgG2, IgG3, and / or IgG4. In specific embodiments, the antibody or antigen-binding fragment can comprise an Fc portion derived from human IgG1.

[0187] As used herein, the term "Fc portion" refers to a sequence that includes or is derived from a portion of an immunoglobulin heavy chain that begins at the hinge region immediately upstream of the papain cleavage site (e.g., residue 216 of native IgG, assuming the first residue of the heavy chain constant region is 114) and ends at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc portion can be a complete Fc portion or a part (e.g., a domain) thereof. In certain embodiments, the complete Fc portion includes the hinge domain, CH2 domain, and CH3 domain (e.g., residues 216-446 of EU). Additional lysine residues (K) may be present at the extreme C-terminus of the Fc portion, but are often cleaved from the mature antibody. Amino acid positions within the Fc portion are numbered according to the Kabat EU numbering system (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 1983 and 1987).

[0188] Amino acid positions of the Fc portion can also be numbered according to the IMGT numbering system (including its unique numbering for C domains and exon numbering) and the Kabat numbering system.

[0189] In some embodiments, the Fc portion comprises at least one of a hinge (e.g., upper, central, and / or lower hinge region) domain, CH2 domain, CH3 domain, or variants, portions, or fragments thereof. In some embodiments, the Fc portion comprises at least the hinge domain, CH2 domain, or CH3 domain. In further embodiments, the Fc portion is a complete Fc portion. The amino acid sequence of an exemplary Fc portion of the human IgG1 isotype is provided in SEQ ID NO: 60. The Fc portion may also include one or more amino acid insertions, deletions, or substitutions as compared to a naturally occurring Fc portion. For example, at least one of, or a portion of, the hinge domain, CH2 domain, or CH3 domain 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.

[0190] The Fc portions of the present disclosure can be modified to have an amino acid sequence different from that of the complete Fc portion of a naturally occurring immunoglobulin molecule while retaining (or enhancing) at least one desired function conferred by the naturally occurring Fc portion. Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Portions of the naturally occurring Fc portion involved in such functions are described in the art.

[0191] For example, to activate the complement cascade, the C1q protein complex can bind to at least two molecules of IgG1 or one molecule of IgM when immunoglobulin molecule(s) is / are bound to an antigen target (Ward ES and Ghetie, V, Ther. Immunol. 1995, 277-94). The heavy chain region containing amino acid residues 318-337 is involved in complement binding (Burton DR, Mol. Immunol. 1985, 22:161-206). Duncan AR and Winter G. (Nature 1988, 332:738-40) reported that site-directed mutagenesis was used to show that Glu318, Lys320, and Lys322 form the binding site to C1q. The role of Glu318, Lys320, and Lys322 residues in C1q binding was confirmed by the ability of short synthetic peptides containing these residues to inhibit complement-mediated lysis.

[0192] For example, FcR binding can be mediated by the interaction of the Fc portion (of an antibody) with an Fc receptor (FcR), a specialized cell surface receptor on cells including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of pathogens coated with antibodies by phagocytosis of immune complexes and the lysis of erythrocytes and various other cell targets (e.g., tumor cells) coated with the corresponding antibody via antibody-dependent cell-mediated cytotoxicity (e.g., (ADCC; Van de Winkel JG and Anderson CL, J. Leukoc. Biol. 1991, 49:511-24). Fc receptors are defined by their specificity for immunoglobulin classes. Fc receptors for IgG antibodies are called FcγRs, Fc receptors for IgE are called FcεRs, Fc receptors for IgA are called FcαRs, etc., 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.

[0193] Cross-linking of receptors by the Fc domain of natural IgG antibodies (FcγR) induces a variety of effector functions, including phagocytosis, antibody-dependent cell cytotoxicity, and the release of inflammatory mediators, as well as the regulation of immune complex clearance and antibody production. Fc moieties (e.g., FcγR) that provide receptor cross-linking are contemplated herein. In humans, three classes of FcγR have been characterized: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) FcγRII (CD32), which binds aggregated IgG with medium to low affinity, is widely expressed particularly on leukocytes, and is thought to play a central role in antibody-mediated immunity, and can be divided into FcγRIIA, FcγRIIB, and FcγRIIA, which perform different functions in the immune system but bind IgG-Fc with similar low affinity and the extracellular domains of these receptors are highly homologous; and (iii) FcγRIII (CD16), which binds IgG with medium to low affinity and is found in two forms: FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells and is thought to mediate ADCC; and FcγRIIB is highly expressed on neutrophils.

[0194] 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 inhibitory process and is found in B cells, macrophages, and mast cells and eosinophils. 75% of all FcγRIIB has been shown to be found in the liver (Ganesan LP et al., Journal of Immunology 2012, 189:4981-8). FcγRIIB is abundantly expressed on liver sinusoidal endothelial cells called LSECs and within Kupffer cells of the liver, and LSECs are the major site of small immune complex clearance (Ganesan et al., 2012, supra).

[0195] In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein include an Fc portion for binding to FcγRIIb, particularly the Fc region, such as an IgG-type antibody. Further, as described by Chu SY et al. (Molecular Immunology 2008, 45: 3926-33), it is possible to engineer the Fc portion to enhance FcγRIIB binding by introducing the mutations S267E and L328F. Thereby, clearance of immune complexes can be enhanced (Chu S et al., Am J Respir Crit, American Thoracic Society International Conference Abstracts 2014). In some embodiments, the antibodies or antigen-binding fragments of the present disclosure include a modified Fc portion having the mutations S267E and L328F, particularly as described by Chu SY et al. (2008, supra).

[0196] On B cells, FcγRIIB appears to function to suppress further immunoglobulin production and isotype switching, for example, to the IgE class. On macrophages, FcγRIIB is thought to inhibit phagocytosis mediated via FcγRIIA. On eosinophils and mast cells, the b form may help to suppress activation of these cells by the binding of IgE to its distinct receptor.

[0197] With respect to FcγRI binding, modification in at least one of E233-G236, P238, D265, N297, A327, and P329 in native IgG reduces binding to FcγRI. The IgG2 residues at positions 233-236 substituted in corresponding positions in IgG1 and IgG4 reduced the binding of IgG1 and IgG4 to FcγRI by 10 3 -fold and eliminated the human monocyte response to antibody-sensitized erythrocytes (Armour KL et al., Eur. J. Immunol. 1999, 29: 2613-2624).

[0198] Regarding FcγRII binding, for example, a decrease in binding to FcγRIIA is found for at least one IgG mutation among E233 - G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

[0199] Regarding FcγRIII binding, for example, a decrease in binding to FcγRIIIA is found for at least one mutation among E233 - G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376.

[0200] The mapping of the binding sites on human IgG1 to Fc receptors, the above-described mutation sites, and the method for measuring binding to FcγRI and FcγRIIA are described in Shields RL et al. (J. Biol. Chem. 2001, 276: 6591 - 6604).

[0201] Regarding binding to FcγRII, two regions of native IgG Fc, namely, (i) the lower hinge region of IgG Fc, particularly the amino acid residues L, L, G, and G (234 - 237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, particularly the loops and strands within the upper CH2 domain adjacent to the lower hinge region, for example, the loops and strands within the region of P331, appear to be involved in the interaction between FcγRII and IgG (Wines BD et al., J. Immunol. 2000, 164: 5313 - 8). Furthermore, FcγRI appears to bind to the same site on IgG Fc, while FcRn and protein A bind to different sites on IgG Fc, which appears to be at the CH2 - CH3 interface (Wines BD et al., 2000, supra).

[0202] Also contemplated are mutations that increase the binding affinity of the Fc portion of the present disclosure to one (i.e., one or more) Fcγ receptors (e.g., compared to a reference Fc portion or antibody that does not contain a mutation (s)). See, for example, Delillo and Ravetch, Cell 2015, 161(5):1035-45, and Ahmed et al., J. Struc. Biol. 2016, 194(1):78, the Fc mutations and techniques of which are incorporated herein by reference. In any of the embodiments disclosed herein, the binding protein can comprise an Fc portion comprising a mutation selected from G236A; S239D; A330L; and I332E; or a combination thereof; for example, S239D / I332E; S239D / A330L / I332E; G236A / S239D / I332E; G236A / A330L / I332E; and G236A / S239D / A330L / I332E.

[0203] In certain embodiments, the Fc portion can comprise or consist of at least a part of the Fc portion involved in binding to FcRn binding. In certain embodiments, the Fc portion comprises one or more amino acid modifications that improve the binding affinity for FcRn, and in some embodiments, thereby extend the in vivo half-life of a molecule comprising the Fc portion (e.g., as compared to a reference Fc portion or an antibody without the modification). In certain embodiments, the Fc portion comprises or is derived from an IgG Fc, and the half-life extending mutations comprise one or more of M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V, T307A, and E380A (EU numbering). In certain embodiments, the half-life extending mutation comprises M428L / N434S. In certain embodiments, the half-life extending mutation comprises M252Y / S254T / T256E. In certain embodiments, the half-life extending mutation comprises T250Q / M428L. In certain embodiments, the half-life extending mutation comprises P257VQ31 II. In certain embodiments, the half-life extending mutation comprises P257I / N434H. In certain embodiments, the half-life extending mutation comprises D376V / N434H. In certain embodiments, the half-life extending mutation comprises T307A / E380A / N434A.

[0204] In certain embodiments, the binding protein comprises an Fc portion comprising the substitution mutations: M428L / N434S and G236A / A330L / I332E. In certain embodiments, the antibody or antigen-binding fragment comprises an Fc portion comprising the substitution mutations: M428L / N434S and G236A / S239D / A330L / I332E.

[0205] In certain embodiments, the binding protein comprises an Fc portion comprising the substitution mutation: G236A / A330L / I332E. In certain embodiments, the antibody or antigen-binding fragment comprises an Fc portion comprising the substitution mutation: G236A / S239D / A330L / I332E.

[0206] Alternatively or additionally, the Fc portion of the binding proteins of the present disclosure can include at least a portion known in the art to be required for protein A binding, and / or the Fc portion of the antibodies of the present disclosure includes at least a portion of an Fc molecule known in the art to be required for protein G binding. In some embodiments, the retained function includes clearance of HBsAg and HBVg. Thus, in certain embodiments, the Fc portion includes at least a portion known in the art to be required for FcγR binding. Thus, as outlined above, the Fc portion can include at least (i) the lower hinge region of native IgG Fc, particularly the amino acid residues L, L, G, and G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of native IgG Fc, particularly the loops and strands within the upper CH2 domain adjacent to the lower hinge region, such as the region of P331, for example, at least 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids around P331 within the upper CH2 domain of native IgG Fc, for example, amino acids 320-340 (EU numbering) of native IgG Fc.

[0207] In some embodiments, the binding proteins according to the present disclosure include an Fc region. As used herein, the term "Fc region" refers to a portion of an immunoglobulin formed by two or more Fc portions of an antibody heavy chain. For example, the Fc region may be a monomeric or "single-chain" Fc region (i.e., scFc region). A single-chain Fc region is composed of Fc portions linked within a single polypeptide chain (e.g., encoded by a single continuous nucleic acid sequence).

[0208] Exemplary scFc regions are disclosed in WO2008 / 143954 A2 and are incorporated herein by reference. The Fc region can be or can include a dimeric Fc region. A "dimeric Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. The dimeric Fc region can be a homodimer of two identical Fc portions (e.g., the Fc region of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc portions (e.g., one Fc monomer of the dimeric Fc region includes at least one amino acid modification (e.g., substitution, deletion, insertion, or chemical modification) not present in the other Fc monomer, or one Fc monomer may be cleaved compared to the other).

[0209] The Fc portion of the present disclosure can include Fc sequences or regions of the same or different classes and / or subclasses. For example, the Fc portion can be derived from an immunoglobulin (e.g., a human immunoglobulin) of the IgG1, IgG2, IgG3, or IgG4 subclass, or any combination thereof. In certain embodiments, the Fc portions of the Fc region are of the same class and subclass. However, the Fc region (or one or more Fc portions of the Fc region) can also be chimeric, and a chimeric Fc region can include Fc portions derived from different immunoglobulin classes and / or subclasses.

[0210] For example, at least two of the Fc portions of a dimeric or single-chain Fc region can be derived from different immunoglobulin classes and / or subclasses. In certain embodiments, the dimeric Fc region can include sequences derived from two or more different isotypes or subclasses; e.g., the SEEDbody ("strand-exchanged domain") (see Davis et al., Protein Eng. Des. Sci. 2010, 23(4):195).

[0211] Additionally or alternatively, the chimeric Fc region may comprise one or more chimeric Fc moieties. For example, the chimeric Fc region or moiety may comprise one or more portions derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG3 subclass), while the remainder of the Fc region or moiety is of a different subclass. For example, the Fc region or moiety of an Fc polypeptide may comprise CH2 and / or CH3 domains derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG4 subclass), and a hinge region derived from an immunoglobulin of a second subclass (e.g., IgG3 subclass). For example, the Fc region or moiety may comprise a hinge and / or CH2 domain derived from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and a CH3 domain derived from an immunoglobulin of a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). For example, the chimeric Fc region may comprise an Fc moiety (e.g., a complete Fc moiety) derived from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and an Fc moiety derived from an immunoglobulin of a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). For example, the Fc region or moiety may comprise a CH2 domain derived from an IgG4 immunoglobulin and a CH3 domain derived from an IgG1 immunoglobulin. For example, the Fc region or moiety may comprise a CH1 domain and a CH2 domain derived from an IgG4 molecule, and a CH3 domain derived from an IgG1 molecule. For example, the Fc region or moiety may comprise a portion of a CH2 domain derived from a particular subclass of an antibody, e.g., EU positions 292-340 of the CH2 domain. For example, the Fc region or moiety may comprise amino acids at positions 292-340 of CH2 derived from an IgG4 portion and the remainder of CH2 derived from an IgG1 portion (alternatively, positions 292-340 of CH2 may be derived from an IgG1 portion and the remainder of CH2 may be derived from an IgG4 portion).

[0212] Furthermore, the Fc region or portion may (additionally or alternatively) include, for example, a chimeric hinge region. For example, a chimeric hinge may be derived, in part, from an IgG1, IgG2, or IgG4 molecule (e.g., the upper and lower central hinge sequences) and, in part, from an IgG3 molecule (e.g., the central hinge sequence). In another example, the Fc region or portion may include a chimeric hinge that is partly derived from an IgG1 molecule and partly derived from an IgG4 molecule. In another example, the chimeric hinge may include upper and lower hinge domains derived from an IgG4 molecule and a central hinge domain derived from an IgG1 molecule. Such a chimeric hinge can be made, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the central hinge domain of the IgG4 hinge region. In some other embodiments, the chimeric hinge can include amino acids from an IgG2 antibody and / or the Ser228Pro mutation at EU positions 233 - 236, and the remaining amino acids of the hinge are from an IgG4 antibody (e.g., a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP (SEQ ID NO: 61)). Further chimeric hinges that can be used in the Fc portion of the antibodies according to the present disclosure are described in US2005 / 0163783A1.

[0213] 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., derived from the Fc region or Fc portion of a human IgG molecule). However, the polypeptide may include one or more amino acids from another mammalian species. For example, a primate Fc portion or primate binding site may be included in the polypeptide of interest. Alternatively, one or more mouse amino acids may be present in the Fc portion or Fc region.

[0214] c.HBC34 antibody In certain embodiments, the anti-HBV antibody is HBC34 or a modified variant thereof. HBC34 is a human antibody against HBsAg with high neutralizing activity. HBC34 binds to the antigen loop of HBsAg with high affinity (in the pM range), recognizes all 10 HBV genotypes and 18 mutants, and binds to globular SVP at low stoichiometry. The activity of HBC34 measured diagnostically by immunoassay is 5000 IU / mg. For comparison, the activity of HBIG is approximately 1 IU / mg.

[0215] As used herein, the terms "HBC34 antibody" and "HBC antibody," unless otherwise specified, can include the wild-type HBC34 antibody or a modified variant thereof (e.g., HBC34 and HBC34 variants described in Table 3).

[0216] Table 3 shows the amino acid sequences of the CDRs, heavy chain variable region (VH), and light chain variable region (VL) of HBC34 and its modified variants. The full-length heavy chain (HC) and light chain (LC) amino acid sequences of exemplary antibodies of the present disclosure are also shown.

[0217]

Table 5-1

[0218]

Table 5-2

[0219]

Table 5-3

[0220]

Table 5-4

[0221]

Table 5-5

[0222] In certain embodiments, the anti-HBV antibody comprises one or more of the amino acid sequences set forth in Table 3. In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure has at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the CDR sequences, VH sequences, VL sequences, HC sequences, and / or LC sequences shown in Table 3 and comprises an amino acid sequence having such identity. In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment may comprise the CDR, V H , V L , HC, and / or LC sequences shown in Table 3. Exemplary methods for synthesizing antibodies having the sequences shown in Table 3, as well as experimental data demonstrating binding and neutralization by AB01, are described in International Application Publication No. WO 2020 / 132091A2, which are incorporated herein by reference.

[0223] In some embodiments, the antibody or antigen-binding fragment of the present disclosure comprises (i) the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 45, or 46, and 47, respectively, and (ii) the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 49, or 50, and 51 or 52, respectively.

[0224] Thus, in some embodiments, CDRH1, CDRH2, and CDRH3 are those set forth in SEQ ID NOs: 44, 45, and 47, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 are those set forth in SEQ ID NOs: 44, 46, and 47, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are those set forth in SEQ ID NOs: 48, 49, and 52, respectively. In some embodiments, CDRL1, CDRL2, and CDRL3 are those set forth in SEQ ID NOs: 48, 50, and 52, respectively.

[0225] It will be understood that the antibodies or antigen-binding fragments of the present disclosure can include any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 44-50 and 52.

[0226] In certain embodiments, the antibody or antigen-binding fragment of the present disclosure comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 45, and 47, respectively, and the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 49, and 52, respectively.

[0227] In certain embodiments, the antibody or antigen-binding fragment of the present disclosure comprises (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 the amino acid sequence set forth in 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 the amino acid sequence set forth in SEQ ID NO: 53.

[0228] In certain embodiments, the antibody or antigen-binding fragment of the present disclosure comprises (a) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 59, and (b) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 57.

[0229] d. Pharmaceutical composition In some embodiments, the antibody or antigen-binding fragment thereof for combination therapy is provided as a pharmaceutical composition comprising an anti-HBV antibody and optionally a pharmaceutically acceptable carrier. In some embodiments, the composition may comprise an anti-HBV antibody, and the antibody may constitute at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of the total protein in the composition. In such a composition, the antibody may be in a purified form.

[0230] The pharmaceutical composition of anti-HBV antibody may contain an antibacterial agent, especially when packaged in a multiple-dose form. They may contain a surfactant, for example, Tween (polysorbate) such as Tween80. When present, the detergent typically exists at a low level, for example, less than 0.01%. The composition may also contain a sodium salt (e.g., sodium chloride) for tonicity. For example, in some embodiments, the pharmaceutical composition contains NaCl at a concentration of 10±2mg / mL.

[0231] Furthermore, the pharmaceutical composition may contain a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) at about 15-30mg / mL (e.g., 25mg / mL), especially when they are lyophilized or contain a material reconstituted from a lyophilized material. The pH of the composition for lyophilization can be adjusted to 5-8, or 5.5-7, or about 6.1 before lyophilization.

[0232] The antibody composition of the present disclosure may also contain one or more immunomodulators. In some embodiments, one or more of the immunomodulators include an adjuvant.

[0233] The method for preparing a pharmaceutical composition of anti-HBV antibody may include (i) a step of preparing the antibody, and (ii) a step of mixing the purified antibody with one or more pharmaceutically acceptable carriers.

[0234] In some embodiments, the pharmaceutical composition containing the anti-HBV antibody (e.g., AB01) described herein contains the antibody at a dose of 100mg, 150mg, 200mg, 250mg, or 300mg. In some embodiments, the pharmaceutical composition containing the anti-HBV antibody (e.g., AB01) described herein contains the antibody at a dose of 100mg to 300mg. In some embodiments, the pharmaceutical composition containing the anti-HBV antibody (e.g., AB01) described herein contains the antibody at a dose of 100mg to 200mg.

[0235] In some embodiments, a pharmaceutical composition comprising an anti-HBV antibody (e.g., AB01) described herein contains the antibody at a dose of 200 mg.

[0236] IV. Methods of Treatment Using Combination Therapy In some embodiments, the present disclosure provides a method for treating HDV infection or an HDV-related disease in a subject.

[0237] In some embodiments, there is provided a method for treating HDV infection or an HDV-related disease in a subject, the method comprising administering to the subject an siRNA and an antibody described herein. In some embodiments, SIRNA01 and AB01 are administered to the subject.

[0238] In some embodiments, provided is a method for treating HDV infection or an HDV-related disease in a subject, the method comprising administering to the subject the siRNA and antibody described herein and administering to the subject a nucleoside / nucleotide reverse transcriptase inhibitor. As used herein, "nucleoside / nucleotide reverse transcriptase inhibitor" or "nucleos(t)ide reverse transcriptase inhibitor" (NRTI) refers to an inhibitor of DNA replication that is structurally similar to a nucleotide or nucleoside and specifically inhibits the replication of HBV cccDNA by inhibiting the action of HBV polymerase and does not significantly inhibit the replication of host (i.e., human) DNA. Such inhibitors include tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil (TD), tenofovir alafenamide (TAF), lamivudine, adefovir, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, famciclovir, N-acetyl-cysteine (NAC), C 1323, Theradigm-HBV, thymosin α, ganciclovir, besifovir (ANA-380 / LB-80380), and tenofovir exaliades (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 disoproxil (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.

[0239] As used herein, "subject" includes animals such as mammals, including any mammal that can be infected with HBV (e.g., humans, non-human primates such as monkeys or chimpanzees), or animals considered to be an acceptable clinical model for HBV infection, an HBV-AAV mouse model (see, e.g., Yang et al., Cell and Mol Immunol 2014, 11:71) or an HBV 1,3xfs transgenic mouse model (Guidotti et al., J. Virol. 1995, 69:6158). In some embodiments, the subject has a hepatitis B virus (HBV) infection. In some other embodiments, the subject has both a hepatitis B virus (HBV) infection and a hepatitis D virus (HDV) infection. In some other embodiments, the subject is a human, e.g., a human having an HBV infection, particularly a chronic hepatitis B virus (CHBV) infection.

[0240] As used herein, the terms "treating" or "treatment" refer to 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, the presence of serum or liver HBV antigens, such as HBsAg or HBeAg, an increase in ALT, an increase in AST (the normal range is typically considered to be about 10 - 34 U / L), the absence or low levels of anti-HBV antibodies; liver damage; cirrhosis; delta hepatitis; acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; hepatic fibrosis; end-stage liver disease, hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, low-grade fever; myalgia; fatigue; disturbances in taste and smell (aversion to food and tobacco); or right upper abdominal pain (intermittent, mild to moderate); hepatic encephalopathy; drowsiness; sleep pattern disturbances; mental confusion; coma; ascites; gastrointestinal bleeding; coagulation disorders; jaundice; hepatomegaly (a slightly enlarged, soft liver); splenomegaly; palmar erythema; spider angiomas; muscle wasting; spider angioma; vasculitis; variceal bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusae); an ALT level higher than the AST level; an increase in gamma-glutamyl transpeptidase (GGT) (the normal range is typically considered to be about 8 - 65 U / L) and alkaline phosphatase (ALP) levels (the normal range is typically considered to be about 44 - 147 IU / L (international units per liter) and is less than 3 times the ULN); a slightly low albumin level; an increase in serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; an increase in the erythrocyte sedimentation rate (ESR); a shortening of the erythrocyte survival period; hemolysis; thrombocytopenia; prolongation of the international normalized ratio (INR); the presence of serum or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), or HBV DNA; an increase in bilirubin levels; hyperglobulinemia; the presence of tissue-nonspecific antibodies, such as anti-smooth muscle antibody (ASMA) or antinuclear antibody (ANA) (10 - 20%); the presence of tissue-specific antibodies, such as antibodies against the thyroid (10 - 20%); high levels of rheumatoid factor (RF); a decrease in the number of platelets and white blood cells; with lobular, degenerative, and regenerative hepatocyte changes, with inflammation;Whether it is detectable or undetectable, it refers to beneficial or desired results including but not limited to the alleviation or improvement of mainly centrilobular necrosis. For example, the possibility of developing liver fibrosis, for example, in an individual having one or more risk factors for liver fibrosis, for example, chronic hepatitis B infection, either does not develop liver fibrosis or develops liver fibrosis at a lower severity compared to a group having the same risk factors and not receiving treatment as described herein. "Treatment" can also mean extending survival compared to the survival expected in the absence of treatment.;

[0241] As used herein, the terms "preventing" or "prevention" refer to not developing a disease, disorder, or condition, or reducing (e.g., to a clinically relevant amount) the development of signs or symptoms associated with such disease, disorder, or condition, or delaying (e.g., by days, weeks, months, or years) the manifestation of signs or symptoms. Prevention may require administration of two or more doses.

[0242] Doses are often expressed in relation to body weight. Thus, even when the term "body weight" is not explicitly mentioned, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) of body weight".

[0243] In some embodiments, the treatment of HBV infection results in a “functional cure” of hepatitis B. As used herein, a functional cure is understood as the clearance of circulating HBsAg and may involve a conversion to a state where HBsAg antibodies are detectable using clinically relevant assays. For example, detectable antibodies can include a signal higher than 10 mIU / mL when measured by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay. A functional cure does not require the clearance of all replication forms of HBV (e.g., cccDNA from the liver). Anti-HBs seroconversion occurs spontaneously in about 0.2–1% / year of chronically infected patients. However, even after anti-HBs seroconversion, low-level persistence of HBV is often observed over decades, indicating that a functional rather than a complete cure has occurred. Without being bound to a particular mechanism, the immune system may be able to continue suppressing HBV in a state where a functional cure has been achieved. A functional cure allows for the interruption of any treatment for HBV infection. However, it is understood that a “functional cure” for HBV infection may not be sufficient to prevent or treat a disease or condition resulting from HBV infection, such as fibrosis, HCC, or cirrhosis. In some particular embodiments, “functional cure” can refer to a sustained decrease in serum HBsAg (e.g., less than 1 IU / mL) for at least 3 months, at least 6 months, or at least 1 year after the start or completion of a treatment regimen.

[0244] As used herein, the terms “hepatitis B virus-related disease” or “HBV-related disease” refer to a disease or disorder caused by or associated with HBV infection or replication. The term “HBV-related disease” includes diseases, disorders, or conditions that benefit from a decrease in HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, delta hepatitis, acute hepatitis B, acute fulminant hepatitis B, chronic hepatitis B, fibrosis, end-stage liver disease, and hepatocellular carcinoma.

[0245] In some embodiments, the HBV-related disease is chronic hepatitis B.

[0246] Chronic hepatitis B is defined by one of the following criteria: (1) serum HBsAg, HBV DNA, or HBeAg is positive on two occasions at least six months apart (any combination of these tests performed at six-month intervals is permitted); (2) immunoglobulin M (IgM) antibody to HBV core antigen (IgM anti-HBc) is negative and the result for one of the following tests: HBsAg, HBeAg, or HBV DNA is positive. Chronic HBV typically includes liver inflammation lasting longer than six months.

[0247] Subjects with chronic HBV are HBsAg positive and have high viremia (>10 4 HBV-DNA copies / mL of blood) or low viremia (<10 3Has any of the following: (HBV-DNA copies / mL of blood). In certain embodiments, the subject has been infected with HBV for at least 5 years. In certain embodiments, the subject has been infected with HBV for at least 10 years. In certain embodiments, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease can be immunotolerant or have an inactive chronic infection without any evidence of active disease, and they are also asymptomatic. Patients with chronic active hepatitis, especially those in a replicative state, can have symptoms similar to acute hepatitis. Subjects with chronic hepatitis B disease may have an active chronic infection with necroinflammatory liver disease, increased hepatocyte turnover in the absence of detectable necroinflammation, or an inactive chronic infection without evidence of active disease and are also asymptomatic. The persistence of HBV infection in chronic HBV subjects is a result of cccHBV DNA. In some embodiments, the subject with chronic HBV is HBeAg positive. In some other embodiments, the subject with chronic HBV is HBeAg negative. Subjects with chronic HBV have a serum HBV DNA level of less than 105 and persistently elevated transaminases, such as ALT, AST, and gamma-glutamyl transferase. Subjects with chronic HBV can have a liver biopsy score of less than 4 (e.g., necroinflammatory score). In some embodiments, the ALT ULN value is 34 IU / mL for females and 43 IU / mL for males.

[0248] In some embodiments, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or delta hepatitis virus (HDV) is a human pathogen. However, this virus is defective and depends on the essential helper function provided by HBV for transmission. Indeed, HDV requires an associated or existing HBV infection for the viral envelope, especially containing the surface antigen of hepatitis B, to become infectious and replicate. HDV can cause severe acute and chronic forms of liver disease associated with HBV. Hepatitis D infection or delta hepatitis is highly prevalent in some African countries, the Amazon region, and the Middle East, but its prevalence is low in industrialized countries outside the Mediterranean.

[0249] HDV infection can occur either through co-infection (simultaneous infection) with HBV or superimposed on chronic hepatitis B or hepatitis B carrier state (superinfection). Both superinfection and co-infection with HDV typically result in more severe complications compared to infection with HBV alone. These complications include a higher likelihood of experiencing liver failure in acute infection and a rapid progression to cirrhosis, with an increased chance of developing liver cancer in chronic infection. In combination with the hepatitis B virus, hepatitis D has the highest mortality rate among all hepatitis infections, which is 20%.

[0250] In some embodiments, the HBV-related disease is acute hepatitis B. Acute hepatitis B involves inflammation of the liver that lasts less than 6 months. Typical symptoms of acute hepatitis B are malaise, anorexia, nausea, and vomiting. Very high aminotransferase levels (above 1000 U / L) and hyperbiliverinemia are often observed.

[0251] Severe cases of acute hepatitis B can rapidly progress to acute liver failure characterized by a decrease in liver synthetic function. This is often 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 can progress to chronic hepatitis B.

[0252] In some embodiments, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B have the symptoms of acute hepatitis, as well as additional symptoms of confusion or coma (due to the liver's inability to detoxify chemicals) and bruising or bleeding (due to a lack of blood clotting factors).

[0253] Subjects with HBV infection, such as those with chronic HBV, can develop liver fibrosis. Thus, 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 (excessive fibrous connective tissue) and a structurally abnormal nodule of normal liver structure.

[0254] 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 can progress as a result of liver fibrosis (i.e., decompensated liver) to the point where the body can no longer compensate for, e.g., a decline in liver function, which can lead to, e.g., mental and neurological symptoms and liver failure.

[0255] Subjects with HBV infection, e.g., chronic HBV, may develop hepatocellular carcinoma (HCC), also known as malignant hepatoma. Thus, in some embodiments, the HBV-related disease is HCC. HCC generally develops in subjects with CHB and can be fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cell), or clear cell.

[0256] "HDV-related disease" or "delta hepatitis virus-related disease" is a disease or disorder associated with the expression of HDV. Exemplary HDV-related 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.

[0257] As used herein, a "therapeutically effective amount" is intended to include an amount of siRNA, anti-HBV antibody, or other active agent (e.g., tenofovir) that is sufficient to achieve treatment of a disease (e.g., by reducing or maintaining an existing disease or one or more symptoms of the disease) when administered to a patient for treating a subject having an HBV infection or an HBV-related disease. The "therapeutically effective amount" can vary depending on the active agent(s), how they are administered, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by HBV gene expression, if present, the type of pretreatment or co-treatment, and other individual characteristics of the patient being treated. A therapeutically effective amount may require administration of two or more doses.

[0258] "Therapeutically effective amount" also includes an amount of siRNA, anti-HBV antibody, or other active agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutic agents (e.g., siRNA, anti-HBV antibody) used in the methods of the present disclosure can be administered in an amount sufficient to provide a reasonable benefit / risk ratio applicable to such treatment.

[0259] As used herein, the term "sample" includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, lymph, urine, saliva, and the like. A tissue sample can include a sample from a tissue, organ, or local area. For example, a sample can be derived from a particular organ, a part of an organ, or a body fluid or cell within those organs. In certain embodiments, the sample can be derived from the liver (e.g., whole liver or a particular segment of the liver or a particular type of cell in the liver, such as hepatocytes, etc.). In certain embodiments, "a sample derived from a subject" refers to blood, or plasma or serum obtained from blood drawn from the subject. In further embodiments, "a sample derived from a subject" refers to liver tissue (or a subcomponent thereof) or blood tissue (or a subcomponent thereof, such as serum) derived from the subject.

[0260] In some embodiments of the methods described herein, the anti-HBV antibody is administered subcutaneously. In some embodiments, the anti-HBV antibody is administered every four weeks. In some embodiments, the anti-HBV antibody is administered every eight 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 subject is administered the anti-HBV antibody for a period of 8 weeks, 24 weeks, 48 weeks, or 88 weeks.

[0261] In some embodiments of the methods described herein, the siRNA is administered subcutaneously. In some embodiments, the siRNA is administered every four weeks. In some embodiments, the siRNA is administered every eight weeks. In some embodiments, the siRNA is administered at a dose of 20 mg to 900 mg. In some embodiments, the siRNA is administered at a dose of 100 mg to 300 mg. In some embodiments, the siRNA is administered at a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg. In some embodiments, the siRNA is administered at a dose of 200 mg. In some embodiments, the subject is administered the siRNA over a period of 8 weeks, 24 weeks, 48 weeks, or 88 weeks.

[0262] 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.

[0263] In some embodiments of the methods described herein, the subject is administered siRNA and the anti-HBV antibody starting on the same day.

[0264] In some embodiments of the methods described herein, the subject is administered siRNA and an anti-HBV antibody starting on the same day for a period of 8 weeks, 24 weeks, 48 weeks, or 88 weeks.

[0265] In some embodiments of the methods described herein, the subject achieves at least one of a reduction in HDV RNA of 2 log10 or more compared to baseline (i.e., pre-treatment), HDV RNA below the LOQ at week 24, and ALT below the upper limit of normal (ULN) at week 24. In some embodiments, the ALT ULN value is 34 IU / mL for females and 43 IU / mL for males.

[0266] The present disclosure also provides an antibody, siRNA, and / or NRTI described herein for use in the methods described above, and a pharmaceutical composition comprising the same. Use of an antibody, siRNA, and / or NRTI.

[0267] V. Kits for combination therapy Kits are also provided herein that include components of a therapy for treating HDV infection or an HDV-related disease. The kit can include siRNA (e.g., SIRNA01), an anti-HBV antibody (e.g., AB01). The kit can include siRNA (e.g., SIRNA01), an anti-HBV antibody (e.g., AB01), and an NRTI (e.g., tenofovir disoproxil fumarate, tenofovir disoproxil). The kit can further include instructions for preparing and / or administering the components of the HBV combination therapy.

[0268] VI. Exemplary embodiments In some embodiments, the present disclosure provides the following.

[0269] 1. A method of treating hepatitis D virus (HDV) infection in a subject in need thereof, the method comprising administering to the subject (a) an anti-HBV antibody, and (b) A method comprising administering an siRNA targeting HBV mRNA.

[0270] 2. A method of treating an HDV-related disease in a subject in need thereof, the method comprising administering to the subject (a) an anti-HBV antibody, and (b) an siRNA targeting HBV mRNA.

[0271] 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.

[0272] 4. The method according to any one of the preceding embodiments, wherein the subject has chronic hepatitis B virus (HBV) / HDV coinfection.

[0273] 5. The method according to any one of the preceding embodiments, wherein the subject has positive serum HBsAg, HBV DNA, or HBeAg at least twice at intervals of at least 6 months before treatment.

[0274] 6. The method according to any one of the preceding embodiments, wherein the subject is tested positive for HDV antibody or HDV RNA before treatment.

[0275] 7. The method according to any one of the preceding embodiments, wherein the subject is tested positive for both HDV antibody and HDV RNA before treatment.

[0276] 8. The method according to any one of the preceding embodiments, wherein the subject is tested positive for HDV antibody for at least 6 months before treatment.

[0277] 9. The method according to any one of the preceding embodiments, wherein the subject has HDV RNA of 500 IU / mL or more before treatment.

[0278] 10. The method according to any one of the preceding embodiments, wherein the subject has an HBsAg level of more than 0.05 IU / mL before treatment.

[0279] 11. The method according to any one of the preceding embodiments, wherein the subject has an HBsAg level of more than 10,000 IU / mL before treatment.

[0280] 12. The method according to any one of the preceding embodiments, wherein the subject has chronic hepatitis.

[0281] 13. The method according to any one of the preceding embodiments, wherein the subject has an alanine aminotransferase (ALT) level that is above the upper limit of normal (ULN) and less than 5 times the ULN before treatment.

[0282] 14. The method according to any one of the preceding embodiments, wherein the subject has an ALT level above the ULN and an aspartate aminotransferase (AST) level above the ULN before treatment.

[0283] 15. The method according to any one of the preceding embodiments, wherein the subject has an ALT level less than 5 times the ULN and an AST level less than 5 times the ULN before treatment.

[0284] 16. The method according to any one of the preceding embodiments, wherein the subject is non-cirrhotic.

[0285] 17. The method according to embodiment 16, wherein the subject has a histological data meta-analysis in viral hepatitis (Meta-Analysis of Histological Data in Viral Hepatitis: METAVIR) F0-F3 or a liver biopsy by Fibroscan (registered trademark) with a liver elastography of less than 12 kilopascals (kPa) within 12 months before treatment.

[0286] 18. The method according to embodiment 16 or embodiment 17, wherein the subject has a creatinine clearance (CLcr) of 30 mL / min or more calculated by the Cockcroft-Gault formula before treatment.

[0287] 19. The method according to any one of Embodiments 1 to 15, wherein the subject has cirrhosis.

[0288] 20. The method according to Embodiment 19, wherein the subject has a liver biopsy of METAVIR F4 or a liver elastography (Fibroscan (registered trademark)) of 12 kilopascals (kPa) or more within 12 months before treatment.

[0289] 21. The method according to Embodiment 19 or Embodiment 20, wherein the subject has a creatinine clearance (CLcr) of 60 mL / min or more calculated by the Cockcroft-Gault formula before treatment.

[0290] 22. The method according to any one of Embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 5 or more before treatment.

[0291] 23. The 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.

[0292] 24. The method according to any one of Embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score of 7 or more before treatment.

[0293] 25. The 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.

[0294] 26. The method according to any one of Embodiments 19 to 21, wherein the subject has a Child-Pugh-Turcotte (CPT) score of more than 10 before treatment.

[0295] 27. The method according to any one of the preceding embodiments, wherein the subject has not been pre-administered an anti-HIV antibody.

[0296] 28. The method according to any one of the preceding embodiments, wherein the subject has not been pre-administered an siRNA targeting HBV mRNA.

[0297] 29. The 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.

[0298] 30. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is a human antibody.

[0299] 31. The method according to any one of the preceding embodiments, wherein the antibody is HBC34 or a non-natural variant of HBC34.

[0300] 32. The anti-HBV antibody is (i) the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 45, or 46, and 47, respectively, and (ii) the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 49, or 50, and 52, respectively, according to the method of any one of the preceding embodiments.

[0301] 33. The anti-HBV antibody is (i) the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 45, and 47, respectively, and (ii) the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 49, and 52, respectively, according to the method of embodiment 32.

[0302] 34. The anti-HBV antibody is (i) the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 46, and 47, respectively, and (ii) the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 50, and 52, respectively, according to the method of embodiment 32.

[0303] 35. The anti-HBV antibody is (a) A light chain variable domain (VL) that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in 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 set forth in SEQ ID NO: 53, the method according to any one of the preceding embodiments.

[0304] 36. The anti-HBV antibody is (a) A light chain variable domain (VL) amino acid sequence set forth in SEQ ID NO: 55, and (b) A heavy chain variable domain (VH) amino acid sequence set forth in SEQ ID NO: 53, the method according to any one of the preceding embodiments.

[0305] 37. The anti-HBV antibody is (a) A light chain that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 59, and (b) A heavy chain that is at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 57, the method according to any one of the preceding embodiments.

[0306] 38. The anti-HBV antibody is (a) A light chain amino acid sequence set forth in SEQ ID NO: 59, and (b) A heavy chain amino acid sequence set forth in SEQ ID NO: 57, the method according to any one of the preceding embodiments.

[0307] 39. The anti-HBV antibody is a monoclonal antibody, the method according to any one of the preceding embodiments.

[0308] 40. The anti-HBV antibody is a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating immune effectors, the method according to any one of the preceding embodiments.

[0309] 41. The method according to embodiment 40, wherein the second specificity stimulates a cytotoxic or vaccine effect.

[0310] 42. The method according to any one of the preceding embodiments, wherein the subject is a human, a therapeutically effective amount of an anti-HBV antibody is administered, and the therapeutically effective amount is from about 3 mg / kg to about 30 mg / kg.

[0311] 43. The method according to any one of the preceding embodiments, wherein the siRNA inhibits the expression of an HBV transcript encoding an HBsAg protein, an HBcAg protein, and an HBx protein, or an HBV DNA polymerase protein.

[0312] 44. The method according to any one of the preceding embodiments, wherein the siRNA comprises a sense strand and an antisense strand that form a double-stranded region, the sense strand comprises at least 15 consecutive nucleotides that differ from nucleotides 1579 to 1597 of SEQ ID NO: 1 by no more than 3 nucleotides, and T is replaced by U.

[0313] 45. The method according to any one of the preceding embodiments, wherein the siRNA comprises a sense strand and an antisense strand, the sense strand comprises nucleotides 1579 to 1597 of SEQ ID NO: 1, and T is replaced by U.

[0314] 46. The method according to any one of the preceding embodiments, wherein the siRNA binds to at least 15 consecutive nucleotides of a target encoded by the P gene, nucleotides 2309 to 3182 and 1 to 1625 of NC_003977.2; the S gene (encoding the L, M, and S proteins), nucleotides 2850 to 3182 and 1 to 837 of NC_003977.2; HBx, nucleotides 1376 to 1840 of NC_003977.2; or the C gene, nucleotides 1816 to 2454 of NC_003977.2.

[0315] 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 of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0316] 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).

[0317] 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).

[0318] 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).

[0319] 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).

[0320] 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).

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] The method according to any one of the preceding embodiments 1, wherein each strand of the RNAi agent has 15 to 30 nucleotides.

[0330] The method according to any one of the preceding embodiments, wherein each strand of the RNAi agent has 19 to 30 nucleotides.

[0331] The method according to any one 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 the sense strand is conjugated to a ligand bound to the 3'-end.

[0332] The method according to embodiment 63, wherein the ligand is one or more GalNAc derivatives bound via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

[0333] The method according to embodiment 63 or 64, wherein the ligand is as follows.

[0334] [Chemistry]

[0335] 66. The siRNA is conjugated to a ligand as shown in the following structure,

[0336] [Chemistry] The method according to embodiment 65, wherein X is O or S.

[0337] 67. The method according to embodiment 66, wherein X is O.

[0338] 68. At least one nucleotide of the siRNA is a deoxynucleotide, 3'-terminal deoxythymidine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, unlocked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, non-natural base containing nucleotide, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing phosphorothioate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, adenosine-glycol nucleic acid, or nucleotide containing 5'-phosphate mimic, a modified nucleotide, the method according to any one of the preceding embodiments.

[0339] 69. The method according to any one of the preceding embodiments, wherein the siRNA comprises a phosphate backbone modification, 2'-ribose modification, 5'-triphosphate modification, or GalNAc conjugation modification.

[0340] 70. The method according to any one of the preceding embodiments, wherein the phosphate backbone modification comprises a phosphorothioate bond.

[0341] 71. The method according to any one of the preceding embodiments, wherein the siRNA comprises a 2'-fluoro or 2'-O-methyl substitution.

[0342] 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.

[0343] 73. The siRNA comprises a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-methyluridine-3'-phosphate, respectively, wherein 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-glycol nucleic acid (GNA), s is a phosphorothioate bond, and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol, according to any one of the preceding embodiments.

[0344] 74. The siRNA comprises a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 7) and an antisense strand comprising 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 8), a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-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, s is a phosphorothioate bond, The method according to any one of the preceding embodiments, wherein L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0345] 75. L96 is conjugated to the sense strand as shown in the following structure,

[0346]

Chemical formula

[0347] 76. The method according to any one of the preceding embodiments, wherein the subject is human, a therapeutically effective amount of siRNA is administered to the subject, and the effective amount of siRNA is about 1 mg / kg to about 8 mg / kg.

[0348] 77. The method according to any one of the preceding embodiments, wherein the siRNA is administered to the subject twice a day, once a day, every two days, every two days, every three days, twice a week, once a week, every other week, every four weeks, or once a month.

[0349] 78. The method according to any one of the preceding embodiments, further comprising administering a nucleoside reverse transcriptase inhibitor (NRTI) to the subject.

[0350] 79. The method according to any one of the preceding embodiments, wherein the subject has been previously administered an NRTI.

[0351] 80. The method according to 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), clevudine, ritonavir, dipivoxil, lobucavir, famciclovir, N-acetyl-cysteine (NAC), PC1323, teradime-HBV, thymosin α, and ganciclovir, besifovir (ANA-380 / LB-80380), or tenofovir-exalides (TLX / CMX157).

[0352] 81. The method according to any one of embodiments 78 to 80, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), or tenofovir disoproxil (TD).

[0353] 82. The method according to any one of embodiments 78 to 81, wherein the NRTI is tenofovir disoproxil fumarate (TDF).

[0354] 83. (a) The anti-HBV antibody comprises or consists of the light chain amino acid sequence set forth in SEQ ID NO: 59 and the heavy chain amino acid sequence set forth in SEQ ID NO: 57, and (b) The siRNA comprises or consists of 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), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-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-glycol nucleic acid (GNA), s is a phosphorothioate bond, The method according to any one of the preceding embodiments, wherein L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline.

[0355] 84. (a) The anti-HBV antibody comprises or consists of the light chain amino acid sequence set forth in SEQ ID NO: 59 and the heavy chain amino acid sequence set forth in SEQ ID NO: 57, (b) The siRNA comprises or consists of 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), a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-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-glycol nucleic acid (GNA), s is a phosphorothioate bond, L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyproline, (c) The method according to any one of embodiments 72 to 76, wherein the NRTI is tenofovir disoproxil fumarate (TDF) or entecavir.

[0356] 85. (a) The anti-HBV antibody comprises the light chain amino acid sequence set forth in SEQ ID NO: 59 and the heavy chain amino acid sequence set forth in SEQ ID NO: 57, (b) The siRNA comprises a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-methyluridine-3'-phosphate, respectively, wherein 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-glycol nucleic acid (GNA), s is a phosphorothioate bond, L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol, and the method according to any one of the preceding embodiments.

[0357] 86. (a) The anti-HBV antibody consists of the light chain amino acid sequence set forth in SEQ ID NO: 59 and the heavy chain amino acid sequence set forth in SEQ ID NO: 57, (b) The siRNA consists of 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), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-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-glycol nucleic acid (GNA), s is a phosphorothioate bond, The method according to any one of the preceding embodiments, wherein L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0358] The method according to any one of the preceding embodiments, wherein the anti-HBV antibody and the siRNA are administered to a subject according to the procedure described in any of FIGS. 1-7C and 12A-19D.

[0359] The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered subcutaneously.

[0360] The method according to any one of the preceding embodiments, wherein the siRNA is administered subcutaneously.

[0361] The method according to any one of embodiments 78-89, wherein the NRTI is administered orally.

[0362] The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered every two weeks.

[0363] The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered every four weeks.

[0364] The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered every eight weeks.

[0365] The method according to any one of the preceding embodiments, wherein the siRNA is administered every four weeks.

[0366] 95. The method according to any one of the preceding embodiments, wherein the siRNA is administered every 8 weeks.

[0367] 96. The method according to any one of embodiments 78 to 95, wherein the NRTI is administered daily.

[0368] 97. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered at a dose of 300 mg.

[0369] 98. The method according to any one of embodiment 1, wherein the siRNA is administered at a dose of 200 mg.

[0370] 99. The method according to any one of embodiments 78 to 98, wherein the NRTI is administered at a dose of 300 mg.

[0371] 100. The method according to any one of embodiments 78 to 98, wherein the NRTI is administered at a dose of 245 mg.

[0372] 101. The method according to any one of the preceding embodiments, wherein the subject is administered siRNA and anti-HBV antibody starting on the same day.

[0373] 102. The method according to any one of the preceding embodiments, wherein the subject is a human.

[0374] 103. The method according to any one of the preceding embodiments, wherein the subject is administered siRNA and anti-HBV antibody for up to 96 weeks, 96 weeks, at least 96 weeks, or 96 weeks or more.

[0375] 104. An anti-HBV antibody and an siRNA targeting HBV mRNA for use in the method according to any one of embodiments 1 to 103.

[0376] 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.

[0377] Use of an anti-HBV antibody in the manufacture of a first medicament and use of an siRNA targeting HBV mRNA in the manufacture of a second medicament, wherein the first medicament and the second medicament are used in combination therapy by the method according to any one of Embodiments 1 to 103, use of the anti-HBV antibody and use of the siRNA.

[0378] An anti-HBV antibody, an siRNA targeting HBV mRNA, and an NRTI for use in the method according to any one of Embodiments 78 to 103.

[0379] Use of an anti-HBV antibody, an siRNA targeting HBV mRNA, and an NRTI in the manufacture of a medicament for use in the method according to any one of Embodiments 78 to 103.

[0380] Use of an anti-HBV antibody in the manufacture of a first medicament, use of an siRNA targeting HBV mRNA in the manufacture of a second medicament, and use of an NRTI in the manufacture of a third medicament, wherein the first, second, and third medicaments are used in combination therapy by the method according to any one of Embodiments 78 to 103, use of the anti-HBV antibody, use of the siRNA, and use of the NRTI.

[0381] 110. A kit comprising: A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient, and A pharmaceutical composition comprising an siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient.

[0382] 111. The kit according to Embodiment 110, further comprising instructions for completing the method according to any one of Embodiments 1 to 103.

[0383] 112. A kit comprising: A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient, A pharmaceutical composition comprising an siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient, and A kit comprising a pharmaceutical composition comprising an NRTI and a pharmaceutically acceptable excipient.

[0384] The kit according to embodiment 104, further comprising instructions for completing the method according to any one of embodiments 78 to 103.

Examples

[0385] Example 1 Clinical evaluation of monotherapy or combination therapy for treating chronic HDV infection In a phase 2 multi-center open-label clinical trial in non-cirrhotic and compensated cirrhotic (CPT-A to CPT-C) human patients with chronic HBV / HDV co-infection who are not currently receiving treatment for HDV, evaluate the safety, efficacy, and tolerability of anti-HBV antibodies in combination with anti-HBV siRNA.

[0386] Background Hepatitis D virus (HDV), the only virus of the deltavirus genus, requires hepatitis B virus (HBV) coinfection for entry into hepatocytes, intrahepatic spread, and seeding. HBV / HDV coinfection is associated with a more rapid and severe course of liver disease compared to other forms of viral hepatitis (Lempp 2016; Lucifora 2020). Based on sequence variation in HDV isolates, eight genotypes have been classified (Le Gal 2017). Genotype 1 is globally distributed, and infections range from fulminant hepatitis to asymptomatic chronic liver disease. HDV genotypes 2–8 have a limited geographic distribution (Niro 2012). The median HDV prevalence in hepatitis B surface antigen (HBsAg) carriers is estimated to be approximately 5%. However, in certain locations, up to 80% of HBsAg carriers have laboratory evidence of current or past HDV infection (Rizzetto 2021). Due to significant knowledge gaps, epidemiological data on HDV prevalence may be grossly underestimated, ranging from 12 million to 72 million (Chen 2019; Miao 2020; Stockdale 2020).

[0387] HDV is a defective RNA satellite virus that does not encode an envelope protein and completes its life cycle depending on HBsAg. Therefore, HDV must either coinfect or superinfect hepatocytes infected with HBV. The HDV virion has an approximately spherical structure of 35 - 43 nm without a distinct nucleocapsid. The ribonucleoprotein contains the only protein encoded by HDV, the 60 large delta antigen and the small δ antigen. Extracellular HDV virions contain genomic HDV RNA (a single-stranded negative-sense covalently closed circular RNA molecule of 1,668 - 1,697 nucleotides, depending on the genotype (Le Gal 2017)). Although HDV does not encode an RNA-dependent RNA polymerase, instead, it promotes RNA-directed RNA synthesis for transcription and replication of its genome in the nucleus of hepatocytes using a double rolling circle mechanism, depending on the host DNA-dependent RNA polymerase (Chang 2008; Modahl 2000; Sureau 2016; Urban 2021).

[0388] HDV virions can be assembled in hepatocytes using all forms of HBsAg derived from cccDNA (in the case of HBV coinfected 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 the HDV Delta antigen (L-HDAg) (Freitas 2014, Shirvani-Dastgerdi 2015). Since the preS1 domain of L-HBsAg mediates the interaction between HBsAg and NTCP, the large form of HBsAg (L-HBsAg) is required to form infectious virions. NTCP is exclusively localized to the basolateral membrane of differentiated mammalian hepatocytes and is a hepatocyte-specific basolateral Na+-dependent bile acid transporter. Therefore, although HDV can efficiently replicate its genome and express hepatitis delta antigen, L-HBsAg is required to form infectious progeny virions.

[0389] HDV infection can occur in two ways: HBV-HDV co-infection and HDV superinfection. Co-infection occurs when HBV and HDV are transmitted simultaneously to HBV-susceptible individuals, while superinfection occurs when HBsAg-positive individuals (typically, those with chronic HBV infection) acquire HDV. HBV / HDV co-infection occurs in 5-15% of cases of HDV infection and often leads to acute hepatitis, which is more severe than acute HBV in many cases. However, progression to chronic HDV infection occurs in only 2-5% (Bahcecioglu 2017, Raimondo 1982, Romeo 2009, Vlachogiannakos 2020). In contrast, HDV superinfection in HBV carriers occurs in approximately 75% of cases, often exacerbates existing liver disease, and fulminant hepatitis develops in 7-15% of cases (Farci 1983).

[0390] Chronic HDV infection is defined by the persistence of HDV RNA or hepatitis delta antigen (HDAg) in serum for at least 6 months after HDV infection. Chronic HBV / HDV infection causes a more severe disease than chronic HBV and has a faster rate of progression of fibrosis (Mathurin 2000; Sagnelli 1989). Additionally, patients with chronic HDV infection are twice as likely to develop liver decompensation or HCC and die compared to patients with HBV mono-infection (Fattovich 2000; Niro 2010; Romeo 2009). Persistent HDV replication is the only factor associated with an increased risk of death (Romeo 2009).

[0391] Considering the HBV-dependence of HDV, strategies to defend against HBV infection (e.g., prophylactic vaccines) also defend against HDV infection. However, several regions of the world have poor access to HBV vaccines (WHO 2021, Hepatitis D). Additionally, vaccine non-responders, immunocompromised individuals, and immunodeficient patients remain vulnerable to the effects of chronic HDV infection. Currently, the only curative treatment available for HDV infection is pegylated interferon α (PEG-IFNα). 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 administering PEG-IFNa to patients with chronic HDV infection for at least 48 weeks (EASL 2017; Sarin 2016; Terrault 2018). PEGIFNa treatment establishes a sustained virological response (SVR, clearance of serum HDV maintained for 6 months after treatment cessation) in approximately 25 - 30% of patients, but recurrence occurs in approximately 50% of patients after long-term follow-up (Patient 2014). In addition to the low SVR rate, adverse reactions associated with PEG-INFa therapy are well documented, and PEG-INFa is contraindicated in patients with autoimmune diseases, several psychiatric syndromes, and Child-Pugh-Turcotte (CPT)-B or CPT-C stage cirrhosis patients (Rizzetto 2015; Sleijfer 2005). Further limiting its usefulness, a decrease in the efficacy of PEG-INFa has been observed when treating cirrhotic patients with chronic HDV (Gunsar 2005). The limitation of not being able to use peg-INFa in cirrhotic HDV patients is particularly notable since 50% of HDV-infected patients are cirrhotic at the time of diagnosis (Fattovich 1987).NRTIs with activity against HBV, 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, nucleoside (tide) reverse transcriptase inhibitors (NRTIs) are used in HBV / HDV co-infected patients because of their ability to regulate HBV replication.

[0392] BLV, an entry inhibitor targeting the NTCP receptor, has recently been granted conditional marketing authorization by the European Medicines Agency under the eligibility of the European Commission and Priority Medicines (PRIME) scheme as the first approved treatment for adults with chronic HDV and compensated liver disease in Europe. Interim results from the Phase 3 MYR301 trial showed that 36.7% of participants with chronic HDV achieved a combination of virological response (percentage of participants with undetectable (below the limit of detection (LOD) of HDV RNA) or a decrease of 2 log10 IU / mL or more from baseline) and biochemical response (ALT normalization) after 24 weeks of a subcutaneous (SC) dose of 2 mg of BLV given daily. Additional planned follow-up trials will test the durability of these responses. Since no participants demonstrated HBsAg loss, lifelong treatment with BLV may be required to manage HDV successfully. Additionally, BLV has not been tested and cannot be administered to people with CPT-B or CPT-C liver impairment, which constitutes a substantial portion of patients with HDV infection (Hepcludex Summary of Product Characteristics 2020).

[0393] Despite the recent introduction of BLV, the methods for treating chronic HDV remain inadequate for several reasons. The efficacy of either PEG-IFNα or BLV is not complete, with only about 17% of PEG-IFNα-treated individuals achieving a long-term virological response, and BLV, while better, still only about 37% of participants meeting the endpoints of HDV RNA suppression and ALT normalization (Wedemeyer 2021). PEG-IFN-α and BLV require once-weekly and daily subcutaneous administration, respectively, which affects treatment compliance. PEG-IFN-α is generally associated with several adverse reactions that further limit compliance, as well as drug-related toxicities that often require intensive laboratory monitoring and dose adjustment. BLV has been associated with treatment discontinuation related to adverse events in 10% of patients in real-world studies (de Ledinghen 2021). Furthermore, several potential drug interactions have been identified for BLV based on its interaction with the NTCP receptor and the hepatic transport proteins OATP1B1 / 3 (Hepcludex product characteristics summary 2020).

[0394] Finally, another area not addressed by current treatments is the treatment of patients with moderate to severe liver dysfunction. This group includes a significant portion of patients with chronic HDV. PEG-IFN-α or BLV cannot be safely prescribed to these patients, and thus, patients remain without treatment options.

[0395] Targets for current and new treatment strategies for HDV include inhibition of HDV RNA transcription, suppression of HBsAg production, or blockade of infection of susceptible hepatocytes (Lok 2021; Yurdaydin 2019). In ongoing trials, the HBV-targeted small interfering ribonucleic acid (siRNA) (SIRNA01) and the HBsAg-targeted monoclonal antibody (mAb) (AB01) have demonstrated the ability to suppress HBsAg in HBV-monoinfected individuals. Additionally, in preclinical models, reducing HBsAg using siRNA (SIRNA01) or the HBsAg-targeted monoclonal antibody (AB01) both resulted in a decrease in HDV viremia (Lempp 2021).

[0396] A single dose of up to 900 mg of SIRNA01 in healthy volunteers and six doses of 200 mg of SIRNA01 administered every four weeks in participants with chronic HBV infection were well tolerated and showed a safety profile that supports continued clinical development. Irrespective of the hepatitis B e-antigen (HBeAg) status, SIRNA01 is associated with a substantial decrease in HBsAg (up to 2 log10 IU / mL) but does not result in serological clearance of HBsAg (Gane 2021). Since HDV replication is dependent on HBsAg, SIRNA01 is expected to decrease (or in some cases eliminate) HDV viremia in parallel with the decrease in HBsAg. Independent of the antiviral activity of SIRNA01, AB01 has the ability to further decrease HBsAg and, as a result, further enhance the suppression of HDV viremia. AB01 is being evaluated in participants with chronic HBV in an ongoing phase 1 trial. Participants received a single dose of AB01. The maximum and most sustained HBsAg reduction (average change of approximately 2.42 log10 IU / mL from baseline HBsAg) was observed in the 300 mg dose cohort. By neutralizing HDV virions, AB01 is designed to inhibit infection of new hepatocytes, and a modified construct of this mAb designed to mobilize immune effector cells should accelerate the elimination of HBV / HDV coinfected hepatocytes.

[0397] The objectives of this trial are to evaluate the safety of SIRNA01 and AB01 in participants with HBV / HDV co-infection, and to evaluate whether monotherapy or combination therapy with the investigational agents, when administered to participants with all degrees of liver disease severity on a monthly or bi-monthly schedule, can permanently suppress HDV replication and normalize ALT.

[0398] Trial Plan Table 4 shows the treatment groups for the trial.

[0399]

Table 6

[0400] The trial scheme for cohort 1 is shown in Figure 1, and the trial schemes for cohorts 2 - 4 are shown in Figure 2.

[0401] Up to 58 participants will be registered for the trial. In cohorts 1a and 1b, a total of approximately 10 participants with HBV / HDV coinfection with histological data in viral hepatitis (METAVIR) fibrosis stages F0 - F3 will be registered with 5 participants per cohort. In cohort 2, a minimum of 12 and a maximum of 22 participants will be registered and will consist of the following three groups: (i) HBV / HDV coinfection with METAVIR fibrosis stages F0 - F3 from cohort 1 (maximum 10 participants); (ii) newly registered participants with METAVIR F0 - F3; and (iii) newly registered participants with METAVIR F4 and mild liver impairment (CPT-A).

[0402] Additionally, up to 8 floater participants may be added to cohort 1 or 2. In cohort 3, a total of 12 participants with HBV / HDV coinfection with METAVIR fibrosis stage F4 and moderate liver impairment (CPT-B) will be registered. In cohort 4, a total of 6 participants with HBV / HDV coinfection with METAVIR fibrosis stage F4 and severe liver dysfunction (CPT-C) will be registered.

[0403] Cohorts 1, 2, 3, and 4 are sequentially registered based on the review of safety data. The cohort may be terminated or interrupted at the discretion of the clinical trial sponsor. Cohort 2 starts with the registration of participants who participated in Cohort 1, after the review of 12-week monotherapy safety data, consultation with the principal investigator of the trial, and re-consent by the participants. Participants in Cohort 1 are eligible to participate in Cohort 2 after the 16th week (or at that time) of the first trial. Cohort 2 also registers additional participants after the safety review of 12-week monotherapy safety data from the first 10 participants in Cohort 1. Cohort 3 starts registration after (1) the review of the safety results of other trials and (2) the review of the safety data in the first 5 participants in Cohort 2 who completed the trial at week 12. Cohort 4 starts registration after (1) the review of the safety results of Cohort 3 of other trials and (2) the review of the safety data in the first 5 participants in Cohort 3 who completed the trial at week 12.

[0404] The total trial period is planned to be up to 102 weeks. For all cohorts, the intervention period consists of two 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 do not transition to the maintenance period or participate in Cohort 2 participate in a follow-up period (48 weeks). Participants who prematurely discontinue the trial treatment during the induction period participate in the follow-up period (48 weeks). Participants who prematurely discontinue the trial treatment during the maintenance period participate in the follow-up period, whichever is earlier, either 48 weeks after the last administration of the trial intervention or at week 96. For Cohorts 2 - 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 do not transition to the maintenance period participate in the follow-up period (48 weeks). Participants who prematurely discontinue the trial treatment during the induction period participate in the follow-up period (48 weeks). Participants who prematurely discontinue the trial treatment during the maintenance period participate in the follow-up period, whichever is earlier, either 48 weeks after the last administration of the trial intervention or at week 96.

[0405] The screening period for all participants shall be a maximum of 42 days.

[0406] The intervention period consists of two periods: introduction and maintenance. Participants in cohorts 1a and 1b receive 3 doses of the test treatment during the introduction period (days 1, 4, and 8 weeks), and if they achieve a decrease in HDV RNA of 2 log10 or more from baseline or HDV RNA below the LOQ and ALT below the upper limit of normal (ULN) (composite endpoint) at the 12-week visit compared to baseline, they can continue for an additional 84-week maintenance period. Participants who do not meet the composite endpoint at 12 weeks can participate in either cohort 2 or the follow-up period.

[0407] Participants in cohorts 2 - 4 receive treatment for 24 or 48 weeks during the introduction period. Participants who meet the composite endpoint at the 24-week visit transition to a 72-week maintenance period and receive the next dose at 32 weeks.

[0408] Participants who do not meet the composite endpoint at the 24-week visit continue the introduction period until 48 weeks. Participants who meet the composite endpoint at the 48-week visit transition to a 48-week maintenance period and receive the next dose at 56 weeks. Participants who do not meet these criteria at 48 weeks end the intervention period and enter the follow-up period. Participants whose HDV RNA rebounds within 1 log10 IU / mL of baseline during the maintenance period return to dosing with SIRNA01 + AB01 every 4 weeks until 88 weeks. In these cases, the next dose should be administered at the next scheduled visit according to the Statement of Activities (SoA) table. If the next scheduled visit does not occur within 8 weeks, the next dose should be given over 4 weeks through the unscheduled visit, and then the dose should be given every 4 weeks until 88 weeks.

[0409] The maximum follow-up period is the earlier of 48 weeks after the last administration of the test intervention or week 96. Participants are included in the follow-up period if they: (1) are enrolled in cohort 1 and do not transition to the maintenance period after meeting the composite endpoint at week 12; (2) are enrolled in cohort 1 and do not participate in cohort 2 after not meeting the composite endpoint at week 12; (3) are enrolled in cohorts 2 - 4 and have not transitioned to the maintenance period at week 48; or (4) are enrolled in any cohort and discontinue the investigational drug prematurely.

[0410] AB01 is provided as a reconstituted lyophilized powder for subcutaneous (SC) administration at 300 mg every 4 or 8 weeks (see Table 4). AB01 (HBC34v35 - MLNS - GAALIE) contains the light chain amino acid sequence of SEQ ID NO: 59 and the heavy chain amino acid sequence of SEQ ID NO: 57. SIRNA01 is provided as a liquid for SC administration at 200 mg every 4 or 8 weeks (see Table 4). SIRNA01 has a sense strand containing the nucleotide sequence of SEQ ID NO: 5 and an antisense strand containing the nucleotide sequence of SEQ ID NO: 6.

[0411] The study objectives and related endpoints are shown in Table 5.

[0412]

Table 7 - 1

[0413]

Table 7 - 2

[0414] The Child - Pugh - Turcotte (CPT) score assessment for liver disease is shown in Table 6.

[0415]

Table 8

[0416] The ULN value of ALT can be, for example, 34 IU / mL for women and 43 IU / mL for men.

[0417] The PK parameters of SIRNA01 and AB01 (free and total PK when applicable) are calculated using standard non-compartmental methods when applicable. The parameters are C max 、C last 、T max 、T last 、AUC inf 、AUC last 、%AUC exp 、t 1 / 2 、λ z 、V z / F, and CL / F may include but are not limited to these.

[0418] Immunogenicity data may include but are not limited to incidence, titer, and neutralization data.

[0419] The activity schedules of the cohorts and follow-up periods ("SoA") are shown in FIGS. 3A - 8C.

[0420] Participant population Participants are male and female participants aged 18 to 70 years old who have chronic HBV / HDV co-infection with all degrees of liver impairment and are currently receiving NRTI therapy. "Chronic HBV infection" for the purposes of this trial is defined as positive serum HBsAg, HBV DNA, or HBeAg in at least two instances separated by at least 6 months, based on previous (within the past 12 months) or current laboratory documentation (any combination of these tests performed 6 months apart is acceptable). Enrollment in each cohort targets baseline HBsAg above 10,000 IU / mL at screening in approximately 40% of participants. Participants have been receiving locally approved NRTI therapy for at least 12 weeks prior to Day 1. Participants also have HBsAg above 0.05 IU / mL at screening, clinical evidence of chronic hepatitis, and positive HDV antibodies for at least 6 months, as well as positive HDV RNA at least 3 months prior to screening. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) above the ULN and less than 5×ULN Participants are above 18 years of age (or the legal age of consent, whichever is older) and less than 70 years old at screening. Participants also have a body mass index (BMI) of 2 18 kg / m

[0421] Additional inclusion criteria include the following. · Female participants must have a negative pregnancy test or confirmed postmenopausal status. Postmenopausal status is defined as 12 months without menstruation without another medical cause. Confirmation of negative follicle-stimulating hormone [FSH] 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 able to breastfeed, and must intend 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 to refrain from egg donation and in vitro fertilization from study drug administration until 48 weeks after the last dose of SIRNA01 or AB01. · Male participants with female partners who may be pregnant must agree to meet one of the following contraception requirements from the time of dosing with the investigational product until 48 weeks after the last dose of SIRNA01 or AB01: documentation of vasectomy or azoospermia, or use of a male condom + use of one of the contraceptive options listed for contraception of the partner's WOCBP. Male participants must also agree not to donate sperm from the time of the first dose of the investigational product until 48 weeks after the last dose of SIRNA01 or AB01. · Be able to give signed informed consent. · Have a 12-lead electrocardiogram (ECG) within normal limits or, when determined by the clinical investigator in charge of the clinical trial, have no clinically significant abnormalities at screening. · Agree not to donate blood during the study period and for an additional 3 months after the last dose of the investigational product. · Inclusion criteria specific to cohorts 1 and 2 ○ Non-cirrhotic ■ Liver biopsy with METAVIR F0 - F3 within 12 months prior to screening or liver elastography (Fibroscan®) less than 12 kilopascals (kPa) (cohorts 1 and 2). ■ Creatinine clearance (CLcr) of 30 mL / min or more calculated by the Cockcroft - Gault formula at screening ○ Cirrhotic ■ Liver biopsy with METAVIR F4 within 12 months prior to screening or liver elastography (Fibroscan®) of 12 kPa or more (cohort 2) ■ ClCr of 60 mL / min or more calculated by the Cockcroft - Gault formula at screening ■ CPT score of 5 or 6 at screening and at the start of the study · Inclusion criteria specific to cohort 3 (moderate impairment of liver function) ○ Apart from liver insufficiency, participants must be sufficiently healthy for study participation based on medical history, physical examination, vital signs, and screening laboratory evaluations, in the opinion of the investigator in charge of the trial. ○ A liver biopsy with METAVIR F4 or a liver elastography (Fibroscan®) of 12 kPa or higher within 12 months before screening ○ CLcr of 60 mL / min or higher calculated by the Cockcroft-Gault formula at screening ○ CPT score of 7 - 9 including at screening and at the start of the trial · Inclusion criteria specific to cohort 4 (severe impairment of liver function) ○ Apart from liver failure, participants must be sufficiently healthy for trial participation based on medical history, physical examination, vital signs, and screening laboratory evaluations, in the opinion of the trial responsible physician. ○ A liver biopsy with a liver elastography (Fibroscan®) reading of METAVIR F4 or 12 kilopascals (kPa) or higher ○ CLcr of 60 mL / min or higher calculated by the Cockcroft-Gault formula at screening and a CPT score of over 10 at screening and at the start of the trial

[0422] Exclusion criteria are as follows. · History of clinically significant liver disease from non-HBV and non-HDV etiologies determined by the trial responsible physician · History of immune complex disease · History of autoimmune disorder · History of HBV-related extrahepatic diseases including, but not limited to, HBV-related rash, arthritis, or glomerulonephritis · History of allergic reaction, hypersensitivity, or intolerance to the investigational drug, its metabolites, or excipients · Anti-HBs over 10 IU / L at screening · Corrected QT interval (QTc) over 450 milliseconds · AST or ALT over 5 × ULN · Total bilirubin over 3 × ULN (cohorts 1 - 3), total bilirubin over 5 × ULN (cohort 4) · Serum albumin less than 28 g / L (cohorts 1 - 3), serum albumin less than 25 g / L (cohort 4) · Absolute neutrophil count less than 1,000 / mm3 · Platelets less than 20,000 / mm3 · Hemoglobin less than 8 g / dL · History of anaphylaxis · History of malignant tumor diagnosed or treated within 5 years (Local treatment of squamous cell or non-invasive basal cell skin cancer is acceptable. Non-invasive cervical cancer is acceptable if appropriately treated prior to screening. Participants under evaluation for malignant tumor are not eligible.) · History of or listed for bone marrow or solid organ transplantation · Known active infection other than chronic HBV and HDV infection, or fever (over 38°C) within 7 days prior to Day 1 or any clinically significant acute condition such as acute respiratory disease · Coinfection 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. Asymptomatic participants who are HAV or HEV immunoglobulin G antibody (IgG) positive are eligible · Any clinically significant medical or psychiatric condition that may interfere with the trial intervention, evaluation, or compliance with the protocol, or otherwise render the participant ineligible for participation in the trial, as determined by the trial responsible physician. Participants with controlled type 2 diabetes are eligible · Use of any therapy known to exacerbate acute or worsening chronic hepatitis, fluctuating or rapidly deteriorating liver function, or liver dysfunction as determined by the opinion of the trial responsible physician · Treatment with immunomodulators, IFN-α (e.g., IFN-α-2a or IFN-α-2b, or pegylated IFN-α-2a or α2b), cytotoxic agents or chemotherapeutic agents, or chronic systemic corticosteroids within 6 months of screening · Administered an HDV active agent (including lonafarnib and brevibertide) within 90 days or within 5 half-lives (if known) before administration of the test drug, or was active during the follow-up period of another clinical trial involving an intervention procedure. Participants must also agree not to participate in any other interventional trial at any time during their participation in this trial, including the follow-up period. · Administration of an oligonucleotide (e.g., siRNA, antisense oligonucleotide) with activity against HBV within 48 weeks before administration of the test drug. · For newly enrolled participants: Administration of AB01 within 24 weeks before Day 1 of the trial. · History or clinical evidence of alcohol or drug abuse within 12 months before screening, or positive drug screening at the time of screening, unless it can be explained by prescription drugs (diagnosis and prescription must be approved by the principal investigator of the clinical trial). The use of marijuana is permitted. · Additional exclusion criteria for participants with liver impairment ○ Participants who require more than one puncture per month. ○ Participants with intractable encephalopathy or serious central nervous system diseases determined by the principal investigator of the clinical trial. ○ History of gastric or esophageal variceal bleeding within the past 6 months. ○ Participants with transjugular intrahepatic portosystemic shunt (TIPS) placement. ○ Presence of hepatopulmonary or hepatorenal syndrome. ○ Presence of primary biliary cholestatic liver disease. ○ Unable or unwilling to follow the dietary recommendations for cirrhosis and liver dysfunction advised by the principal investigator of the clinical trial and the lifestyle considerations outlined in this protocol.

[0423] Combination therapy · Combination therapy was not permitted during the trial. · In the opinion of the principal investigator and the sponsor of the clinical trial, unless the drug interferes with the clinical trial, trial participants must refrain from taking prescription and over-the-counter medications (including vitamins, recreational drugs, and dietary or herbal supplements) other than those prescribed by the treating physician within 7 days before the start of the trial intervention (or 14 days if the drug is a potential enzyme inducer) or 5 half-lives (whichever is longer), until the completion of follow-up visits. · Participants with liver dysfunction associated with co-existing diseases requiring medications must have been taking the medications without dose changes for more than 3 months prior to screening. All concomitant medications must be approved by the medical monitor before trial registration. · The use of any of the following systemic medications is prohibited within 14 days before the administration of the investigational drug and throughout the trial: ■ Chronic systemic steroids (prednisone equivalent > 10 mg / day) or other immunosuppressants (Note: Administration of corticosteroids for the treatment of immune-mediated AEs and short-term courses of corticosteroids for chronic obstructive pulmonary disease or asthma exacerbation are permitted). ■ Paracetamol (acetaminophen) > 3 g / day ■ Isoniazid · Additionally, the administration of any potentially hepatotoxic drugs during the trial should only be considered when treatment options cannot be identified and after careful consideration of the potential risks and benefits to the participants. Drugs that are potentially hepatotoxic or 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-clavulanic acid; minocycline; nitrofurantoin; sulfamethoxazole / trimethoprim; erythromycin; rifampin; azole antifungals; and herbs or natural remedies.

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NCI Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0 Released on 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 / CTCAE_v5_Quick_Reference_8.5xl1.pdf. Niro GA, Smedile A. Current concept in the pathophysiology of hepatitis delta infection. Curr Infect Dis Rep. February 2012; 14(1): 9-14. doi:10.1007 / sl1908-011-0233-5. PMID:22161240. Niro GA, Smedile A, Ippolito AM, Ciancio A, Fontana R, Olivero A, Valvano MR, Abate ML, Gioffreda D, Caviglia GP, Rizzetto M, Andriulli A. Outcome of chronic delta hepatitis in Italy: a long-term cohort study. J. Hepatol. November 2010; 53(5): 834 - 40. doi:10.1016 / j.jhep.2010.06.008. Epub July 29, 2010. PMID:20800919. Petrillo R, Buti M, Durand F, Charlton M, Gadano A, Cantisani G, et al. Entecavir and hepatitis B immune globulin in patients undergoing liver transplantation for chronic hepatitis B. Liver Transpl. August 2013; 19(8): 887 - 95. Polaris Observatory Collaborators. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. Lancet Gastroenterol Hepatol. June 2018; 3(6): 383 - 403. doi:10.1016 / S2468 - 1253(18)30056 - 6. Epub March 27, 2018. PMID:29599078. Raimondo G, Smedile A, Gallo L, Balbo A, Ponzetto A, Rizzetto M. Multicentre study of prevalence of HBV-associated delta infection and liver disease in drug-addicts. Lancet. January 30, 1982; 1(8266): 249-51. doi:10.1016 / s0140-6736(82)90976-x. PMID:6120275. Reed WD, Eddleston AL, Cullens H, Williams R, Zuckerman AJ, Peters DK, Williams DG, Maycock WA. Infusion of hepatitis-B antibody in antigen-positive active chronic hepatitis. Lancet. December 15, 1973; 2(7842):1347-51. doi:10.1016 / s0140-6736(73)93321-7. PMID:4128052. Reilly MC, Zbrozek AS, Dukes EM. The validity and reproducibility of a work productivity and activity impairment instrument. Pharmacoeconomics. November 1993; 4(5): 353-65. doi:10.2165 / 00019053-199304050-00006. PMID:10146874. Rizzetto M. Hepatitis D Virus: Introduction and Epidemiology. Cold Spring Harb Perspect Med. July 1, 2015; 5(7): a021576. doi:10.1101 / cshperspect.a021576. PMID:26134842; PMCID:PMC4484953. Rizzetto M, Hamid S, Negro F. The changing context of hepatitis D. J Hepatol. May 2021; 74(5):1200 - 1211. doi:10.1016 / j.jhep.2021.01.014. Epub Jan 20, 2021. PMID:33484770. Romeo R, Del Ninno E, Rumi M, Russo A, Sangiovanni A, de Franchis R, Ronchi G, Colombo M. A 28 - year study of the course of hepatitis Delta infection: a risk factor for cirrhosis and hepatocellular carcinoma. Gastroenterology. May 2009; 136(5):1629 - 38. doi:10.1053 / j.gastro.2009.01.052. Epub Jan 29, 2009. PMID:19208358. 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 chronic hepatitis. Liver. Aug 1989; 9(4):229 - 34. doi:10.1111 / j.l 600 - 0676.1989.tb00404.x. PMID:2770435. Sarin SK, Kumar M, Lau GK, Abbas Z, Chan HL, Chen CJ, Chen DS, Chen HL, Chen PJ, Chien RN, Dokmeci AK, Gane E, Hou JL, Jafri W, Jia J, Kim JH, Lai CL, Lee HC, Lim SG, 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-Pacific clinical practice guidelines on the management of hepatitis B: 2015 update. Hepatol Int. January 2016; 10(1):1 - 98. doi:10.1007 / sl2072 - 015 - 9675 - 4. Epub November 13, 2015. PMID:26563120; PMCID:PMC4722087. Shirvani - Dastgerdi E, Tacke F. Molecular interactions between hepatitis B virus and delta virus. World J Virol. 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April 2018; 67(4):1560-1599. doi:10.1002 / hep.29800. PMID:29405329; PMCID:PMC5975958. 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, Aikata H, Ochi H, Hayes CN, Chayama K. Antiviral effects of anti-HBs immunoglobulin and vaccine on HBs antigen seroclearance for chronic hepatitis B infection. J. Gastroenterol. November 2016; 51(11):1073-1080. doi:10.1007 / s00535-016-1189-x. Epub March 4, 2016. PMID:26943168. Urban S, Neumann-Haefelin C, Lampertico P. Hepatitis D virus in 2021: virology, immunology and new treatment approaches for a difficult-to-treat disease. Gut. September 2021; 70(9):1782-1794. doi:10.1136 / gutjnl-2020-323888. Epub June 8, 2021. PMID:34103404; PMCID:PMC8355886. Vlachogiannakos J, Papatheodoridis GV. New epidemiology of hepatitis delta. Liver Int. February 2020; 40 Suppl 1:48-53. doi:10.111 l / liv.14357. PMID:32077599. Wedemeyer H, et al. Treatment with bulevirtide improves patient reported outcomes in patients with chronic hepatitis delta (CHD): an interim exploratory analysis at week 24. The Liver Meeting, abstract 680, 2021. Wedemeyer H, Yurdaydin C, Dalekos GN, Erhardt A, Qakaloglu Y, Degertekin H, Gurel S, Zeuzem S, Zachou K, Bozkaya H, Koch A, Bock T, Dienes HP, Manns MP; HID IT Study Group. Peginterferon plus adefovir versus either drug alone for hepatitis delta. N Engl J Med. January 27, 2011; 364(4): 322-31. doi:10.1056 / NEJMoa0912696. PMID:21268724. WHO (World Health Organization). Hepatitis D. https: / / www.who.int / news-room / factsheets / detail / hepatitis-d. Published in July 2021. Accessed on April 10, 2022. WHO (World Health Organization). Hepatitis B. https: / / www.who.int / news-room / factsheets / detail / hepatitis-b. Published in July 2021. Accessed on April 10, 2022. Ye X, Tateno C, Thi EP, Kakuni M, Snead NM, Ishida Y, Barnard TR, Sofia MJ, Shimada T, Lee ACH. 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[0425] Example 2 Neutralization of HDV by antibodies targeting HBsAg Background Chronic hepatitis B virus (HBV) infection is a major global public health burden affecting approximately 296 million people worldwide, with an estimated 820,000 deaths annually (Polaris Observatory Collaborators, 2018; WHO, Hepatitis B, 2021).

[0426] SIRNA01 is an investigational siRNA therapeutic targeting the HBx region of the HBV genome and exhibits potent in vitro and in vivo antiviral activity. The sense strand of SIRNA01 is conjugated to an N-acetylgalactosamine (GalNAc) ligand to enable targeted delivery to the liver. AB01 is an investigational neutralizing monoclonal antibody targeting the antigenic loop of HBsAg with pan-genotypic neutralizing activity in vitro. Treatment with murine AB01 inhibits viral spread and results in elimination of HBsAg in vivo. The mAb has a modified Fc that extends the serum half-life (LS mutation) and increases binding to activating FcγRs (FcγRIIa and GAALIE) but decreases binding to inhibitory FcγRIIb (XX 2 / Illa mutation).

[0427] Method Using primary human hepatocytes (PHH) infected with HBV (genotype D), inhibition of HBV entry during AB01 treatment was confirmed. HBeAg in the cell culture supernatant was quantified by chemiluminescent immunoassay (CLIA) using it as a marker of infection.

[0428] To evaluate the in vitro activity of AB01 against eight different HBV genotypes, hepatitis D virus (HDV) (a satellite virus of HBV found in 5% of patients with chronic HBV (WHO, Hepatitis D, 2021)) was used. Since HDV requires HBV envelope proteins for entry into hepatocytes and secretion from hepatocytes, it can be used as a tool to study HBsAg from different HBV genotypes (Wang et al., 2021). Here, neutralization by AB01 was evaluated in Huh7-NTCP cells infected with HDV pseudotyped with HBsAg from eight different HBV genotypes (A - H).

[0429] To evaluate the in vitro antiviral activity of SIRNA01 against representative HBV genotypes A - D, a HBV1.3 over-length genome system in which all viral RNAs are transcribed under the regulation of authentic HBV promoters was used. Huh7 cells were transfected with plasmids containing HBV1.3 genomic sequences from 13 isolates of HBV representing genotypes A - D, and then transfected with SIRNA01 or control siRNA. HBsAg was used as a readout.

[0430] To evaluate the combinatorial activity of SIRNA01 and AB01 against monotherapy, two in vivo studies were conducted using well-established mouse models of HBV infection: C57BL / 6 mice transduced with AAV8-HBV (genotype D) or human liver-chimeric PXB-mice (genotype C) infected with HBV. Mice were treated with SIRNA01, HBC34-mu (mouse version of AB01), entecavir (ETV, only in the AAV8-HBV test), or combinations of the drugs at different concentrations. Antiviral activity was determined by evaluation of viral serum / plasma markers including HBV DNA, HBsAg, and HBeAg.

[0431] Results AB01 and SIRNA01 exhibit potent activity against viruses carrying all HBsAg genotypes tested. Primary human hepatocytes (PHH) were infected with HBV (genotype D) in the presence of AB01, preS1-targeting Mai8 / 7mAb, or polyclonal hepatitis B immunoglobulin (HBIG). HBV neutralizing activity was evaluated by quantifying HBeAg secreted 7 days after infection as a marker of infection (Figure 9). To evaluate the neutralizing capacity of AB01 against different HBV genotypes in relation to infection, HDV enveloped with HBsAg from different HBV genotypes was utilized. Neutralization by AB01 was evaluated in Huh7-NTCP cells infected with HDV pseudotyped with HBsAg from eight different HBV genotypes (A - H) (Figure 10).

[0432] The activity of SIRNA01 against HBV genotypes A - D was evaluated in HBV genome-transfected human hepatocellular carcinoma cells (Huh7) (Figure 11).

[0433] References Polaris Observatory Collaborators. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. Lancet Gastroenterol Hepatol 2018; 3(6): 383 - 403. Hepatitis B. World Health Organization. July 27, 2021. Accessed April 14, 2022. https: / / www.who.int / news-room / fact-sheets / detail / hepatitis-b. Hepatitis D. World Health Organization. July 28, 2021. Accessed April 29, 2022. https: / / www.who.int / news-room / fact-sheets / detail / hepatitis-d Wang W. et. al.: Assembly and infection efficacy of hepatitis B virus surface protein exchanges in 8 hepatitis D virus genotype isolates; J. Hepatol. August 2021; 75(2): 311 - 323. doi:10.1016 / j.jhep.2021.03.025

[0434] Example 3 Clinical evaluation of monotherapy or combination therapy for treating chronic HDV infection Evaluate the safety, efficacy, and tolerability of anti - HBV antibodies in combination with anti - HBV siRNA in a phase 2 multi - center open - label clinical trial in non - cirrhotic and compensated cirrhotic (CPT - A) human patients with chronic HBV / HDV co - infection against nucleoside reverse transcriptase inhibitor (NRTI) therapy.

[0435] The purpose of this study is to evaluate the safety of SIRNA01 and AB01 in participants with chronic HBV / HDV coinfection, and to assess whether monotherapy or combination therapy with the investigational drugs can permanently suppress HDV replication and normalize ALT when administered to participants with varying degrees of hepatic fibrosis and compensated cirrhosis on a biweekly, monthly, or bimonthly schedule.

[0436] Study Plan Table 6 shows the treatment groups for the study. The study consists of cohorts receiving either SIRNA01 or AB01 monotherapy or combination therapy. For cohorts 1a and 1b, the intervention period with SIRNA01 and AB01 monotherapy consists of two periods: induction (12 weeks) and maintenance (up to 84 weeks). For cohorts 2a, 2b1, 2b2, 2c, and 3, the intervention period with SIRNA01 and / or AB01 consists of the treatment period only (up to 96 weeks). Participants in cohort 4 have a 12-week treatment delay before starting combination therapy with SIRNA01 and AB01 for up to 96 weeks. Cohorts 2 - 4 are opened after review of 12-week safety and efficacy data from cohorts 1a and 1b. The study also includes three optional substudies that collect (1) liver tissue, (2) fine needle aspirates of the liver, and (3) blood samples for pharmacokinetic (PK) studies.

[0437] [Table 9] SC = subcutaneous * The composite endpoint is defined as undetectable HDV RNA (< LOD) or a ≥ 2 log 10 decrease in HDV RNA from baseline and ALT < ULN. a Participants who achieve the composite endpoint at week 12 receive 10 additional doses every 8 weeks. b Participants who do not achieve the composite endpoint at week 12 may enter cohort 2c on day 1 or during the follow-up period. c Participants who achieve the composite endpoint at week 48 continue monotherapy until week 96. d Participants who have not achieved the composite endpoint at week 48 may participate during the follow-up period or initiate combination therapy with SIRNA01 + AB01 and follow the investigational product (IP) administration in the Cohort 2c activity schedule (SoA) from week 52 to week 96. e Participants who meet the virological non-response criteria at week 24 may initiate combination therapy with SIRNA01 and follow the IP administration in the Cohort 2c SoA from week 28 to week 96. f New participants, including those from Cohort 4 who achieve the composite endpoint at week 48, continue combination therapy until week 96. g New participants, including those from Cohort 4 who do not achieve the composite endpoint at week 48, participate during the follow-up period. h Participants delay treatment for 12 weeks before transitioning to Day 1 of Cohort 2c.

[0438] The trial scheme for Cohort 1 is shown in Figure 12A, and the trial schemes for Cohorts 2, 3, and 4 are shown in Figure 12B. The activity schedules (「SoA」) for the cohorts and follow-up periods are provided in Figures 13A - 20B.

[0439] Up to approximately 118 participants will be enrolled. This includes a pool of up to 24 floater participants that can be added to any group(s). Participants are planned to be enrolled at sites worldwide. The participants in the cohorts are as follows. · Cohorts 1a and 1b: Approximately 10 participants with HBV / HDV co-infection having a METAVIR F0 - F3 hepatic fibrosis stage in histological data in viral hepatitis: Approximately 5 participants per cohort.

[0440] · Cohort 2 consists of four groups that enrolled up to approximately 78 participants, each having either METAVIR F0-F3 or METAVIR F4, CPT-A liver disease. Approximately 50% of the participants in each cohort have METAVIR F4, CPT-A liver disease. Each cohort enrolls approximately 50% of the participants with baseline HBsAg less than 5000 IU / mL. ■ Cohort 2a: Planned enrollment of approximately 12 participants, and a maximum of 36 including floaters. ■ Cohort 2b1: Planned enrollment of approximately 12 participants, and a maximum of 36 including floaters. ■ Cohort 2b2: Planned enrollment of approximately 12 participants, and a maximum of 36 including floaters. ■ Cohort 2c: Planned enrollment of up to approximately 30 new participants (if applicable) including participants migrating from Cohort 4. Participants migrating from Cohort 1a or Cohort 1b do not contribute to the planned enrollment. Total cohort size of up to 54 including floaters. · Cohort 3: Planned enrollment of up to approximately 30 participants, and a maximum of 54 including floaters. Approximately 50% of the participants in this cohort have METAVIR F4, CPT-A liver disease. · Cohort 4: Planned enrollment of up to approximately 12 participants. Approximately 50% of the participants in this cohort have METAVIR F4, CPT-A liver disease.

[0441] The trial starts with Cohort 1. Participants in Cohort 1 who do not meet the composite endpoint at Week 12 may, at the discretion of the treating physician and the participant, participate in the first day of Cohort 2c at the first scheduled visit at Week 16, provided that the participant's Week 12 data meets the inclusion / exclusion (I / E) criteria of Cohort 2. The groups within Cohort 2 may be started separately based on the available safety and antiviral data from Cohort 1. Cohort 3 is started after review of the 12-week safety and available antiviral data from Cohort 1b. Enrollment of new non-cirrhotic participants into Cohorts 2c and 4 is started only after review of the safety and antiviral data for at least 12 weeks from Cohorts 1a (approx. n = 5) and 1b (approx. n = 5). Enrollment of CPT-A cirrhotic participants into Cohorts 2a, 2b1 or 2b2, and 3 is started only after review of the safety and antiviral data for at least 12 weeks from approximately 10 participants who have received SIRNA01 monotherapy (approx. n = 5) or AB01 monotherapy (approx. n = 5), and other available safety data. Enrollment of CPT-A cirrhotic participants into Cohorts 2c and 4 is started only after review of the safety and antiviral data for at least 12 weeks and other available safety data from approximately 5 non-cirrhotic participants in Cohort 2c. The cohorts may be paused, closed, or interrupted.

[0442] The maximum total study period is planned to be up to 118 weeks. 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 may (1) transition to the maintenance period, (2) enroll in Cohort 2c, or (3) enroll in the follow-up period at the next visit (week 16). Participants who discontinue the study treatment early during the induction period will have an Early Termination (ET) visit and then enroll in the follow-up period 4 weeks later. Participants who discontinue the study treatment early during the maintenance period will have an ET visit and then enroll in the follow-up period at the earlier of 48 weeks after the last dose of the study intervention or week 96. For Cohorts 2a, 2b1, 2b2, 2c, and 3, this includes a screening period (up to 6 weeks) and a treatment period (48 - 96 weeks). Participants who discontinue the study treatment early will have an ET visit and then enroll in the follow-up period at the earlier of 48 weeks after the last dose of the study intervention or week 96. For Cohort 4, this includes a screening period (up to 6 weeks) and a 12-week delayed treatment (while continuing NRTI).

[0443] The screening period for all participants is up to 42 days.

[0444] The intervention period consists of two periods, induction and maintenance. Participants in Cohorts 1a and 1b will receive 3 doses of the study treatment during the induction period (day 1, week 4, and week 8) and at the week 12 visit, the composite endpoint (undetectable HDV RNA [< LOD] or a 2 log decrease in HDV RNA from baseline) 10If the above decrease and ALT below ULN are achieved, the participant can continue into an additional 84-week maintenance period. Participants who do not meet the composite endpoint at week 12 can participate in either the follow-up period at week 16 or cohort 2c, day 1 (must meet the I / E criteria of cohort 2 at week 12). Participants in maintenance period cohorts 1a and 1b whose HDV RNA rebounds within 2 log10 IU / mL of baseline return to dosing with SIRNA01 or AB01 every 4 weeks until week 92. In these cases, the participant should follow the activities in the SoA of cohort 2a or 2b (see Figures 15A-D and 16A-D), and the next dose should be administered within 4 weeks. Participants in cohort 2a (SIRNA01 monotherapy), cohort 2b1 or 2b2 (AB01 monotherapy), and cohort 2c (SIRNA01 + AB01 combination therapy) receive monthly investigational treatment for up to 96 weeks during the treatment period. Participants in cohort 2a who do not achieve the composite endpoint at week 48 can participate in the follow-up period or start combination therapy by adding AB01 to SIRNA01 at the visit at week 52 and follow the administration of the investigational product (IP) in the SoA of cohort 2c (Figures 16A-16D) from week 52 to week 96. Participants in cohort 2b1 or 2b2 who do not achieve the composite endpoint at week 48 can participate in the follow-up period or start combination therapy with SIRNA01 in addition to AB01 at the visit at week 52 and follow the IP administration in the SoA of cohort 2c (Figures 16A-16D) from week 52 to week 96. At week 24, at least 1 log 10Participants in cohort 2b1 who have not achieved an HDV RNA reduction of IU / mL may initiate combination therapy and follow IP administration in the SoA of cohort 2c (Figs. 15A - D and 16A - D) from week 28 to week 96. Participants newly enrolled in cohort 2c who do not meet the composite endpoint at week 48 discontinue the study treatment and participate in the follow - up period. Participants in cohort 3 receive AB01 monotherapy every other week up to week 96 during the treatment period. Participants who do not achieve the composite endpoint at week 48 may either participate in the follow - up period or initiate combination therapy with SIRNA01 added to AB01 and follow IP administration in the SoA of cohort 2c (Figs. 16A - 16D) from week 52 to week 96. Participants in cohort 3 who have not achieved an HDV RNA reduction of at least 1 log 10 IU / mL by week 24 may initiate combination therapy and follow IP administration in the SoA of cohort 2c (Figs. 15A - D and 16A - D) from week 28 to week 96. Participants in cohort 4 delay treatment for 12 weeks, continue NRTI standard treatment, and are then re - assigned to cohort 2c.

[0445] The maximum period of the follow - up period is the earlier of 48 weeks after the last administration of the study intervention or week 96. Participants participate in the follow - up period in the following cases: When enrolled in cohort 1 and do not transition to the maintenance period after meeting the composite endpoint at week 12; When enrolled in cohort 1 and do not participate in cohort 2c after not meeting the composite endpoint at week 12; When enrolled in cohort 2a, 2b1, 2b2, or 3 and do not participate in combination therapy after not achieving the composite endpoint at week 48; When enrolled in cohort 2b1 or 3 and do not participate in combination therapy after meeting the virological non - response criteria at week 24; When enrolled in cohort 2c and do not achieve the composite endpoint at week 48; When transitioning from cohort 4 to cohort 2c and do not achieve the composite endpoint at week 48; or When enrolled in any cohort and discontinue the study drug early. Those who continue until week 96 do not have additional follow - up visits.

[0446] Any optional liver biopsy sub-study is conducted in the selected country and facility when available. All participants newly enrolled in Cohort 2 at the selected facility are eligible to participate in the sub-study. The target enrollment is approximately 18 participants across all Cohort 2 groups. Pretreatment liver tissue samples are collected during the screening window or by week 2 of the study. If a participant had a liver biopsy in the past 12 months and the tissue block is available and considered usable for research purposes, this sample can be used as the pretreatment sample. Follow-up liver tissue samples are collected at treatment times of approximately 48 ± 2 weeks and / or 96 ± 2 weeks. The tissue samples are used to directly assess changes in liver fibrosis during treatment with the study drug. The samples are also used for assays to evaluate HBV and HDV replication in hepatocytes and for exploratory studies.

[0447] Participants with baseline HBsAg above 3000 IU / mL in Cohort 2b1 or 2b2, and 2c may participate in an optional AB01 PK sub-study. This sub-study has up to 2 additional study visits to collect AB01 PK samples. The first visit is conducted 5 - 7 days after the 3rd, 4th, or 5th dose of AB01, and the second optional visit is conducted 5 - 7 days after the 7th, 8th, or 9th dose of AB01. Participants transitioning from Cohort 1a or 1b to Cohort 2c are excluded from this sub-study. In addition to PK, HBsAg, HDV RNA, HBV DNA, and liver function tests are also collected at the same visit. This optional PK sub-study ends after 30 participants have been enrolled in the sub-study.

[0448] Any liver fine needle aspiration sub-study will be conducted at selected UK facilities. All participants newly registered in cohorts 2b1 / 2b2, 2c, and 3 at the selected facilities will be eligible to participate in the sub-study. The target enrollment is up to approximately 10 participants. Liver FNA and peripheral blood mononuclear cell (PBMC) samples will be collected at pretreatment, at week 24 ± 2, and at any visit between week 48 and 96 at the discretion of the investigator. This FNA sub-study will provide information on changes in the intrahepatic environment and detailed immunological and virological data in participants treated with a combination of siRNA and mAb targeting HDV replication.

[0449] AB01 is provided as a reconstituted lyophilized powder to be administered subcutaneously (SC) at 300 mg every 4 or 8 weeks (see Table 6). AB01 (HBC34v35-MLNS-GAALIE) contains the light chain amino acid sequence of SEQ ID NO: 59 and the heavy chain amino acid sequence of SEQ ID NO: 57. SIRNA01 is provided as a liquid to be administered SC at 200 mg every 4 or 8 weeks (see Table 6). SIRNA01 has a sense strand containing the nucleotide sequence of SEQ ID NO: 5 and an antisense strand containing the nucleotide sequence of SEQ ID NO: 6.

[0450] The study objectives and related endpoints are shown in Table 7.

[0451]

Table 10-1

[0452]

Table 10-2

[0453]

Table 10-3

[0454] The Child-Pugh-Turcotte (CPT) score assessment for liver disease is shown in Table 8.

[0455] [Table 11] * Grade 0: Normal consciousness, personality, neurological examination electroencephalogram Grade 1: Restless, sleep disorder, irritability / excitability, tremor, handwriting disorder, 5 cycles / second wave Grade 2: Somnolence, time-disorientation, inappropriate, asterixis, ataxia, slow triphasic wave Grade 3: Subsomnolence, coma, disorientation to place, hyperreflexia, rigidity, slower waves Grade 4: Unarousable coma, no personality / behavior, paralysis, slow 2 - 3 cycles per second δ-type activity Source: FDA 2003 Evaluation: In the case of 5 or 6 points, Class A (mild liver function impairment); in the case of 7 - 9 points, B (moderate liver function impairment), and in the case of 10 - 15 points, C (severe liver function impairment).

[0456] The ULN value of ALT can be, for example, 34 IU / mL for women and 43 IU / mL for men.

[0457] Collect PK samples as shown in Figures 21A - 21G and calculate the PK parameters (free and total PK if applicable) of SIRNA01 and AB01. The parameters include, but are not limited to, C max , C last , T max , T last , AUC inf , AUC last , %AUC exp , t 1 / 2 , λ z , V z / F, and CL / F.

[0458] Immunogenicity data can include, but are not limited to, the presence / absence and titer of anti-drug antibodies (ADA) and neutralization data.

[0459] Participant population This trial enrolls male and female participants aged 18 to 70 years with chronic HBV / HDV co-infection (both non-cirrhotic and cirrhotic up to METAVIR-F4 / CPT-A) who are currently receiving NRTI therapy. "Chronic HBV infection" for the purposes of this trial is defined as positive serum HBsAg, HBV DNA, or HBeAg in at least two instances separated by at least 6 months, based on previous (within the past 12 months) or current laboratory documentation (any combination of these tests performed 6 months apart is acceptable). Participants have been receiving locally approved NRTI therapy for at least 12 weeks prior to Day 1. Participants also 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; and serum alanine aminotransferase (ALT) above ULN and < 5×ULN.

[0460] Participants are aged 18 years (or legal age of consent, whichever is older) or older and < 70 years at screening. Participants also have a body mass index (BMI) of ≥ 18 kg / m 2 ² to ≤ 40 kg / m 2 ².

[0461] Additional inclusion criteria include the following. · Female participants must have a negative pregnancy test or confirmed postmenopausal status. Postmenopausal status is defined as 12 months without menses without another medical cause. 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 able to breastfeed, and must be willing to use a highly effective method of contraception from 14 days prior to study drug administration until 48 weeks after the last dose of SIRNA01 or AB01. Female participants must also agree to refrain from egg donation and in vitro fertilization from study drug administration until 48 weeks after the last dose of SIRNA01 or AB01. Male participants with female partners who may be pregnant must agree to meet one of the following contraceptive requirements from the time of dosing with the investigational product until 48 weeks after the last dose of SIRNA01 or AB01: documentation of vasectomy or azoospermia, or use of a male condom + use of one of the contraceptive options listed for contraception of the partner's WOCBP. Male participants must also agree not to donate sperm from the time of the first dose of the investigational product until 48 weeks after the last dose of SIRNA01 or AB01. · Be able to give signed informed consent. · A 12-lead electrocardiogram (ECG) within normal limits; or, as determined by the principal investigator of the clinical trial, no clinically significant abnormalities at the time of screening. · Agree not to donate blood during the study period and for an additional 3 months after the last dose of the investigational product.

[0462] Inclusion criteria specific to cohort 1 · Non-cirrhotic ○ Liver biopsy by METAVIR F0-F3 or liver elastography (e.g., Fibroscan) less than 12 kilopascals (kPa) within 12 months before screening ○ Creatinine clearance (CLcr) of 30 mL / min or more calculated by the Cockcroft-Gault formula at the time of screening ○ Platelet count greater than 150,000 cells / mm 3 ( / μL) Inclusion criteria specific to cohorts 2a, 2b1, 2b2, 2c, 3, and 4 · Non-cirrhotic ○ Liver biopsy by METAVIR F0-F3 or liver elastography (e.g., Fibroscan®) less than 12 kPa within 12 months before screening ○ CLcr of 30 mL / min or more calculated by the Cockcroft-Gault formula at the time of screening ○ Platelet count greater than 150,000 cells / mm 3 ( / μL) · CPT-A cirrhotic ○ Within 12 months before screening, METAVIR F4 or liver elastography of 12 kPa or more (e.g., liver biopsy by Fibroscan®) ○ CLcr of 60 mL / min or more calculated by the Cockcroft-Gault formula at the time of screening ○ 90,000 cells / mm 3 ( / μL) or more platelet count ○ 5 or 6 CPT scores including at the time of screening and at the start of the test · As an alternative to Fibroscan®, for example, 2D shear wave elastography may be acceptable.

[0463] Exclusion criteria are as follows. · History of clinically significant liver disease from non-HBV and non-HDV etiologies · History of clinically significant immune complex disease · History of clinically significant autoimmune disorder · History of HBV-related extrahepatic diseases including but not limited to HBV-related rash, arthritis, or glomerulonephritis. · History of allergic reaction, hypersensitivity, or intolerance to the test drug, its metabolites, or excipients · Anti-HBs at screening of more than 10 mIU / mL · Corrected QT interval (QTc) of more than 450 milliseconds · ALT or AST of 5 × ULN or more · Total bilirubin of more than 2.0 mg / dL · Serum albumin of less than 30 g / L · Absolute neutrophil count of less than 1,000 / mm 3 ( / μL) · International normalized ratio (INR) of more than 1.5 · Hemoglobin of less than 8 g / dL · History of anaphylaxis · History of malignant tumor diagnosed or treated within 5 years (local treatment of squamous cell or non-invasive basal cell skin cancer is acceptable. Non-invasive cervical cancer is acceptable if appropriately treated before screening. Participants under evaluation for malignant tumor are not eligible.) · Has a history of or is on the list for bone marrow or solid organ transplantation. · Known active infection other than chronic HBV and HDV infection, or fever (> 38°C) within 7 days prior to Day 1 or any clinically significant acute condition s...

Claims

1. A pharmaceutical composition for use in a method of treating hepatitis D virus (HDV) infection or HDV-related disease in a target population, (a) Anti-HBV antibody, or (b) containing siRNA that targets HBV mRNA, The above method applies to the above target, (a) Anti-HBV antibody, and (b) comprising administering siRNA targeting HBV mRNA, A pharmaceutical composition in which the subject has a Child's Pew Turcott (CPT) score of 5 or higher before treatment.

2. The pharmaceutical composition 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 pharmaceutical composition according to claim 1, wherein the subject has chronic hepatitis B virus (HBV) / HDV co-infection.

4. The pharmaceutical composition according to claim 1, wherein the subject has positive serum HBsAg, HBV DNA, or HBeAg at least twice at intervals of six months prior to treatment.

5. The pharmaceutical composition according to claim 1, wherein the subject has been tested for HDV antibody or HDV RNA before treatment.

6. The pharmaceutical composition according to claim 1, wherein the subject has been tested for positive results for HDV antibody and HDV RNA before treatment.

7. The pharmaceutical composition according to claim 1, wherein the subject has been tested for positive HDV antibodies for at least six months prior to treatment.

8. The pharmaceutical composition according to claim 1, wherein the subject has 500 IU / mL or more of HDV RNA before treatment.

9. The pharmaceutical composition according to claim 1, wherein the subject has an HBsAg level greater than 0.05 IU / mL before treatment.

10. The pharmaceutical composition according to claim 1, wherein the subject has an HBsAg level of more than 10,000 IU / mL before treatment.

11. The pharmaceutical composition according to claim 1, wherein the subject has chronic hepatitis.

12. The pharmaceutical composition according to claim 1, wherein the subject has an alanine aminotransferase (ALT) level that is above the upper limit of normal (ULN) and less than five times the ULN before treatment.

13. The pharmaceutical composition according to claim 1, wherein the subject has an ALT level above the upper limit of normal (ULN) and an aspartate aminotransferase (AST) level above ULN before treatment.

14. The pharmaceutical composition according to claim 1, wherein the subject has an ALT level of less than 5 times the ULN level and an AST level of less than 5 times the ULN level before treatment.

15. The pharmaceutical composition according to claim 1, wherein the subject has had a liver biopsy of METAVIR F4 or 12 kilopascals (kPa) or higher liver elastography (Fibroscan®) within 12 months prior to treatment.

16. The pharmaceutical composition according to claim 1, wherein the subject has a creatine clearance (CLcr) of 60 mL / min or more, calculated by the Cockcroft-Gault formula before treatment.

17. The pharmaceutical composition according to claim 1, wherein the subject has a Child's Pew Turcott (CPT) score of 5 or 6 before treatment.

18. The pharmaceutical composition according to claim 1, wherein the subject has a Child's Pew Turcott (CPT) score of 7 or higher before treatment.

19. The pharmaceutical composition according to claim 1, wherein the subject has a Child's Pew Turcott (CPT) score of 7 to 9 before treatment.

20. The pharmaceutical composition according to claim 1, wherein the subject has a Child's Pew Turcott (CPT) score of 10 or more before treatment.

21. The pharmaceutical composition according to claim 1, wherein the subject has not been previously administered an anti-HBV antibody or siRNA targeting HBV mRNA.

22. The pharmaceutical composition according to claim 1, wherein the anti-HBV antibody is a human antibody.

23. The pharmaceutical composition according to claim 1, wherein the antibody is HBC34 or a non-natural variant of HBC34.

24. The aforementioned anti-HBV antibody (i) The CDRH1, CDRH2, and CDRH3 amino acid sequences described in SEQ ID NOs. 44, 45, 46, and 47, respectively, and (ii) The pharmaceutical composition according to claim 1, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 48, 49, or 50, and 52, respectively.

25. The aforementioned anti-HBV antibody (i) The CDRH1, CDRH2, and CDRH3 amino acid sequences described in Sequence ID Nos. 44, 45, and 47, respectively, and (ii) The pharmaceutical composition according to claim 24, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 48, 49, and 52, respectively.

26. The aforementioned anti-HBV antibody (i) The CDRH1, CDRH2, and CDRH3 amino acid sequences described in SEQ ID NOs. 44, 46, and 47, respectively, and (ii) The pharmaceutical composition according to claim 24, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences described in Sequence ID Nos. 48, 50, and 52, respectively.

27. The aforementioned anti-HBV antibody The pharmaceutical composition according to claim 1, comprising (a) a light chain variable domain (VL) which is at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO: 55, and (b) a heavy chain variable domain (VH) which is at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO:

53.

28. The aforementioned anti-HBV antibody The pharmaceutical composition according to claim 1, comprising (a) a light chain that is at least 90%, at least 95%, or 100% identical to the amino acid sequence described 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 described in SEQ ID NO:

57.

29. The pharmaceutical composition according to claim 1, wherein the siRNA comprises a sense strand and an antisense strand forming a double-stranded region, and the sense strand comprises at least 15 consecutive nucleotides that differ from nucleotides 1579 to 1597 of SEQ ID NO: 1 by three or fewer nucleotides, and T is substituted with U.

30. The pharmaceutical composition according to claim 1, wherein the antisense strand of the siRNA contains or consists of the nucleotide sequence 5'-UGUGAGCGGAAGUGCAACUCUU-3' (SEQ ID NO: 4).

31. The pharmaceutical composition according to claim 30, wherein the sense strand of the siRNA contains or consists of the nucleotide sequence 5'-GUGUGCCACUUCGCCUUCACA-3' (SEQ ID NO: 3).

32. The pharmaceutical composition according to claim 31, wherein at least one strand of the siRNA includes a 3' overhang of at least one nucleotide.

33. The pharmaceutical composition according to claim 31, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs long.

34. The pharmaceutical composition according to claim 31, wherein each chain of the RNAi agent has 15 to 30 nucleotides.

35. Substantially all nucleotides of the sense strand of the siRNA and substantially all nucleotides of the antisense strand of the siRNA are modified nucleotides. The pharmaceutical composition according to claim 31, wherein the sense chain is conjugated to a ligand bound to its 3' end.

36. The pharmaceutical composition according to claim 35, wherein the ligand is one or more GalNAc derivatives linked via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

37. The pharmaceutical composition according to claim 36, wherein the ligand is as follows. 【Chemistry 1】

38. The siRNA is conjugated to the ligand as shown in the following structure, 【Chemistry 2】 The pharmaceutical composition according to claim 37, wherein X is O or S in the formula.

39. The pharmaceutical composition according to claim 38, wherein X is O.

40. The pharmaceutical composition according to claim 31, wherein at least one nucleotide of the siRNA is a modified nucleotide comprising a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a stereostructure-restricted nucleotide, a restricted ethyl nucleotide, a base-free nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramide, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing 5'-phosphate, an adenosine glycol nucleic acid, or a nucleotide containing a 5'-phosphate mimetic.

41. The pharmaceutical composition according to claim 31, wherein the siRNA includes phosphate backbone modification, 2'-ribose modification, 5'-triphosphate modification, or GalNAc conjugation modification.

42. The pharmaceutical composition according to claim 31, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand of the siRNA are modified nucleotides.

43. The siRNA comprises a sense strand containing 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand containing 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), 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 glycol nucleic acid (GNA), s is a phosphorothioate bond, L96 is 【Transformation 3】 The pharmaceutical composition according to claim 1.

44. The L96 is conjugated to the sense chain as shown in the following structure, 【Chemistry 4】 The pharmaceutical composition according to claim 43, wherein X is O in the formula.

45. A pharmaceutical composition for use in a method of treating hepatitis D virus (HDV) infection or HDV-related disease in a subject requiring treatment, (a) anti-HBV antibody, (b) siRNA targeting HBV mRNA, or (c) Contains a nucleoside(tide) reverse transcriptase inhibitor (NRTI), The above method applies to the above target, (a) anti-HBV antibody, (b) siRNAs that target HBV mRNA, and (c) The administration of a nucleoside(tide) reverse transcriptase inhibitor (NRTI), A pharmaceutical composition in which the subject has a Child's Pew Turcott (CPT) score of 5 or higher before treatment.

46. The pharmaceutical composition according to claim 1, wherein the subject has been administered NRTI in advance.

47. The pharmaceutical composition according to claim 45, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil (TD), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), terbivudine, AGX-1009, emtricitabine (FTC), klevudine, ritonavir, dipivoxil, lobucavir, famvir, N-acetylcysteine ​​(NAC), PC1323, teradigm-HBV, thymosin-α, and ganciclovir, besifovir (ANA-380 / LB-80380), or tenofovir exaliades (TLX / CMX157).

48. The pharmaceutical composition according to claim 45, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), or tenofovir disoproxil (TD).

49. (a) The anti-HBV antibody contains or comprises the light chain amino acid sequence described in SEQ ID NO: 59 and the heavy chain amino acid sequence described in SEQ ID NO: 57, and (b) The siRNA comprises or consists of a sense strand containing or comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand containing or comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), 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 glycol nucleic acid (GNA), s is a phosphorothioate bond, L96 is 【Transformation 5】 The pharmaceutical composition according to claim 1.

50. (a) The anti-HBV antibody comprises or consists of the light chain amino acid sequence described in SEQ ID NO: 59 and the heavy chain amino acid sequence described in SEQ ID NO:

57. (b) The siRNA comprises or consists of a sense strand containing or comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5) and an antisense strand containing or comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), 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 glycol nucleic acid (GNA), s is a phosphorothioate bond, L96 is 【Transformation 6】 And, (c) The pharmaceutical composition according to claim 45, wherein the NRTI is tenofovir disoproxil fumarate (TDF) or entecavir.

51. The pharmaceutical composition according to claim 1, wherein the anti-HBV antibody is administered every two weeks, every four weeks, or every eight weeks.

52. The pharmaceutical composition according to claim 1, wherein the siRNA is administered every four weeks or every eight weeks.

53. The pharmaceutical composition according to claim 45, wherein the NRTI is administered daily.

54. The pharmaceutical composition according to claim 1, wherein the anti-HBV antibody is administered in a dose of 300 mg.

55. The pharmaceutical composition according to claim 1, wherein the siRNA is administered in a dose of 200 mg.

56. The pharmaceutical composition according to claim 45, wherein the NRTI is administered in a dose of 300 mg.

57. The pharmaceutical composition according to claim 45, wherein the NRTI is administered in a dose of 245 mg.

58. The pharmaceutical composition according to claim 1, wherein the subject is administered the siRNA and the anti-HBV antibody starting on the same day.

59. The pharmaceutical composition according to claim 1, wherein the subject is administered the siRNA and the anti-HBV antibody for a maximum of 96 weeks, 96 weeks, at least 96 weeks, or for 96 weeks or more.

60. The pharmaceutical composition according to claim 1, wherein the subject is a human.

61. A kit for use in a method for treating hepatitis D virus (HDV) infection or HDV-related disease in a subject requiring treatment, A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient, and A kit comprising a pharmaceutical composition containing an siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient.

62. A kit for use in a method for treating hepatitis D virus (HDV) infection or HDV-related disease in a subject requiring treatment, A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient, A pharmaceutical composition comprising an siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient, and A kit comprising a pharmaceutical composition containing NRTI and a pharmaceutically acceptable excipient.

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