Compositions and methods for treating hepatitis B virus (HBV) infection and HBV-related diseases

Abstract

The present disclosure provides methods for treating HBV infection using combination therapies, as well as related kits and compositions for use. The components of the combination therapy may include one or more of an anti-HBV antibody, an siRNA targeting HBV mRNA, interferon-α, and a nucleos(t)ide reverse transcriptase inhibitor (NRTI).

Description

Technical Field

[0001] Reference to Electronic Sequence Listing The content of the electronic sequence listing (441WO_SeqListing.xml; size: 70.5 KB; and creation date: May 4, 2023) is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] 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, incomplete DNA (relaxed-circular DNA, rcDNA) and has four open reading frames encoding seven proteins: HBcAg (HBV core antigen, the 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 (HBV x antigen, which requires transcriptional regulation 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).

[0003] 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 chromatin 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 of new virions, which is secreted without causing cytotoxicity. In addition to infectious virions, infected hepatocytes secrete a large number of subviral particles without genomes, which can exceed 10,000 times the number of secreted virions (Seeger et al., 2015, see above). Similarly, random integration of the virus into the host genome can also occur, which is a mechanism contributing to the transformation of hepatocytes (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).

[0004] Hepatitis B infection is characterized by serological viral markers and antibodies. In acute resolving infections, the virus is cleared by effective innate and adaptive immune responses, including the induction of cytotoxic T cells, which cause the death of infected hepatocytes, and B cells, which produce neutralizing antibodies that prevent viral spread (Bertoletti A, Adaptive immunity in HBV infection, Journal of Hepatology 2016, 64(1): S71 - S83, Maini MK et al., The role of innate immunity in the immunopathology and treatment of HBV infection, Journal of Hepatology 2016, 64(1): S60 - S70, Li Y et al., Genome - wide association study identifies 8p21.3 associated with persistent hepatitis B virus infection among Chinese, Nature Communications 2016, 7:11664). In contrast, chronic infection is associated with T - and B - cell dysfunction 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). Thus, the continuous expression and secretion of viral proteins due to the persistence of cccDNA in hepatocytes is considered an important step in the host's inability to eliminate the infection.

[0005] Chronic HBV infection continues to be an important global public health problem with significant morbidity and mortality (Trepo C, A brief history of hepatitis milestones, Liver Int. 2014, Feb; 34 Suppl 1: 29 - 37). Chronic HBV infection is a dynamic process characterized by the interaction between viral replication and the host immune response. Patients can be classified into different disease stages based on the levels of hepatitis B e antigen (HBeAg), HBV DNA, alanine aminotransferase (ALT), and liver inflammation (European Association for the Study of the Liver, EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection, J Hepatol. 2017 Aug, 67(2): 370 - 398, Sarin SK et al., Asian - Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update, Hepatol Int. 2016 Jan, 10(1): 1 - 98, Terrault NA et al., Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance, Hepatology 2018 Apr, 67(4): 1560 - 1599). Among the 300 million patients chronically infected with HBV, HBeAg - negative patients represent the largest subgroup. These patients range from those who are inactive carriers to those with chronic active hepatitis who can progress to severe liver complications including hepatocellular carcinoma (HCC). Inactive carriers are HBeAg - negative and anti - HBe - positive, with persistently low levels of HBV DNA (less than 2,000 IU / mL) and normal ALT maintained for at least one year.These patients have a good long-term prognosis, a low histological progression rate, a low risk of cirrhosis or HCC, and a high long-term hepatitis B surface antigen (HBsAg) clearance rate (EASL, 2017, see above, Invernizzi F et al., The prognosis and management of inactive HBV carriers, Liver Int. 2016 Jan, 36 Suppl 1:100-4; Terrault et al., 2018, see above, Yeo YH et al., Incidence, Factors, and Patient-Level Data for Spontaneous HBsAg Seroclearance: A Cohort Study of 11,264 Patients, Clin Transl Gastroenterol 2020Sep, 11(9):e00196). Inactive carriers lack indications for currently available treatments aimed at HBV DNA suppression, but they have the highest potential to achieve functional cure, thereby becoming non-infectious and having a lower risk of disease reactivation.

[0006] Among non-cirrhotic patients with chronic HBV infection, treatment is currently recommended for subsets with high levels of viremia (HBV DNA) and elevated levels of alanine aminotransferase (ALT) (EASL, 2017, supra; Sarin et al., 2016, supra; Terrault et al., 2018, supra). Current treatment options for chronic HBV infection are limited to nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and peginterferon-alpha-2a (PEG-IFNα-2a or "PEG-IFNa-2a") (Liang TJ et al., Present and future therapies of hepatitis B: From discovery to cure, Hepatology 2015 Dec, 62(6):1893-908). Long-term NRTI therapy can suppress HBV DNA but does not eliminate cccDNA or integrated DNA. In contrast to NRTIs, PEG-IFNa can induce long-term viral control but only in a small percentage (less than 10%) of patients and only after 48 weeks of treatment (Konerman MA et al., Interferon Treatment for Hepatitis B, Clin Liver Dis. 2016 Nov, 20(4):645-665). Thus, there remains an unmet need for better treatment options that can achieve functional cure.

[0007] None of the currently available treatments restore immunological control of HBV in the majority of patients. Thus, there remains a need for an effective treatment for HBV infection that can inhibit viral replication and restore immunological control in the majority of patients. SUMMARY OF THE INVENTION

[0008] In some aspects, the present disclosure provides a method of doing so in a subject in need of treating hepatitis B virus (HBV) infection or an HBV-related disease, the method comprising administering to the subject, (a) an anti-HBV antibody, and (b) an siRNA targeting HBV mRNA, and (c) a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI), wherein the subject is HBeAg-negative, has an HBV DNA level of 2000 IU / mL or less prior to treatment, is non-cirrhotic, has an alanine transaminase (ALT) level at or below the upper limit of normal (ULN), has not been previously administered an NRTI, has not received an NRTI within 24 weeks prior to treatment, has not been previously administered an anti-HIV antibody, and / or has not been previously administered an siRNA targeting HBV mRNA.

[0009] In some aspects, the present disclosure provides a method of doing so in a subject in need of treating hepatitis B virus (HBV) infection or an HBV-related disease, the method comprising administering to the subject, (a) an anti-HBV antibody, and (b) an siRNA targeting HBV mRNA, and (c) interferon-α, and (d) a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI), A method is provided where the subject is HBeAg-negative or HBeAg-positive, the subject has an HBV DNA level of more than 2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level above the upper limit of normal (ULN) and up to 5 times the ULN, the subject has not been previously administered an NRTI, the subject has not received an NRTI within 24 weeks before treatment, the subject has not been previously administered an anti-HIV antibody, the subject has not been previously administered an siRNA targeting HBV mRNA, the subject has not been previously administered interferon-α, the subject is HBsAg-positive before treatment, and / or the subject has an HBsAg level of more than 10 IU / mL before treatment.

[0010] In some embodiments, compositions for use in treatment, compositions for use in the manufacture of a medicament, and kits are provided.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0012] The present disclosure provides methods and compositions for use in the treatment of hepatitis B virus (HBV) infection or HBV-related diseases, wherein a subject is administered one or more of an anti-HBV antibody, anti-HBV siRNA, interferon alpha, and an NRTI, as well as related kits. In some embodiments, such combination therapies are used to treat chronic hepatitis B. In some embodiments, such combination therapies are used to treat hepatitis D virus (HDV) infection.

[0013] I. Glossary The following section provides a detailed description of combination therapies for treating HBV infection or HBV-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 to facilitate understanding of the present disclosure. Additional definitions are provided throughout the present disclosure.

[0014] As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise indicated.

[0015] The term "comprise" (and similar terms such as "comprising of" and "comprised of") means the presence of the described features, integers, steps, or components recited in the claims, but does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. The term "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the basic and novel features of the claimed invention.

[0016] As used herein, the terms "a" and "an" are to be understood as referring to "one or more" of the recited components. The use of an alternative (e.g., "or") is to be understood as meaning either one of the alternatives, both, or any combination thereof, 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 construed as non-limiting or open-ended.

[0017] The term "substantially" does not exclude "completely"; for example, a composition "substantially free of" Y may not completely exclude Y. Optionally, the term "substantially" may be omitted from the definitions provided herein.

[0018] As used herein, the term "disease" is generally intended to be synonymous and is used interchangeably with the terms "disorder" and "condition" (in the context of "medical condition"), all reflecting an abnormal condition of one of the human or animal body or a part thereof that impairs normal function, typically manifested by characteristic signs and symptoms, and causing a reduction in the lifespan or quality of life of a human or animal.

[0019] As used herein, the terms "peptide", "polypeptide", and "protein", and variations of these terms, each refer to a molecule, particularly a protein that includes a peptide, oligopeptide, polypeptide, or fusion protein, which contains at least two amino acids joined to each other by normal peptide bonds or by modified peptide bonds such as, for example, 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 that are linked to each other by normal peptide bonds ("classical" polypeptides). A peptide, polypeptide, or protein can be composed of L-amino acids and / or D-amino acids. In particular, the terms "peptide", "polypeptide", and "protein" also include "peptidomimetics" defined as peptide analogs that contain non-peptidic structural elements such that the peptide is capable of mimicking or antagonizing the biological action of the native parent peptide. Peptidomimetics lack classical peptide features such as enzymatically cleavable peptide bonds. In particular, a peptide, polypeptide, or protein can contain amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids, or can 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 terminal ends. In particular, a peptide or polypeptide can be branched after ubiquitination, or can 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 particularly also include modified peptides, polypeptides, and proteins.For example, peptide, polypeptide, or protein modifications 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 crosslinking, cyclization, disulfide bond formation, demethylation, glycosylation including PEGylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfation, addition of amino acids such as arginylation, or ubiquitination. Such modifications are fully detailed in the literature (Proteins Structure and Molecular Properties, 2nd Ed., 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.

[0020] As used herein, "(poly)peptide" includes a single chain of amino acid monomers linked by peptide bonds as described above. "Protein", as used herein, includes 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.

[0021] The term "recombinant", as used herein, (e.g., recombinant antibodies, recombinant proteins, recombinant nucleic acids, etc.) refers to any molecule (antibodies, proteins, nucleic acids, siRNA, etc.) that is prepared, expressed, produced, or isolated by recombinant means and does not occur 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.

[0022] 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 passages. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or adventitious mutations. Mutant progeny having the same function or biological activity as screened in the originally transformed cell are included. When a particular name is intended, it will be apparent from the context.

[0023] As used herein, the term "array variant" refers to any array having one or more changes compared to a reference array, where the reference array is any one 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 sequence is also a nucleotide sequence, while for array variants in the context of amino acid sequences, the reference sequence 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 arrays listed in this application) unless otherwise specified. The percentage identity, when referred to herein, can be determined using BLAST with the default parameters specified, for example, 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]. An "array variant" in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more of the 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, an "array variant" of a nucleotide sequence can either result in a change in the respective reference amino acid sequence, i.e., an amino acid "array variant", or not. In certain embodiments, a nucleotide sequence variant is a mutant 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 give rise to 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 of the amino acids are deleted, substituted, or inserted. As a result of the change, 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 changes, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids of the reference sequence is "at least 90% identical" to the reference sequence.

[0024] It is possible to have non-conservative amino acid substitutions, but in certain embodiments, the substitutions are conservative amino acid substitutions where the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the reference sequence. Examples of conservative amino acid substitutions include substitution of one aliphatic or hydrophobic amino acid, e.g., alanine, valine, leucine, and isoleucine, for another, substitution of one hydroxyl-containing amino acid, e.g., serine and threonine, for another, substitution of one acidic residue, e.g., glutamic acid or aspartic acid, for another, replacement of one amide-containing residue, e.g., asparagine and glutamine, for another, replacement of one aromatic residue, e.g., phenylalanine and tyrosine, for another, replacement of one basic residue, e.g., lysine, arginine, and histidine, for another, and replacement with another of one small amino acid, e.g., alanine, serine, threonine, methionine, and glycine.

[0025] Amino acid insertions include amino-terminal fusions and / or carboxyl-terminal fusions in the range of lengths from one 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.

[0026] Unless otherwise stated, changes in sequence variants do not impair the functionality of the respective reference sequences, e.g., in this case, the functionality of the sequences of anti-HBV antibodies or siRNAs that sufficiently neutralize HBV infection or sufficiently reduce HBV protein expression, respectively. Guidance for determining 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.

[0027] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide, or protein refers to the source of the nucleic acid, peptide, polypeptide, or protein. 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. "Corresponding" particularly refers to the same functionality. 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 skilled in the art. Similarly, sequences "derived from" other sequences are generally identifiable to those skilled in the art as having their source in the sequence.

[0028] In some embodiments, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein can 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 can also have one or more mutations compared to the starting nucleic acid, peptide, polypeptide, or protein from which it is derived. In particular, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein can be a functional sequence variant of the starting nucleic acid, peptide, polypeptide, or protein from which it is derived. For example, in a peptide / protein, one or more amino acid residues can be substituted with other amino acid residues, or one or more insertions or deletions of amino acid residues can be made.

[0029] 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. For example, a mutation as compared to a genomic sequence can be, e.g., a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation induced, e.g., by an enzyme, a chemical, or radiation, or a mutation obtained by site-directed mutagenesis (a molecular biology method for creating specific and intentional changes in a nucleic acid sequence and / or an amino acid sequence). Thus, it is understood that the term "mutation" or "mutating" also includes, e.g., 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, e.g., by changing the codon of a nucleic acid molecule encoding one amino acid, e.g., by site-directed mutagenesis, to yield a codon encoding a different amino acid, or by synthesizing sequence variants by, e.g., 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 mutant of the polypeptide without mutating one or more nucleotides of the nucleic acid molecule.

[0030] 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.

[0031] As used herein, the term "expression" refers to any step involved in the production of a polypeptide, including transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0032] 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 or immunogen. The antigen or immunogen can be derived from any material suitable for vaccination. For example, the antigen or immunogen can be derived from pathogens such as bacterial or viral particles, or from tumors or cancerous tissues. 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.

[0033] 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 is part of subviral particles that are produced in very large excess compared to virion particles, in addition to decorating the envelope of HBV, 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 has been associated with the development of liver cancer.

[0034] Nine genotypes of HBV designated 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. As used herein, the term “HBV” also refers to naturally occurring DNA sequence variations of the HBV genome, namely genotypes A-J and variants thereof.

[0035] In some embodiments, the present disclosure provides a combination therapy for treating HBV that includes an anti-HBV siRNA. The 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 frequently 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 broken down into siRNA by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 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, enabling 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).

[0036] 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, as used herein, refer to at least a partial reduction in the expression of the HBV gene as represented by a reduction in the amount of HBV mRNA, the HBV gene is transcribed, treated with an inhibitor of HBV gene expression, and thereby substantially identical to a first cell or cell population but a second cell or cell population (control cell) that has or has not been so treated, compared to, the expression of the HBV gene is inhibited, represented by a reduction in the amount of HBV mRNA that can be isolated or detected from the first cell or cell population. The degree of inhibition can be measured, for example, as the difference obtained by subtracting the degree of mRNA expression in the treated cells from the degree of mRNA expression in the control cells. Alternatively, the degree of inhibition can be obtained in terms of a parameter that is functionally related to HBV gene expression, for example, the amount of protein encoded by the HBV gene, or a particular phenotype, for example, the reduction in the number of cells exhibiting an HBV infection phenotype. In principle, HBV gene silencing can be determined by any appropriate assay in any cell that expresses the HBV gene, for example, an HBV-infected cell or a cell engineered to express the HBV gene.

[0037] 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 evaluating mRNA expression, such as the rtPCR method provided in Example 2 of International Application Publication No. 2016 / 077321 (A1) and U.S. Patent Application Publication No. 2017 / 0349900 (A1), which are incorporated herein by reference. In some embodiments, the expression level of an HBV gene (e.g., total HBV RNA, HBV transcript, e.g., HBV 3.5 kb transcript) in a sample 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 using RNA extraction techniques including, for example, acid phenol / guanidine 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 assay, RT-PCR, RNase protection assay (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. 2012 / 177906 (A1) and U.S. Patent Application Publication No. 2014 / 0275211 (A1), which are incorporated herein by reference.

[0038] 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 will be at least long enough to function as a substrate for RNAi-specific cleavage at or near that portion. For example, target sequences are 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, target sequences can be 15 to 30 nucleotides, 15 to 26 nucleotides, 15 to 23 nucleotides, 15 to 22 nucleotides, 15 to 21 nucleotides, 15 to 20 nucleotides, 15 to 19 nucleotides, 15 to 18 nucleotides, 15 to 17 nucleotides, 18 to 30 nucleotides, 18 to 26 nucleotides, 18 to 23 nucleotides, 18 to 22 nucleotides, 18 to 21 nucleotides, 18 to 20 nucleotides, 19 to 30 nucleotides, 19 to 26 nucleotides, 19 to 23 nucleotides, 19 to 22 nucleotides, 19 to 21 nucleotides, 19 to 20 nucleotides, 20 to 30 nucleotides, 20 to 26 nucleotides, 20 to 25 nucleotides, 20 to 24 nucleotides, 20 to 23 nucleotides, 20 to 22 nucleotides, 20 to 21 nucleotides, 21 to 30 nucleotides, 21 to 26 nucleotides, 21 to 25 nucleotides, 21 to 24 nucleotides, 21 to 23 nucleotides, or 21 to 22 nucleotides.

[0039] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide that includes a strand of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0040] 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 would be understood by one of ordinary skill in the art. Such conditions can be, for example, stringent conditions, which can include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, and incubation at 50°C or 70°C for 12 to 16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered within an organism, can also be applied. One of ordinary skill in the art will be able to determine the most appropriate set of conditions for testing the complementarity of the two sequences, depending on the ultimate use of the hybridized nucleotides.

[0041] 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 may be referred to herein as "perfectly complementary" to each other. However, as used herein, when it is mentioned that a first sequence is "substantially complementary" to a second sequence, the two sequences may be perfectly complementary or, while retaining the ability to hybridize under conditions most relevant to their ultimate use, e.g., inhibition of gene expression via the RISC pathway, form one or more, but generally 5, 4, 3, or 2 or fewer mismatched base pairs upon hybridization for double-strands up to 30 base pairs. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches with respect to the determination of complementarity. For example, an siRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "perfectly complementary" herein for the purposes described.

[0042] As used herein, "complementary" sequences may also include, or consist entirely of, base pairs formed from non-Watson-Crick base pairs and / or non-natural and modified nucleotides, so long as the above requirements regarding the ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogsteen base pairing.

[0043] 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 siRNA, or between the antisense strand of an siRNA agent and a target sequence, as would be understood from the context in which they are used.

[0044] As used herein, a polynucleotide that is "substantially complementary" to at least a portion of an mRNA refers to a polynucleotide that is substantially complementary to a continuous portion of the target mRNA (e.g., the mRNA encoding an HBV protein). For example, a polynucleotide is complementary to at least a portion of HBV mRNA if the sequence is substantially complementary to an uninterrupted portion of HBV mRNA.

[0045] 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 allows for the specific degradation of a desired target RNA through 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, without limitation, any sub-range therebetween including 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, 21-23 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.

[0046] 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 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 (referred to herein as a "hairpin loop") separated by a single strand of nucleotides 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, and 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 connected, but can be covalently connected. When the two strands are covalently connected by means other than a hairpin loop, the connecting structure is referred to as a "linker".

[0047] 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.

[0048] The term "antisense strand" or "guide strand" refers to a 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, for example, the target sequence as defined herein. When 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, for example, the 5' end and / or the 3' end.

[0049] As used herein, the term "sense strand" or "passenger strand" refers to the strand of siRNA that includes a region that is substantially complementary to the region of the antisense strand as defined herein.

[0050] The term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules that occur naturally or are 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" may 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 duplexes. By way of non-limiting example, RNA molecules can also include, but are not limited to, 2'-O-methyl modified nucleosides, nucleosides containing 5' phosphorothioate groups, terminal nucleosides linked to cholesteryl derivatives or didodecylamid groups of dodecanoic acid, 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, and can include at least one modified ribonucleoside. In another example, the RNA molecule can consist of 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 largest 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, for example, one or more deoxynucleosides containing a deoxynucleotide overhang, or one or more deoxynucleosides within the duplex portion of the siRNA.However, the term "siRNA" as used herein does not include a complete DNA molecule.

[0051] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of the siRNA. For example, when the 3' end of one strand of the siRNA extends beyond the 5' end of the other strand, or vice versa, a nucleotide overhang is present. The siRNA can include an overhang of at least 1 nucleotide, or alternatively, the overhang can include at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, or more. The nucleotide overhang can include or consist of nucleotide / nucleoside analogs that include deoxynucleotides / nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotides 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.

[0052] The term "blunt" or "blunt-ended" as used herein in connection with siRNA 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. Such molecules are often double-stranded over their entire length.

[0053] 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 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 D virus.

[0054] 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 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 the antibody of the 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.

[0055] As used herein, a "neutralizing antibody" is an antibody that can neutralize, i.e., prevent, inhibit, reduce, interfere with, or disrupt, the ability of a pathogen to initiate and / or perpetuate infection in a host. The terms "neutralizing antibody" and "antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used alone or in combination as prophylactic or therapeutic agents upon appropriate formulation, in connection with active vaccination, as diagnostic tools, or as production tools described herein.

[0056] 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 products during immunization. The introduction of a 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 the improved EBV-B cell immortalization described by Traggiai E et al. (Nat Med. 2004, 10(8):871-5). As used herein, the term “human antibody” also includes antibodies, e.g., antibodies having modified variable regions, which are engineered to produce the characteristics described herein.

[0057] The antibody for combination therapy can be of any isotype (e.g., IgA, IgG, IgM, i.e., κ, γ, or μ 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 κ or λ 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 through the FcRN-IgG receptor. Thus, the IgG-type HBsAg-specific antibody can bind intracellularly, thereby blocking the release of HBV virions and HBsAg.

[0058] As used herein, "variable region" (variable region of a light chain (VL), variable region of a heavy chain (V HThe term "()" includes complementarity determining regions ("CDR") and framework regions ("FR"), and refers to a portion of an antibody light chain (LC) or heavy chain (HC) (typically, about 105 - 120 amino-terminal amino acids of a mature antibody heavy or light chain) that is directly involved in the binding of the antibody to an antigen. The terms "complementary determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids within the variable region of an antibody that confer antigen specificity and / or binding affinity. Generally, for example, an antibody has three CDRs in each variable region of the immunoglobulin binding protein, and the VH and VL regions generally contain six CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3). Immunoglobulin sequences can be aligned against a numbering scheme (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). In certain embodiments, it will be understood that 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.

[0059] 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 antibody, Fab, Fab’, F(ab’)2, Fv, or scFv. In some places, the present disclosure may explicitly refer to antigen-binding fragments of the antibody, antibody fragments, variants, and / or derivatives, but as used herein, the term “antibody” or “antibody of the combination therapy” includes all categories of antibodies, i.e., antigen-binding fragments of the antibody, antibody fragments, variants, and derivatives.

[0060] Antibody fragments can be obtained from antibodies by methods including digestion with enzymes such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments 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 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, e.g., single-chain Fv in which the heavy and light chain variable domains are joined by a peptide linker.

[0061] The antibody fragments of the present disclosure can confer monovalent or multivalent interactions and can be incorporated into various structures as described above. For example, scFv molecules can be synthesized to create trivalent "triabodies" or tetravalent "tetrabodies". The scFv molecule can include domains of the Fc region that result in a bivalent minibody. In addition, 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).

[0062] Antibodies according to the present disclosure can be provided in a purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition, usually less than 60% by weight, more usually less than 50% by weight, will be composed of other polypeptides.

[0063] The antibodies and antigen-binding fragments of the present disclosure can be, in embodiments, multispecific (e.g., bispecific, trispecific, tetravalent, etc.) and can 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), the bispecific formats and methods for making the same are incorporated herein by reference, for example, bispecific T cell engager (BiTE), DART, Knobs-Into-Hole (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 (DAF) (two-in-one or four-in-one), DutaMabs, DT-IgG, charge pair, Fab arm exchange, SEED body, Triomab, LUZ-Y assembly, Fcab, κλ body, orthogonal Fab, 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, Zy body, and DVI-IgG (four-in-one). Bispecific or multispecific antibodies can include those that combine the HBV- and / or HDV-specific binding domains of the present disclosure with another such binding domain of the present disclosure, or with different binding domains that specifically bind to HBV and / or HDV (e.g., at the same or different epitopes).

[0064] II. siRNA Targeting HBV In some embodiments, the disclosure provides a method of treatment comprising administering an siRNA that targets HBV mRNA, as well as related compositions and kits.

[0065] In some embodiments, the siRNA that targets HBV mRNA is SIRNA01. SIRNA01 is a synthetic chemically modified siRNA that targets HBV RNA and has a covalently attached branched trisaccharide N-acetyl-galactosamine (GalNAc) ligand that enables specific uptake by hepatocytes. SIRNA01 targets mRNA encoded by regions of the HBV genome that are common to all HBV viral transcripts and are 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 may have multiple antiviral effects, including degradation of pgRNA and thus inhibition of viral replication, and degradation of all viral mRNA transcripts, thereby preventing the expression of viral proteins. This may result in 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.

[0066] SIRNA01 targets the mRNA encoded by the HBV genome described in NCBI reference sequence NC_003977.2 (GenBank accession number GI:21326584) (SEQ ID NO:1) and inhibits its expression. More specifically, SIRNA01 targets the mRNA encoded by a portion of the HBV genome that includes 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 causes a 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. 2020 / 036862 (A1), which is hereby incorporated by reference into this specification.

[0067] 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 trisubstituted N-acetyl-galactosamine (GalNAc) ligand at the 3’ end of the sense strand to facilitate delivery to hepatocytes through the asialoglycoprotein receptor (ASGPR). Including the modifications, the sense strand of SIRNA01 contains 5’-gsusguGfcAfCfUfucgcuucacaL96-3’ (SEQ ID NO:5), and the antisense strand contains 5’-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3’ (SEQ ID NO:6), with the modifications abbreviated as shown in Table 1.

[0068]

Table 1

[0069] In some embodiments, the siRNA used in the methods, compositions, or kits described herein is SIRNA01. In some embodiments, the siRNA used in the methods, compositions, or kits described herein comprises a sequence variant of SIRNA01. In certain embodiments, a portion of the HBV transcript targeted by the sequence variant of SIRNA01 overlaps with a portion of the HBV transcript targeted by SIRNA01.

[0070] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein (1) the sense strand comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence that differs from SEQ ID NO: 3 or SEQ ID NO: 5 by 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides, respectively, or (2) the antisense strand comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or a sequence that differs from SEQ ID NO: 4 or SEQ ID NO: 6 by 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides, respectively.

[0071] In some embodiments, shorter double-strands are used that have a sequence with only a few nucleotides subtracted from one or both ends of the sequence of SEQ ID NO: 4 or SEQ ID NO: 6. Thus, siRNAs having a partial sequence 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 having an ability to inhibit the expression of the HBV gene that differs by 5, 10, 15, 20, 25, or 30% or less from that of an siRNA comprising 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 SIRNA01 are provided.

[0072] 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 four or fewer, three or fewer, two or fewer, or one 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 four or fewer, three or fewer, two or fewer, or one or fewer nucleotides, respectively.

[0073] In some embodiments, shorter double-strands having a sequence with only a few nucleotides removed from one or both ends of the sequence of SEQ ID NO: 8 are used. Thus, siRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from SEQ ID NO: 8 and having an ability to inhibit HBV gene expression that differs from that of the siRNA containing the full sequence by 5, 10, 15, 20, 25, or 30% or less 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.

[0074] 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 to within the last 5 nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide siRNA strand that is complementary to a region of the HBV gene, the RNA strand may not contain a mismatch within the central 13 nucleotides. It can be determined whether an siRNA containing a mismatch to the target sequence is effective in inhibiting HBV gene expression using the methods described herein or methods known in the art.

[0075] In some embodiments, the siRNA used in the methods, compositions, and kits described herein comprises 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 known in the art, the complementary sequences of siRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0076] In some embodiments, single-stranded antisense RNA molecules comprising the antisense strand of the siRNA 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.

[0077] In some embodiments, single-stranded antisense RNA molecules comprising the antisense strand of SIRNA01 or its sequence variant 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.

[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 may 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 hereby incorporated by reference herein. Examples of such modifications are described in more detail below.

[0079] In some embodiments, substantially all or all nucleotides of the sense strand of the siRNA, and substantially all or all nucleotides of the antisense strand, are modified nucleotides. The nucleotides can be modified as described below.

[0080] a. Modified siRNA Modifications disclosed herein include, for example, (a) sugar modifications (e.g., at the 2'- or 4'-position) or replacement of the sugar; (b) backbone modifications including modification or replacement of the phosphodiester bond; (c) base modifications, e.g., replacement of a base with a base that pairs with an expanded repertoire of partners, a stabilized base, a destabilized base, or a conjugated base, removal of a base (abasic nucleotide), or a conjugated base; and (d) terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, inverted linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.). Some specific examples of modifications that can be incorporated into the siRNAs of the present application are shown in Table 1.

[0081] The modification includes a substituted sugar moiety. The siRNAs taken up in this specification may include, at the 2'-position, one of the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In some other embodiments, the siRNA may include, at the 2'-position, one of the following: C1-C 10 lower alkyl, substituted lower alkyl, aralkyl, aralkyl, O-aralkyl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, cycloalkyl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of siRNA, or group for improving the pharmacodynamic properties of siRNA, and other substituents having similar properties. In some embodiments, the modification includes 2'-O-(2-methoxyethyl) or 2'-methoxyethoxy also known as 2'-MOE, 2'-O-CH2CH2OCH3 (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'-DMAOE, and 2'-dimethylaminoethoxyethoxy (in the art also known as 2 * -O-dimethylaminoethoxyethyl or 2 * -DMAEOE), i.e., 2* -O-CH2-O-CH2-N(CH2)2. Other exemplary modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also 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 2'-5'-linked siRNA, and at the 5'-position of the 5'-terminal nucleotide. Modifications can also include sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars.

[0082] Representative U.S. patents teaching 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 teachings related to methods of preparing such modifications.

[0083] Examples of modified RNA backbones include phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonate (including 3'-alkylene phosphonate and chiral phosphonate), phosphinate, phosphoramidate (including 3'-aminophosphoramidate and aminoalkyl phosphoramidate), thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and normal 3'-5' linkages, 2'-5' linkage analogs, and boranophosphates having reverse 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.

[0084] Representative U.S. patents that teach the preparation of the above 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; 7321029; and U.S. Patent Reissue No. 39464, each of which is hereby incorporated by reference herein for the teachings related to methods of preparing such modifications.

[0085] 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 that does not contain a phosphorus atom therein has a backbone 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; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl 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 parts.

[0086] Representative U.S. patents teaching the preparation of the above 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 the teachings related to the methods of preparing such modifications.

[0087] 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 units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the azanitrogen atoms of the amide portion 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).

[0088] Some embodiments taken up in the techniques described herein include RNAs having a phosphorothioate backbone and oligonucleosides having a heteroatom backbone, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as the 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 [where 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.

[0089] The modifications of the siRNAs disclosed herein may 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 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 (daazaadenine), and other synthetic and natural nucleobases such as 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 (Herdewijn P, ed., Wiley-VCH, 2008), those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering (pages 858-859, Kroschwitz JL, ed., John Wiley & Sons, 1990), those disclosed by Englisch et al. (Angewandte Chemie, International Edition, 30, 613, 1991), and those disclosed by Sanghvi YS (Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke ST and Lebleu B, ed., CRC Press, 1993). Certain of these nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds taken up in the techniques described herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2 °C (Sanghvi YS et al., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, pp. 276-278, 1993), and is a particularly exemplary base substitution when combined with 2'-O-methoxyethyl sugar modification.

[0090] Representative U.S. patents that teach the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is incorporated herein by reference for teachings related to methods of preparing such modifications.

[0091] siRNA can also be modified to include one or more glycol nucleic acids, such as adenosine-glycol nucleic acid (GNA). Descriptions 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 teachings related to methods of preparing GNA modifications.

[0092] 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, wherein the ribose moiety includes an extra bridge connecting the 2'-carbon and the 4'-carbon. 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 et al., Nucleic Acids Research 2005, 33(1):439-47, Mook OR et al., Mol Cancer Ther 2007, 6(3):833-43, Grunweller A et al., Nucleic Acids Research 2003, 31(12):3185-93).

[0093] 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.

[0094] 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 beryllium-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), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J 1991, 10:1111-18, Kabanov et al., FEBS Lett. 1990, 259:327-30, Svinarchuk, e.g., 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., Tetrahedron Lett. 1995, 36:3651-54, Shea et al., Nucl. Acids Res. 1990, 18:3777-83), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides 1995, 14:969-73), or adamantane acetic 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 hexylamino-carbonyloxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277:923-37).

[0095] 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 cell or organ compartment, tissue, organ, or body region, compared to species in which such a ligand is absent. In such embodiments, the ligand will not be involved in duplex pairing in the double-stranded nucleic acid.

[0096] 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 synthetic polymer, such as a recombinant or synthetic molecule 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 alpha-helical peptides.

[0097] The ligand may also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody that binds to a specific cell type such as a liver cell. 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, polyglutamate, polyaspartate, 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., p-soralene, 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, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption enhancer (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.

[0098] 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.

[0099] 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, for example, 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.

[0100] In some embodiments, the ligand is a moiety taken up by a target cell, such as a hepatocyte, for example, a vitamin. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamins B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells such as hepatocytes. HSA and low density lipoprotein (LDL) are also included.

[0101] In some embodiments, the ligand conjugated to the siRNA described herein acts as a pharmacokinetic (PK) modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. Examples of PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. 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, and the like. 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 modulating ligands). In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0102] (i) Lipid conjugates. In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. The lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to 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). The HSA-binding ligand enables 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, nephroxin 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 more strongly to HSA are less likely to target the kidney and thus less likely to be excreted from the body. To target the conjugate to the kidney, lipids or lipid-based ligands that do not bind too strongly to HSA can be used.

[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-kidney 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 antennopedia. When the agent is a peptide, it can be modified to include peptidomimetics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. In some embodiments, the helical agent is an alpha-helical agent. In certain embodiments, the helical agent has a lipophilic phase and a lipophobic phase.

[0107] "Cell-permeable peptides" are capable of permeating cells, such as microbial cells like bacterial or fungal cells, or mammalian cells like human cells. Microbial cell-permeable peptides can be, for example, alpha-helix linear peptides (such as LL-37 or seropin PI), disulfide bond-containing peptides (such as alpha-defensin, beta-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 capable of folding into a defined three-dimensional structure similar to that of a natural peptide. The binding of peptides and peptidomimetics to siRNA can affect the pharmacokinetic distribution of RNAi, such as 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 from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWK (SEQ ID NO: 12)) have been found to be capable of functioning as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, 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 HIV-1 gp41 and the NLS of the 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 use in in vivo therapies. As used herein, "carbohydrate" refers to a compound that is itself composed of one or more monosaccharide units having at least six carbon atoms (which can be linear, branched, or cyclic) with oxygen, nitrogen, or sulfur atoms attached to each carbon atom, or a compound having as part of it a carbohydrate moiety composed of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched, or cyclic) with oxygen, nitrogen, or sulfur atoms attached 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 having C5 or more (in some embodiments, C5-C8), and disaccharides and trisaccharides include sugars having two or three monosaccharide units (in some embodiments, C5-C8).

[0112] In some embodiments, the carbohydrate conjugate is selected from the group consisting of:

[0113]

Chemical formula

[0114]

Chemical formula

[0115]

Chemical formula

[0116]

Chemical formula

[0117]

Chem.

[0118]

Chem.

[0119] Another exemplary carbohydrate conjugate for use in the embodiments described herein includes

[0120]

Chem.

[0121] (Formula XXII), wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0122] In some embodiments, the carbohydrate conjugate further comprises another ligand such as, but not limited to, a PK regulator, an endosome-degradable ligand, or a cell-penetrating peptide.

[0123] (iv) Linker. In some embodiments, the conjugates described herein can be attached to the siRNA oligonucleotide by various linkers that can be cleavable or non-cleavable.

[0124] 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, a 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, containing a chain of atoms, where 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 heterocyclic, where 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.

[0125] The cleavable linking group is sufficiently stable extracellularly but is cleaved upon entry into the target cell to release the two moieties that the linker holds together. In certain embodiments, the cleavable linking 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.

[0126] The cleavable linking group is sensitive to a cleaving agent, e.g., pH, redox potential, or the presence of a degradable molecule. Generally, the cleaving agent is more prevalent or found at a higher level or activity intracellularly than in serum or blood. Examples of such degrading agents include, for example, redox agents selected for or having substrate specificity for a particular substrate, such as an oxidase or reductase present in the cell or a reducing agent containing a mercaptan, that can decompose a redox-cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that produce a pH of 5 or less; enzymes, peptidases (which can be substrate-specific), and phosphatases that can hydrolyze or decompose an acid-cleavable linking group by acting as a general acid. Cleavable linking groups such as disulfide bonds can be sensitive to pH. 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 a cleavable linking group that is cleaved at a particular pH, thereby releasing a cationic lipid from an intracellular ligand or into a desired compartment of the cell.

[0127] The linker may include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver-targeting ligand may be linked to a cationic lipid via a linker containing an ester group. Liver cells are rich in esterases, and thus the linker will be cleaved more efficiently in liver cells 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.

[0128] Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synovial cells.

[0129] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with 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 be an indicator of cleavage in the target cell and the second condition is selected to be an indicator of 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).

[0130] 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 candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use, for example, with a particular RNAi moiety and a particular targeting agent, reference can be made to the methods described herein. For example, a candidate can be evaluated by incubation with other reducing agents using dithiothreitol (DTT), or a reagent known in the art that mimics the cleavage rate observed in cells, such as 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 by 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 intracellular media, compared to conditions selected to mimic extracellular media.

[0131] Phosphate-based cleavable linkers are cleaved by agents that decompose or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups intracellularly are enzymes such as intracellular phosphatases. 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-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. 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-, -O-P(0)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-Ρ(O)(Η)-O-, -O-P(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 a particular embodiment, the phosphate linker is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to the above methods.

[0132] 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. Acid-cleavable groups can have the general 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.

[0133] Ester-based cleavable linking groups are cleaved by enzymes such as intracellular esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0134] Peptide-based cleavable linkers are cleaved by enzymes such as peptidases and proteases within cells. Peptide-based cleavable linkers are peptide bonds formed between amino acids that result in 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 results in peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that result in 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 methods similar to the above methods.

[0135] Representative carbohydrate conjugates with linkers include, but are not limited to, the following

[0136]

Chemical formula

[0137]

Chemical formula

[0138] In certain embodiments of the compositions and methods described herein, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives conjugated via a divalent or trivalent branched linker. For example, in some embodiments, siRNA is conjugated to a GalNAc ligand as shown in the following schematic diagram,

[0139] [Chemical formula] In the formula, X is O or S. In some of these embodiments, X is O.

[0140] In some embodiments, the combination therapy comprises siRNA conjugated to a divalent or trivalent branched linker selected from the group of structures shown in any of formulas (XXXI)-(XXXIV).

[0141] [Chemical formula]

[0142] [Chemical formula] In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent 0 to 20 for each occurrence, and the repeating units can be the same or different. 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 do not exist or are CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O for each occurrence. Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B and Q 5Cis, independently for each occurrence, absent, or is alkylene, or substituted alkylene, and one or more methylenes are interrupted or terminated by one or more of O, S, S(O), SO2, N(R N ), C(R’)=C(R’’),

Chem.

[0143]

Chem.

[0144]

Chem.

[0145] Examples of suitable divalent and trivalent branched linker groups for conjugation to a GalNAc derivative include, but are not limited to, the structures listed above as formulas I, VI, X, IX, and XII.

[0146] Representative U.S. patents teaching the preparation of RNA conjugates include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 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 hereby incorporated by reference herein for teachings related to such preparation methods.

[0147] In certain instances, the RNA of the siRNA can be modified by non-ligand groups. Some non-ligand molecules have been conjugated to siRNAs in order to enhance the activity, cellular distribution, or cellular uptake of the siRNA, and procedures for performing such conjugation are available in the scientific literature.Such non-ligand moieties include lipid moieties such as cholesterol (Kubo T. et al., Biochem. Biophys. Res. Comm. 2007, 365(1):54-61, Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. 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), fatty chains such as 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 such as di-hexadecyl-rac-glycerol or triethylammonium l,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 adamantane acetic 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., J. Pharmacol. Exp. Ther. 1996, 277:923).

[0148] Typical conjugation protocols involve the synthesis of RNA carrying an amino linker at one or more positions of the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out either while the RNA is still attached to a solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0149] b. Pharmaceutical Compositions and Delivery of siRNA In some embodiments, pharmaceutical compositions are provided that contain the siRNA described herein and a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions containing siRNA 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.

[0150] "Pharmaceutically acceptable carrier" or "excipient" is any other pharmacologically inert vehicle for delivering a drug, such as a pharmaceutically acceptable solvent, suspending agent, or one or more nucleic acids, to an animal. Excipients can be liquid or solid and are selected considering the planned mode of administration so as to provide the desired bulk, viscosity, etc. when combined with the drug (e.g., nucleic acid) and other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers or excipients include, but are not limited to, binders (e.g., pregelatinized corn starch, 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).

[0151] Organic or inorganic pharmaceutically acceptable excipients that do not react detrimentally with nucleic acids can also be used to formulate siRNA compositions. Pharmaceutically acceptable carriers suitable for use in formulations for non - oral delivery include, but are not limited to, water, saline solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0152] 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. Organic or inorganic pharmaceutically acceptable excipients that do not react detrimentally with nucleic acids can be used.

[0153] In some embodiments, administration of the pharmaceutical compositions and formulations described herein can be local (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 can be intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subcutaneous administration (e.g., via an implanted device); or intracranial administration (e.g., parenchymal, intrathecal, or intraventricular administration).

[0154] 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.

[0155] In some embodiments, the pharmaceutical composition containing the siRNA described herein is administered in a dosage sufficient to inhibit the expression of the HBV gene. In some embodiments, the dosage of the 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 daily, or can be administered as two, three, or more partial doses at appropriate intervals throughout the day, or can also be administered using delivery through continuous infusion or controlled release formulations. In that case, the siRNA contained in each partial dose needs to be correspondingly smaller in order to achieve the total daily dose. The dosage unit can also be formulated using conventional sustained release formulations that provide for delivery over several days, for example, providing for sustained release of the siRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for the delivery of drugs at specific sites that 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.

[0156] In some embodiments, the pharmaceutical composition containing the siRNA targeting the HBV mRNA described herein (e.g., SIRNA01) contains the siRNA at a dosage of 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg, or 15 mg / kg.

[0157] In some embodiments, the 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, the pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 20 mg to 900 mg. In some embodiments, the pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 100 mg to 300 mg.

[0158] In some embodiments, the 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.

[0159] In some embodiments, the pharmaceutical composition comprising the siRNA described herein (e.g., SIRNA01) contains siRNA at a dose of 200 mg.

[0160] III. Anti-HBV Antibodies The present disclosure also provides anti-HBV antibodies for use in combination therapies for treating HBV or HBV-related diseases.

[0161] a. Antibodies that Bind to HBV Proteins In some embodiments, the anti-HBV antibody or antigen-binding fragment thereof in combination therapy binds to the antigen loop region of HBsAg. The envelope of hepatitis B virus contains three "HBV envelope proteins" (also known as "hepatitis B surface antigen"): the S protein (representing "small", also referred to as S-HBsAg), the M protein (representing "medium", also referred to as M-HBsAg), and the L protein (representing "large", also referred to as L-HBsAg). S-HBsAg, M-HBsAg, and L-HBsAg share the same C-terminal tip (also referred to as 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 through the Golgi apparatus. The S domain contains four predicted transmembrane (TM) domains, whereby both the N-terminal and C-terminal ends of the S domain are exposed to the lumen. Both transmembrane domains TM1 and TM2 are required for co-translational protein incorporation 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 the 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).

[0162] 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.

[0163] [Table 2] (SEQ ID NO: 13; amino acids 101-172 are underlined).

[0164] For example, the expression "amino acids 101-172 of the S domain" refers to the amino acid residues from the polypeptide at positions 101-172 set forth in SEQ ID NO: 13. However, one of ordinary skill in the art will understand that mutations or variations (e.g., including, but not limited to, substitutions, deletions, and / or additions in different genotypes of HBsAg or different HBsAg mutants 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 mutants (natural or artificial mutants).

[0165] As used herein, the expressions "corresponding array fragment" or "corresponding fragment" refer to fragments located at equal positions of an array when the arrays are subjected to an optimized alignment, i.e., when the arrays are 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 either present on the inner surface of the viral particle (cytoplasmic side of the ER) and play an important role in viral assembly, or are present on the outer surface (lumen side of the ER) and are available for interaction with target cells, and may be either necessary for viral infectivity. 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" (SVP) that are released from the cell by secretion.

[0166] All three HBV envelope proteins, S-HBsAg, M-HBsAg, and L-HBsAg, contain an S domain and therefore also contain an "antigen loop region". Thus, 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.

[0167] Furthermore, in some embodiments, the anti-HBV antibody or antigen-binding fragment thereof in 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 in the combination therapy neutralizes infection by hepatitis D virus. In other words, the antibody or antigen-binding fragment thereof can reduce the viral infectivity of hepatitis D virus (see below for further description of hepatitis D virus as an obligate satellite of hepatitis B virus).

[0168] To study and quantify (or "neutralize") viral infectivity in the laboratory, those skilled in the art are aware of various standard "neutralization assays". In a neutralization assay, an animal virus is typically grown in cells and / or cell lines. In the context of the present disclosure, in a neutralization assay, cultured cells can be incubated with a certain 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 an HBV neutralization assay, cultured cells, such as HepaRG cells, particularly differentiated HepaRG cells, are incubated with a certain amount of HBV, for example, at 37°C for 16 hours, in the presence or absence of the antibody being tested. The incubation can be carried out in a medium supplemented with (for example, 4% PEG8000). After incubation, the cells can be washed and further cultured. To measure viral infectivity, after infection, for example, the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant from day 7 to day 11 can be determined by enzyme-linked immunosorbent assay (ELISA). Additionally, HBcAg staining can be evaluated in an immunofluorescence assay.

[0169] 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 a very low concentration of antibody is required for 50% neutralization of HBV. Specificity and potency can be measured using standard assays known to those of skill in the art.

[0170] 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.

[0171] In some embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure promote the clearance of HBsAg and HBV. In particular, the antibodies or antigen-binding fragments thereof according to the present disclosure can promote the clearance of both HBV and subviral particles (SVP) of hepatitis B virus. The clearance of HBsAg or subviral particles can be evaluated, for example, by measuring the level of HBsAg in a blood sample, for example, 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, for example, from a hepatitis B patient.

[0172] 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 subviral particles (SVP) composed solely of HBV envelope protein (HBsAg) in the form of relatively small spheres and filaments of variable length. Subviral particles have been shown to strongly enhance intracellular viral replication and gene expression of HBV (Bruns M et al., J Virol 1998, 72(2):1462 - 8). Also, since infectivity depends on the number of not only the virus but also SVP, this is important in the context of the infectivity of sera containing HBV (Bruns et al., 1998, see above). Furthermore, excess subviral particles can function as decoys by absorbing neutralizing antibodies and thus can delay the clearance of infection. Therefore, typically, the achievement of hepatitis B surface antigen (HBsAg) loss is considered the ideal endpoint of treatment and the closest outcome for curing chronic hepatitis B (CHB). Thus, in some embodiments, the antibodies or antigen - binding fragments thereof according to the present disclosure that promote clearance of HBsAg, particularly of subviral particles of hepatitis B virus and HBV, enable 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 the SVP that function as decoys. In addition, in certain embodiments, the antibodies or antigen - binding fragments thereof according to the present disclosure promote the clearance of subviral particles of hepatitis B virus and reduce the infectivity of HBV in serum.

[0173] HBV is classified into multiple genotypes according to its genomic sequence. To date, eight well-known genotypes (A - H) of the HBV genome have been defined. Additionally, 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 disease progression, and differences between genotypes in response to antiviral therapy have been determined. For example, genotype A has a tendency to become chronic, while 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 AB117758 (HBV-C1 Cambodia), GenBank accession number AB205192 (HBV-E Ghana), GenBank accession number X69798 (HBV-F4 Brazil), GenBank accession number AF160501 (HBV-G USA), GenBank accession number AY090454 (HBV-H Nicaragua), GenBank accession number AF241409 (HBV-I Vietnam), and GenBank accession number AB486012 (HBV-J Borneo). The amino acid sequences of the antigen loop regions of the S domain of HBsAg of different genotypes are shown in Table 2 (SEQ ID NOs: 14 - 42).

[0174]

Table 3-1

[0175]

Table 3-2

[0176] 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 residues are indicated in the name).

[0177]

Table 4

[0178] 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.

[0179] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to an epitope comprising at least 1, at least 2, at least 3, or at least 4 amino acids in the antigen loop region of HBsAg, and at least 2, at least 3, or at least 4 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 the above-mentioned HBsAg that is present in all three HBV envelope proteins S-HBsAg, M-HBsAg, and L-HBsAg.

[0180] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to an epitope in the antigen loop region of HBsAg, whereby the epitope is formed by one or more amino acids located at positions selected from amino acids 115-133, amino acids 120-133, or amino acids 120-130 of the S domain of HBsAg.

[0181] As used herein in the context of an epitope, "formed by" means that the epitope to which the 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, if the epitope is a linear epitope and contains two or more amino acids located at positions selected from amino acids 115-133 or amino acids 120-133 of the S domain of HBsAg, the amino acids included by 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 by" 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 the antibody or antigen-binding fragment thereof of the present disclosure binds is formed only by amino acids selected from amino acids 115-133, 120-133, or 120-130 of the S domain of HBsAg. In certain embodiments, (additional) amino acids located outside positions 115-133, 120-133, or 120-130 are not required to form the epitope to which the antibody or antigen-binding fragment thereof of the present disclosure binds.

[0182] 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 acids 115 to 133, amino acids 120 to 133, or amino acids 120 to 130 of the S domain of HBsAg. In certain embodiments, the epitope within 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 acids 115 to 133, amino acids 120 to 133, and amino acids 120 to 130 of the S domain of HBsAg. In some embodiments, the epitope 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 acids 115 to 133, amino acids 120 to 133, or amino acids 120 to 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 of the antigen loop region of HBsAg selected from amino acids 115 to 133 of the S domain of HBsAg, amino acids 120 to 133 of the S domain of HBsAg, or amino acids 120 to 130 of the S domain of HBsAg. 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 of 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 to 133 or amino acids 120 to 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).

[0183] 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, wherein 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 at least two, at least three, or at least four amino acids are not located at adjacent positions (in the primary structure).

[0184] 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.

[0185] 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.

[0186] 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, with the first residue of the heavy chain constant region being 114) and ends at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc portion can be a complete Fc portion or a portion (e.g., domain) thereof. In certain embodiments, the complete Fc portion includes the hinge domain, CH2 domain, and CH3 domain (e.g., EU amino acids 216-446). Additional lysine residues (K) are sometimes present at the C-terminal end 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). Amino acid positions of the Fc portion can also be numbered according to the IMGT numbering system (including unique numbering for C domains and exon numbering) and the Kabat numbering system.

[0187] In some embodiments, the Fc portion comprises at least one of a hinge (e.g., upper, central, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or variants, portions, or fragments thereof. In some embodiments, the Fc portion comprises at least a hinge domain, a CH2 domain, or a CH3 domain. In further embodiments, the Fc portion is a complete Fc portion. 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 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.

[0188] 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.

[0189] 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 the immunoglobulin molecules are bound to an antigenic 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 fixation (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 the 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.

[0190] 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 antibody-coated pathogens by phagocytosis of immune complexes and the lysis of various other cell targets (e.g., tumor cells) coated with the corresponding antibody via erythrocytes and antibody-dependent cell cytotoxicity (ADCC; Van de Winkel JG and Anderson CL, J. Leukoc. Biol. 1991, 49:511-24). FcRs are defined by their specificity for immunoglobulin classes, and Fc receptors for IgG antibodies are designated as FcγRs, for IgE as FcεRs, for IgA as FcαRs, etc., and the neonatal Fc receptor is designated as 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.

[0191] Cross-linking of receptors by the Fc domain (FcγR) of natural IgG antibodies induces a wide variety of effector functions, including phagocytosis, antibody-dependent cell cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. Fc moieties (e.g., FcγR) that provide receptor cross-linking are contemplated herein. In humans, there are three classes of FcγRs: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) FcγRII, which binds complexed 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 classified into FcγRIIA, FcγRIIB, and FcγRIIC, which perform different functions in the immune system, bind with low affinity similar to IgG-Fc, 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: on NK cells, macrophages, eosinophils, and some monocytes and T cells and is thought to mediate ADCC; and FcγRIIIB, which is highly expressed on neutrophils.

[0192] 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 on 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 on Kupffer cells of the liver, and LSECs are the major site of small immune complex clearance (Ganesan et al., 2012, supra).

[0193] In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein include an Fc portion for binding to FcγRIIb, particularly an Fc region such as an IgG-type antibody. 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, the clearance of immune complexes can be enhanced (Chu S et al., Am J Respir Crit, American Thoracic Society International Conference Abstracts 2014). In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure includes an engineered Fc portion having the mutations S267E and L328F, particularly as described by Chu SY et al. (2008, supra).

[0194] 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 through FcγRIIA. On eosinophils and mast cells, the b form may help to suppress the activation of these cells through the binding of IgE to its respective receptor.

[0195] 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 reduce 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).

[0196] Regarding FcγRII binding, for example, a reduction in binding to FcγRIIA has been found for at least one IgG mutation among E233 - G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

[0197] Regarding FcγRIII binding, for example, a reduction in binding to FcγRIIIA has been 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.

[0198] Methods for mapping the binding sites on human IgG1 to Fc receptors, the above - mentioned mutation sites, and measuring the binding to FcγRI and FcγRIIA are described in Shields RL et al. 2001, 276:6591 - 6604).

[0199] 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 upper CH2 domain adjacent to the region of P331, such as the loop and strand in the upper CH2 domain adjacent to the lower hinge region, 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, see above).

[0200] (For example, mutations that increase the binding affinity of the Fc portion of the present disclosure for one or more Fcγ receptors compared to a reference Fc portion or antibody that does not contain a mutation are also contemplated. 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 may comprise an Fc portion comprising a mutation selected from G236A, S239D, A330L, and I332E, or combinations thereof, such as S239D / I332E, S239D / A330L / I332E, G236A / S239D / I332E, G236A / A330L / I332E, and G236A / S239D / A330L / I332E.)

[0201] In certain embodiments, the Fc portion can comprise or consist of at least a portion 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 the molecule comprising the Fc portion (e.g., as compared to a reference Fc portion or antibody that does not comprise the modification). In certain embodiments, the Fc portion comprises or is derived from IgG Fc, and the half-life extending mutations comprise any 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 P257I / Q311I. In certain embodiments, the half-life extending mutation comprises P257I / N434H. In certain embodiments, the half-life extending mutation comprises D376V / N434H. In certain embodiments, the half-life extending mutation comprises T307A / E380A / N434A.

[0202] 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.

[0203] 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.

[0204] Alternatively or additionally, the Fc portion of the disclosed binding proteins can include at least a portion known in the art to be required for protein A binding, and / or the Fc portion of the disclosed antibodies 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 includes 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 upper CH2 domain adjacent to the lower hinge region, e.g., the upper CH2 domain of native IgG Fc around P331, for example, a region of at least 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids, such as the loop and strand in amino acids 320-340 (EU numbering) of native IgG Fc.

[0205] 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 can 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 in a single continuous nucleic acid sequence). Exemplary scFc regions are disclosed in International Publication No. WO 2008 / 143954 (A2), which is incorporated herein by reference. The Fc region can be or include a dimeric Fc region. "Dimeric Fc region" or "dcFc" refers to a dimer formed by 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., at least one amino acid modification (e.g., substitution, deletion, insertion, or chemical modification) that is not present in the other Fc monomer is included in one Fc monomer of the dimeric Fc region, or one Fc monomer can be shorter compared to the other).

[0206] 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 portion of the Fc region is of the same class and subclass. However, the Fc region (or one or more Fc portions of the Fc region) can also be chimeric, whereby the chimeric Fc region can include Fc portions derived from different immunoglobulin classes and / or subclasses. 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 from two or more different isotypes or subclasses; e.g., a SEEDbody (a "strand-exchanged domain") (see Davis et al., Protein Eng. Des. Sel. 2010, 23(4):195).

[0207] 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., an 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., an IgG1, IgG2, or IgG4 subclass) and a hinge region from an immunoglobulin of a second subclass (e.g., an 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., an IgG4 subclass) and a CH3 domain from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, the chimeric Fc region may comprise an Fc moiety (e.g., a complete Fc moiety) from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and an Fc moiety from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, the Fc region or moiety may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or moiety may comprise a CH1 domain and a CH2 domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. For example, the Fc region or moiety may comprise a portion of the CH2 domain from a particular subclass of antibody, e.g., EU positions 292-340 of the CH2 domain. For example, the Fc region or moiety may comprise amino acids 292-340 of CH2 derived from an IgG4 portion and the remainder of CH2 derived from an IgG1 portion (alternatively, 292-340 of CH2 may be derived from an IgG1 portion and the remainder of CH2 may be derived from an IgG4 portion).

[0208] 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, for example, 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 derived in part from an IgG1 molecule and in part from an IgG4 molecule. In another example, a chimeric hinge may include the upper and lower hinge domains from an IgG4 molecule and the central hinge domain 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 may include amino acids from an IgG2 antibody at EU positions 233-236 and / or the Ser228Pro mutation, with the remaining amino acids of the hinge being 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 an antibody according to the present disclosure are described in U.S. Patent Application Publication No. 2005 / 0163783 (A1).

[0209] 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., 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 subject polypeptide. Alternatively, one or more mouse amino acids may be present in the Fc portion or Fc region.

[0210] c.HBC34 antibody In certain embodiments, the anti-HBV antibody is HBC34 or an engineered variant thereof. HBC34 is a human antibody against HBsAg with high neutralizing activity. HBC34 binds to the antigen loop of HBsAg with high affinity (in the pM range), recognizes all 10 HBV genotypes and 18 mutants, and binds to spherical SVP at low stoichiometry. The activity of HBC34 measured diagnostically by immunoassay is 5000 IU / mg. By comparison, the activity of HBIG is approximately 1 IU / mg.

[0211] As used herein, the terms "HBC34 antibody" and "HBC antibody" may include, unless otherwise stated, the wild-type HBC34 antibody or an engineered variant thereof (e.g., HBC34 and HBC34 variants described in Table 3).

[0212] 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 engineered variants. Also shown are the full-length heavy chain (HC) and light chain (LC) amino acid sequences of exemplary antibodies of the present disclosure.

[0213]

Table 5-1

[0214]

Table 5-2

[0215]

Table 5-3

[0216] In certain embodiments, the anti-HBV antibody comprises one or more of the amino acid sequences described in Table 3. In certain embodiments, an antibody or an antigen-binding fragment thereof according to the present disclosure has the CDR sequences shown in Table 3, V H sequence, V LIt includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an array, HC array, and / or LC array. In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment is the CDR, V H , V L , HC, and / or LC sequences may be included. 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. 2020 / 132091 (A2), and this method and data are incorporated herein by reference.

[0217] 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.

[0218] 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.

[0219] It will be understood that the antibody or antigen-binding fragment of the present disclosure may 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.

[0220] 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.

[0221] In certain embodiments, the antibody or antigen-binding fragment of the present disclosure comprises or consists of (a) a light chain variable domain (V L ) 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.

[0222] In certain embodiments, the antibody or antigen-binding fragment of the present disclosure comprises (a) a light chain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59, and (b) a heavy chain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

[0223] 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.

[0224] A pharmaceutical composition of anti-HBV antibody may contain an antibacterial agent, especially when packaged in a multiple-dose form. They may contain a detergent, such as Tween (polysorbate) like 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 (such as sodium chloride) for tonicity. For example, in some embodiments, the pharmaceutical composition contains NaCl at a concentration of 10±2 mg / ml.

[0225] Furthermore, the pharmaceutical composition may contain a sugar alcohol (such as mannitol) or a disaccharide (such as sucrose or trehalose) at, for example, about 15 - 30 mg / ml (such as 25 mg / ml), especially when lyophilized or when containing 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.

[0226] 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.

[0227] A method for preparing a pharmaceutical composition of anti-HBV antibody may include the steps of (i) preparing the antibody, and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

[0228] In some embodiments, a pharmaceutical composition containing the anti-HBV antibody described herein (such as AB01) contains the antibody at a dose of 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg. In some embodiments, a pharmaceutical composition containing the anti-HBV antibody described herein (such as AB01) contains the antibody at a dose of 100 mg - 300 mg. In some embodiments, a pharmaceutical composition containing the anti-HBV antibody described herein (such as AB01) contains the antibody at a dose of 100 mg - 200 mg.

[0229] 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.

[0230] IV. Methods of treatment using combination therapy In some embodiments, the present disclosure provides a method for treating HBV infection or HBV-related diseases in a subject.

[0231] In some embodiments, a method for treating HBV infection or HBV-related diseases in a subject is provided, 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.

[0232] In some embodiments, a method for treating HBV infection or HBV-related diseases in a subject is provided, 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 (e.g., 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), PC1323, Theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380 / LB-80380), and tenofvir-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.In some embodiments of the method, the subject is HBeAg-negative, has an HBV DNA level of 2000 IU / mL or less before treatment, has an HBV DNA level of less than 2000 IU / mL before treatment, and / or has an alanine aminotransferase (ALT) level at or below the upper limit of normal (ULN), and / or has an alanine aminotransferase (ALT) level of less than the upper limit of normal (ULN). In some embodiments, the ALT ULN value is 34 IU / mL for females and 43 IU / mL for males. In some embodiments of the method, the subject has not been previously administered one or more elements of the combination therapy (e.g., the subject has not been previously administered an NRTI, the subject has not been previously administered an anti-HIV antibody, and / or the subject has not been previously administered an siRNA targeting HBV mRNA). In some embodiments of the method, the subject has not been previously administered an NRTI within 24 weeks prior to treatment with the combination therapy or has never received it before.

[0233] In some embodiments, a method for treating HBV infection or HBV-related disease in a subject is provided, the method comprising administering to the subject the siRNA and antibody described herein, and also administering to the subject a nucleoside / nucleotide reverse transcriptase inhibitor and interferon α (e.g., PEG-IFNα). In some embodiments of the method, the subject is HBeAg-negative or HBeAg-positive, has an HBV DNA level of more than 2000 IU / mL before treatment, and / or has an alanine aminotransferase (ALT) level above the upper limit of normal (ULN) and up to 5 times the ULN. In some embodiments of the method, the subject has not been previously administered one or more elements of the combination therapy (e.g., the subject has not been previously administered an NRTI, the subject has not been previously administered interferon α, the subject has not been previously administered an anti-HIV antibody, and / or the subject has not been previously administered an siRNA targeting HBV mRNA). In some embodiments, the ALT ULN value is 34 IU / mL for females and 43 IU / mL for males.

[0234] As used herein, "subject" refers to any mammal that can be infected with HBV, such as mammals including primates (humans, non-human primates such as monkeys or chimpanzees, etc.), or animals considered to be an acceptable clinical model of HBV infection, HBV-AAV mouse models (see, e.g., Yang et al., Cell and Mol Immunol 2014, 11:71), or animals such as HBV 1.3 xfs transgenic mouse models (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 such as a human having an HBV infection, particularly a chronic hepatitis B virus (CHBV) infection.

[0235] As used herein, the term "treating" or "treatment" includes, but is not limited to, 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, elevated ALT, elevated AST (normal range is typically considered to be about 10 - 34 U / L), low levels of anti-HBV antibodies or their absence; liver damage; cirrhosis; delta hepatitis; acute hepatitis B; fulminant acute hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, low-grade fever; myalgia; easy fatigability; disturbances in taste acuity and sense of smell (aversion to food and tobacco); or intermittent, mild to moderate pain in the upper right abdomen and upper stomach; hepatic encephalopathy; drowsiness; disrupted sleep patterns; mental confusion; coma; ascites; gastrointestinal bleeding; coagulation disorders; jaundice; hepatomegaly (a slightly enlarged, soft liver); splenomegaly; palmar erythema; spider nevi; muscle wasting; spider angiomas; vasculitis; variceal bleeding; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); an ALT level higher than the AST level; elevated gamma-glutamyl transpeptidase (GGT) (normal range is typically considered to be about 8 - 65 U / L) and elevated alkaline phosphatase (ALP) levels (normal range is typically considered to be about 44 - 147 IU / L (international units per liter) and less than 3 times the ULN); slightly low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened erythrocyte survival period; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); the presence of serum or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), or HBV DNA; increased 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%); an increase in the rheumatoid factor (RF) level; low platelet and white blood cell counts; refers to beneficial or desirable results including remission or improvement of one or more signs or symptoms associated with lobules having degenerative and regenerative hepatocyte changes, with inflammation, mainly centrilobular necrosis, whether detectable or not. For example, the likelihood of developing liver fibrosis is reduced if, for example, an individual having one or more risk factors for liver fibrosis, such as chronic hepatitis B infection, does not develop liver fibrosis, or develops liver fibrosis at a lower severity compared to a population having the same risk factors and not having received the treatment described herein. "Treatment" can also mean extending survival compared to survival expected in the absence of treatment.;

[0236] As used herein, the term "preventing" or "prevention" refers 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 exhibiting a delay (e.g., of days, weeks, months, or years) in the development of signs or symptoms. Prevention may require administration of more than one dose.

[0237] Doses are often expressed in relation to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) refers to [g, mg, or other unit] "per kg of body weight (or g, mg, etc.)" even when the term "body weight" is not explicitly mentioned.

[0238] 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 clearance of circulating HBsAg and may involve conversion to a state where HBsAg antibodies are detectable using clinically relevant assays. For example, detectable antibodies may contain a signal higher than 10 mIU / ml as measured by a chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay. A functional cure does not require clearance of all forms of HBV replication (e.g., cccDNA from the liver). Anti-HBs seroconversion occurs spontaneously in about 0.2–1% of chronically infected patients per year. However, even after anti-HBs seroconversion, low-level persistence of HBV is often observed over decades, indicating that a functional cure rather than a complete cure has occurred. Without being bound to a particular mechanism, the immune system may be able to continue to block HBV under conditions where a functional cure has been achieved. A functional cure allows for discontinuation 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 diseases or conditions resulting from HBV infection, such as liver fibrosis, HCC, or cirrhosis. In some particular embodiments, “functional cure” may refer to a sustained reduction, such as less than 1 IU / mL of serum HBsAg, for at least 3 months, at least 6 months, or at least 1 year after the start or completion of a treatment regimen.

[0239] As used herein, the terms “hepatitis B virus-related disease” or “HBV-related disease” are diseases or disorders caused by or associated with HBV infection or replication. The term “HBV-related disease” includes diseases, disorders, or conditions that would benefit from a reduction in HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0240] In some embodiments, the HBV-related disease is chronic hepatitis B. Chronic hepatitis B is defined by one of the following criteria: (1) serum HBsAg, HBV DNA, or HBeAg is positive at two time points separated by at least six months (any combination of these tests performed six months apart is allowed); or (2) immunoglobulin M (IgM) antibody to HBV core antigen (IgM anti-HBc) is negative and the result of one of the following tests: HBsAg, HBeAg, or HBV DNA is positive. Chronic HBV typically includes liver inflammation that persists for more than six months. Subjects with chronic HBV are HBsAg positive and have either high viremia (more than 10 4 HBV-DNA copies / ml of blood) or low viremia (less than 10 3 HBV-DNA copies / ml of blood). In certain embodiments, the subject has been infected with HBV for at least five years. In certain embodiments, the subject has been infected with HBV for at least ten years. In certain embodiments, the subject was infected with HBV at birth. Subjects with chronic hepatitis B disease may be immunotolerant or have an inactive chronic infection without any evidence of active disease and may also be asymptomatic. Patients with chronic active hepatitis, especially those in a replicative state, may have symptoms similar to those of acute hepatitis. Subjects with chronic hepatitis B disease may have an active chronic infection with necroinflammatory liver disease, with increased hepatocyte turnover in the absence of detectable necroinflammation, or may have an inactive chronic infection without any evidence of active disease and may also be asymptomatic. The persistence of HBV infection in chronic HBV subjects is a result of cccHBV DNA. In some embodiments, subjects with chronic HBV are HBeAg positive. In some other embodiments, subjects with chronic HBV are HBeAg negative. Subjects with chronic HBV have a serum HBV DNA level of less than 105 and have persistently elevated transaminases, such as ALT, AST, and gamma-glutamyltransferase. Subjects with chronic HBV may have a liver biopsy score (e.g., necroinflammatory score) of less than 4.

[0241] 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 functions provided by HBV for transmission, and indeed, HDV requires an associated HBV infection or an existing HBV infection for the viral envelope containing the hepatitis B surface antigen, in particular, to become infectious and proliferate. HDV can result in severe acute and chronic forms of liver disease associated with HBV. Hepatitis D infection or delta hepatitis is highly endemic in several African countries, the Amazon region, and the Middle East, but its prevalence is low in developed countries except in the Mediterranean.

[0242] Transmission of HDV 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 by HBV alone. These complications include a higher likelihood of experiencing liver failure in acute infection and a rapid progression to cirrhosis, and an increased chance of developing liver cancer in chronic infection. In combination with hepatitis B virus, hepatitis D has the highest mortality rate among all hepatitis infections, which is 20%.

[0243] In some embodiments, the HBV-related disease is acute hepatitis B. Acute hepatitis B includes inflammation of the liver that persists for less than six months. Typical symptoms of acute hepatitis B are malaise, anorexia, nausea, and vomiting. In many cases, very high aminotransferase levels (above 1000 U / L) and hyperbilirubinemia are observed. 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.

[0244] 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 mental confusion or coma (due to the liver's inability to detoxify chemicals) and bruising or bleeding (due to a lack of blood clotting factors).

[0245] HBV infection, e.g., in subjects with chronic HBV, can develop liver fibrosis. Thus, in some embodiments, the HBV-related disease is liver fibrosis. Liver fibrosis or cirrhosis is defined histologically as a diffuse hepatic process characterized by fibrosis (excessive fibrous connective tissue) and the conversion of normal liver architecture into structurally abnormal nodules.

[0246] HBV infection, e.g., in subjects with chronic HBV, can develop end-stage liver disease. Thus, in some embodiments, the HBV-related disease is end-stage liver disease. For example, liver fibrosis can progress to the point where the body can no longer compensate for, e.g., reduced liver function, resulting in, e.g., mental and neurological symptoms, and liver failure.

[0247] HBV infection, e.g., in subjects with chronic HBV, can 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.

[0248] "HDV-related disorder" or "delta hepatitis virus-related disorder" is a disease or disorder associated with the expression of HDV. Exemplary HDV-related disorders include hepatitis B virus infection, acute hepatitis B, acute delta hepatitis; acute fulminant delta hepatitis; chronic delta hepatitis; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0249] As used herein, "therapeutically effective amount" is intended to include an amount of siRNA, anti-HBV antibody, or other active agent (e.g., PEG-IFNα, tenofovir) that, when administered to a subject having HBV infection or an HBV-related disease, is sufficient to achieve treatment of the disease (e.g., by reducing or maintaining an existing disease, or one or more symptoms of the disease). The "therapeutically effective amount" can vary depending on the active agent, 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 prior or concurrent treatment, and other individual characteristics of the patient being treated. A therapeutically effective amount may require administration of more than one dose.

[0250] "Therapeutically effective amount" also includes an amount of siRNA, anti-HBV antibody, or other active agent that produces a somewhat desirable 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 produce a reasonable benefit / risk ratio applicable to such treatment.

[0251] 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 a 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 the fluid or cells within those organs. In certain embodiments, the sample can be derived from the liver (e.g., the 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 taken from a subject, or plasma or serum obtained from the blood. In further embodiments, "a sample derived from a subject" refers to liver tissue (or a partial component thereof) or blood tissue (or a partial component thereof, such as serum) derived from a subject.

[0252] 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 8 to 12 weeks, every 8 weeks, or every 12 weeks. In some embodiments, the subject is administered the anti-HBV antibody for a period of 44 weeks. In some embodiments, the subject is administered the anti-HBV antibody for a period of 48 weeks. In some embodiments, the subject is administered the anti-HBV antibody for a period of at least 44 weeks, 44 weeks, 44 weeks or more, at least 48 weeks, 48 weeks, or 48 weeks or more. In some embodiments, the subject is administered the anti-HBV antibody for a period of 20 weeks, 40 weeks, 44 weeks, or 48 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.

[0253] 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 subject is administered siRNA for a period of 20 weeks, 44 weeks, or 48 weeks. In some embodiments, the subject is administered siRNA for a period of 44 weeks. In some embodiments, the subject is administered siRNA for a period of 48 weeks. In some embodiments, the subject is administered siRNA for a period of at least 44 weeks, 44 weeks, 44 weeks or more, at least 48 weeks, 48 weeks, or 48 weeks or more. 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.

[0254] In some embodiments of the methods described herein, interferon-α is administered subcutaneously. In some embodiments, interferon-α is administered once a week. In some embodiments, the subject is administered interferon-α for a period of 44 weeks. In some embodiments, the subject is administered interferon-α for a period of 48 weeks. In some embodiments, the subject is administered interferon-α for a period of at least 44 weeks, 44 weeks, 44 weeks or more, at least 48 weeks, 48 weeks, or 48 weeks or more. In some embodiments, interferon-α is administered at a dose of at least 120 mcg. In some embodiments, interferon-α is administered at a dose of 120 mcg to 180 mcg. In some embodiments, interferon-α is administered at a dose of 180 mcg.

[0255] 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 subject is administered the NRTI for a period of 44 weeks. In some embodiments, the subject is administered the NRTI for a period of 48 weeks. In some embodiments, the subject is administered the NRTI for a period of at least 44 weeks, 44 weeks, 44 weeks or more, at least 48 weeks, 48 weeks, or 48 weeks or more. In some embodiments, the NRTI is administered at a dose of 300 mg. In some embodiments, the NRTI is administered at a dose of 245 mg.

[0256] In some embodiments of the methods described herein, the subject is administered siRNA and an anti-HBV antibody starting on the same day.

[0257] In some embodiments of the methods described herein, the subject is administered siRNA and an anti-HBV antibody for a period of 20 weeks, 44 weeks, or 48 weeks starting on the same day.

[0258] In some embodiments of the methods described herein, the subject is HBsAg negative after treatment, e.g., at week 24, week 48, or later. In some embodiments of the methods described herein, the subject achieves functional cure after treatment, i.e., has undetectable HBsAg (is HBsAg negative) and has sustained suppression of HBV DNA (HBV DNA is not detected). In some embodiments of the methods described herein, the subject has undetectable HBeAg and / or achieves anti-HBe seroconversion after treatment, e.g., at week 24, week 48, or later. In some embodiments of the methods described herein, the subject has normal alanine aminotransferase (ALT) levels after treatment, e.g., at week 24, week 48, or later.

[0259] The present disclosure also provides the antibodies, siRNAs, NRTIs, and / or interferons described herein for use in the methods described above, and pharmaceutical compositions comprising them. Also provided is the use of the antibodies, siRNAs, NRTIs, and / or interferons described herein in the manufacture of a medicament for use in the methods described above.

[0260] V. Kits for Combination Therapy Also provided herein are kits comprising components of a therapy for treating HBV infection or HBV-related diseases. 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 include siRNA (e.g., SIRNA01), an anti-HBV antibody (e.g., AB01), PEG-IFNα, and an NRTI (e.g., tenofovir disoproxil fumarate, tenofovir disoproxil). Additionally, the kit can include instructions for preparing and / or administering the components of the HBV combination therapy.

[0261] VI. Exemplary Embodiments In some embodiments, the present disclosure provides the following: 1. A method of doing so in a subject in need of treating hepatitis B virus (HBV) infection or an HBV-related disease, the method comprising administering to the subject (a) an anti-HBV antibody, and (b) an siRNA targeting HBV mRNA, and (c) a nucleoside (thio)phosphate reverse transcriptase inhibitor (NRTI). 2. The method of embodiment 1, wherein the subject is HBeAg-negative. 3. The method of embodiment 1 or 2, wherein the subject has an HBV DNA level of 2000 IU / mL or less prior to treatment. 4. The method of embodiment 1 or 2, wherein the subject has an HBV DNA level of less than 2000 IU / mL prior to treatment. 5. The method according to any one of embodiments 1 to 4, wherein the subject is non-cirrhotic and / or has alanine aminotransferase (ALT) levels below the upper limit of normal (ULN). 6. The method according to any one of embodiments 1 to 4, wherein the subject is non-cirrhotic and / or has alanine aminotransferase (ALT) levels below the upper limit of normal (ULN). 7. The method according to any one of embodiments 1 to 6, wherein the subject has not been previously administered an NRTI. 8. The method according to any one of embodiments 1 to 6, wherein the subject has not received an NRTI within 24 weeks prior to treatment. 9. The method according to any one of embodiments 1 to 8, wherein the subject has not been previously administered an anti-HIV antibody. 10. The method according to any one of embodiments 1 to 9, wherein the subject has not been previously administered an siRNA targeting HBV mRNA. 11. A method of doing so in a subject in need of treating hepatitis B virus (HBV) infection or an HBV-related disease, the method comprising administering to the subject (a) an anti-HBV antibody, and (b) an siRNA targeting HBV mRNA, and (c) interferon-α, and (d) a nucleoside(tide) reverse transcriptase inhibitor (NRTI). 12. The method according to embodiment 11, wherein the subject is HBeAg-negative. 13. The method according to embodiment 11, wherein the subject is HBeAg-positive. 14. The method according to any one of embodiments 11 to 13, wherein the subject has an HBV DNA level of more than 2000 IU / mL prior to treatment. 15. The method according to any one of embodiments 11 to 14, wherein the subject has alanine aminotransferase (ALT) levels above the upper limit of normal (ULN) and up to 5 times the ULN. 16. The method according to any one of embodiments 11 to 15, wherein the subject has not been previously administered an NRTI. 17. The method according to any one of embodiments 11 - 16, wherein the subject has not been previously administered an anti - HIV antibody. 18. The method according to any one of embodiments 11 - 17, wherein the subject has not been previously administered an siRNA targeting HBV mRNA. 19. The method according to any one of embodiments 11 - 18, wherein the subject has not been previously administered interferon - α. 20. The method according to any one of embodiments 1 - 19, wherein the subject is HBsAg - positive before treatment. 21. The method according to any one of embodiments 11 - 20, wherein the subject has an HBsAg level of greater than 10 IU / mL before treatment. 22. The method according to any one of embodiments 1 - 21, wherein the anti - HBV antibody recognizes HBV genotypes A, B, C, D, E, F, G, H, I, and J. 23. The method according to any one of embodiments 1 - 22, wherein the anti - HBV antibody is a human antibody. 24. The method according to any one of embodiments 1 - 23, wherein the antibody is HBC34 or a non - natural variant of HBC34. 25. The anti - HBV antibody is (i) respectively, the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 45, or 46, and 47, and (ii) respectively, the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 49, or 50, and 52, and the method according to any one of embodiments 1 - 24. 26. The anti - HBV antibody is (i) respectively, the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 45, and 47, and (ii) respectively, the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 49, and 52, and the method according to embodiment 25. 27. The anti - HBV antibody is (i) respectively, the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 44, 46, and 47, and (ii) The method according to embodiment 25, each comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 48, 50, and 52, respectively. 28. 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 embodiments 1 to 27. 29. The anti-HBV antibody is (a) the light chain variable domain (VL) amino acid sequence set forth in SEQ ID NO: 55, and (b) the heavy chain variable domain (V H ) amino acid sequence set forth in SEQ ID NO: 53, the method according to any one of embodiments 1 to 28. 30. 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 embodiments 1 to 29. 31. The anti-HBV antibody is (a) the light chain amino acid sequence set forth in SEQ ID NO: 59, and (b) the heavy chain amino acid sequence set forth in SEQ ID NO: 57, the method according to any one of embodiments 1 to 30. 32. The anti-HBV antibody is a monoclonal antibody, the method according to any one of embodiments 1 to 31. 33. The anti-HBV antibody is a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating an immune effector, the method according to any one of embodiments 1 to 32. 34. The method according to embodiment 33, wherein the second specificity stimulates a cytotoxic or vaccine effect. 35. The subject is a human, and a therapeutically effective amount of the anti-HBV antibody is administered, and the therapeutically effective amount is from about 3 mg / kg to about 30 mg / kg, the method according to any one of embodiments 1 to 34. 36. The method according to any one of embodiments 1 to 35, wherein the siRNA inhibits the expression of HBV transcripts encoding HBsAg protein, HBcAg protein, and HBx protein, or HBV DNA polymerase protein. 37. The method according to any one of embodiments 1 to 36, wherein the siRNA comprises a sense strand and an antisense strand forming 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 three or fewer nucleotides, and T is replaced by U. 38. The method according to any one of embodiments 1 to 37, 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. 39. The method according to any one of embodiments 1 to 38, 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. 40. The method according to any one of embodiments 1 to 39, 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). 41. The method according to any one of embodiments 1 to 40, wherein the antisense strand of the siRNA comprises at least 19 consecutive nucleotides of the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4). 42. The method according to any one of embodiments 1 to 41, wherein the antisense strand of the siRNA comprises the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4). 43. The method according to any one of Embodiments 1 to 42, wherein the antisense strand of the siRNA consists of nucleotides of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4). 44. The method according to any one of Embodiments 1 to 43, wherein the sense strand of the siRNA comprises a nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3). 45. The method according to any one of Embodiments 1 to 44, wherein the sense strand of the siRNA consists of a nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3). 46. The method according to any one of Embodiments 1 to 45, wherein at least one strand of the siRNA comprises a 3'-overhang of at least one nucleotide. 47. The method according to any one of Embodiments 1 to 46, wherein at least one strand of the siRNA comprises a 3'-overhang of at least two nucleotides. 48. The method according to any one of Embodiments 1 to 47, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs in length. 49. The method according to any one of Embodiments 1 to 48, wherein the double-stranded region of the siRNA is 17 to 23 nucleotide pairs in length. 50. The method according to any one of Embodiments 1 to 49, wherein the double-stranded region of the siRNA is 17 to 25 nucleotide pairs in length. 51. The method according to any one of Embodiments 1 to 50, wherein the double-stranded region of the siRNA is 23 to 27 nucleotide pairs in length. 52. The method according to any one of Embodiments 1 to 51, wherein the double-stranded region of the siRNA is 19 to 21 nucleotide pairs in length. 53. The method according to any one of Embodiments 1 to 52, wherein the double-stranded region of the siRNA is 21 to 23 nucleotide pairs in length. 54. The method according to any one of Embodiments 1 to 40, wherein each strand of the RNAi agent has 15 to 30 nucleotides. 55. The method according to any one of Embodiments 1 to 54, wherein each strand of the RNAi agent has 19 to 30 nucleotides. 56. The method according to any one of embodiments 1 to 55, 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 attached to the 3'-end. 57. The method according to embodiment 56, wherein the ligand is one or more GalNAc derivatives attached via a monovalent linker, a divalent branched linker, or a trivalent branched linker. 58. The method according to embodiment 56 or 57, wherein the ligand is as follows.

[0262]

Chemical formula

[0263]

Chemical formula

[0264]

Chemical formula

Example

[0265] Example 1 Clinical evaluation of combination therapy for treating HBeAg-negative participants with chronic HBV infection with low viral load Evaluate the safety and efficacy of an anti-HBV antibody, optionally in combination with anti-HBV siRNA and / or the nucleoside / nucleotide reverse transcriptase inhibitor tenofovir, in a phase 2 multi-center open-label clinical study in HBeAg-negative non-cirrhotic human patients with low-load chronic HBV infection.

[0266] Background Chronic HBV infection continues to be an important global public health problem with significant morbidity and mortality (Trepo C, A brief history of hepatitis milestones, Liver Int. 2014 Feb, 34 Suppl 1:29-37). Chronic HBV infection is a dynamic process characterized by the interaction between viral replication and the host immune response. Patients can be classified into different disease stages based on the levels of hepatitis B e antigen (HBeAg), hepatitis B (HBV) DNA, alanine aminotransferase (ALT), and liver inflammation (European Association for the Study of the Liver, EASL 2017 Clinical Practice Guidelines on the management of Hepatitis B virus infection, J Hepatol. 2017 Aug, 67(2):370-398, Sarin SK et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update, Hepatol Int. 2016 Jan, 10(1):1-98, Terrault NA et al., Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance, Hepatology 2018 Apr, 67(4):1560-1599).

[0267] Chronic HBV infection is a dynamic process that depends on the relationship between viral replication and host immune response. Patients can vary between different stages of the disease, and continuous monitoring of biomarkers such as HBV DNA, ALT, and HBV antigens helps to determine the natural course of the disease. Among the 300 million patients chronically infected with HBV, HBeAg-negative patients represent the largest subgroup. These patients range from those with inactive immunity (also referred to as "inactive carriers" or "inactive hepatitis") to those with chronic active hepatitis who can progress to severe liver complications including hepatocellular carcinoma (HCC). Inactive carriers are HBeAg-negative and anti-HBe-positive, with persistently low levels of HBV DNA (≤2,000 IU / mL) and normal ALT maintained for at least one year. These patients have a favorable long-term prognosis, a low histological progression rate, a low risk of cirrhosis or HCC, and a high long-term HBsAg clearance rate (EASL, 2017, supra; Invernizzi et al., 2016, supra; Terrault et al., 2018, supra; Yeo et al., 2020, supra). Inactive carriers lack indication for currently available treatments aimed at HBV DNA suppression, but they have the highest potential to achieve functional cure, thereby becoming non-infectious and having a lower risk of disease reactivation.

[0268] AB01 is a monoclonal antibody (mAb) that targets hepatitis B surface antigen (HBsAg) and has multiple potential mechanisms of action, including potent neutralizing activity and enhanced immunological activity resulting from Fc domain engineering. AB01 (HBC34v35-MLNS-GAALIE) contains the light chain amino acids of SEQ ID NO: 59 and the heavy chain amino acids of SEQ ID NO: 57.

[0269] In patients with chronic HBV infection, previous studies have shown that the restoration of HBV-specific CD8+ T cell function by PD-1 blockade in immunoinactive patients is associated with T cell differentiation (Bengsch B et al., Restoration of HBV-specific CD8+ T cell function by PD-1 blockade in inactive carrier patients is linked to T cell differentiation, J Hepatol. 2014 Dec, 61(6):1212-9). Reports from this study suggest that the potential to revive HBV-specific T cells in the immunoinactive population may be achievable by the proposed mechanism of AB01 as a vaccine mAb with T cell engagement activity. AB01 provides a new strategy for the treatment of chronic HBV infection by neutralizing HBV viral and subviral particles through targeting of HBsAg and inhibition of viral entry into hepatocytes. Additionally, the Fc region of AB01 has been engineered to increase its binding affinity for the neonatal Fc receptor (FcRn) and to promote its Fc-gamma receptor (FcγR) binding profile to activating receptors. These modifications can extend the serum half-life, increase the potency, and induce a "vaccine" effect (induction of antigen-specific T cell responses). The normal serum half-life of IgG is approximately 21 days and is regulated by the balance of FcRn-mediated endocytosis and recycling versus endosomal degradation.To produce a product with sustained activity against HBV, the well-characterized LS modification (M428L and N434S) (Gaudinski MR et al., Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults, PLoS Med. 2018 Jan 24, 15(1):e1002493, Ko SY et al., Enhanced neonatal Fc receptor function improves protection against primate SHIV infection, Nature 2014 Oct 30, 514(7524):642-5, Zalevski J et al., Enhanced antibody half-life improves in vivo activity, Nat Biotechnol. 2010 Feb, 28(2):157-9) was included in the Fc region of AB01. The LS modification increases IgG1 binding to FcRn only in the acidic pH endosomal compartment, thereby increasing the recycling of IgG to circulation. Monoclonal antibodies containing the LS mutation have been previously studied in humans (Gaudinski et al., 2018, supra). For example, VRC01LS, an mAb against the CD4 binding site of the HIV-1 glycoprotein, was safe and well tolerated at doses of 5-40 mg / kg IV and 5 mg / kg SC in healthy volunteers. Anti-drug antibodies (ADA) were not detected for 48 weeks. AB01 is similarly expected to have an extended half-life in humans, resulting in an extended exposure duration. In vitro data for AB01 suggest that the LS mutation is not expected to interfere with further Fc modifications described below.The Fc region of AB01 was also engineered to contain modifications that regulate binding to human FcγRs by enhancing binding to activating receptors FcγRIIa and FcγRIIIa while decreasing binding to the inhibitory receptor FcγRIIb. This Fc modification was designed to enhance ADCP and antigen presentation of HBsAg and HBV virions, and as a result, to promote the induction of T cell responses (the "vaccine effect"). The impact of similar modifications on mAb potency has been studied in a FcγR humanized lymphoma mouse model. In this model, the anti-CD20 antibody promoted direct killing of tumor cells via engagement of FcγRIIIa on macrophages and monocytes. In addition, anti-CD20 immune complexes induced CD8+ T cell responses via FcγRIIa-dependent presentation of tumor antigens by dendritic cells. Overall, Fc engineering of anti-CD20 antibodies (GASDALIE mutation) to increase FcγRIIa and FcγRIIIa binding had excellent therapeutic activity (DiLillo DJ et al., Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect, Cell 2015 May 21, 161(5):1035-1045). Parallel reduction of binding to the inhibitory FcγRIIb has the potential to further enhance this vaccine effect (DiLillo et al., 2015, supra).

[0270] Available data on the use of HBsAg-specific antibodies in the treatment of patients with chronic HBV infection suggest that these molecules have the potential to reduce HBsAg levels while maintaining an acceptable safety and tolerability profile. GC1102, a fully human anti-HBsAg mAb under development for the prevention of chronic HBV infection and recurrent HBV after liver transplantation, effectively reduced HBsAg by 2 to 3 log10 IU / mL, was well tolerated in a phase 1 study in patients with chronic HBV infection, and there was no evidence of severe sequelae such as immune complex disease (Lee HW et al., A prospective, open-label, dose-escalation, single-center, phase 1 study for GC1102, a recombinant human immunoglobulin for chronic hepatitis B patients, American Association for the Study of Liver Diseases, The Liver Meeting 2018, Abstract 453). HBV-ABXTL (HepeX-B), a mixture of two human anti-HBsAg mAbs, was administered to 27 patients with HBsAg levels in the range of approximately 20 to 85,000 IU / mL. HBV-ABXTL was found to have a favorable safety and tolerability profile at a maximum dose of 80 mg administered weekly over 4 doses, and no signs of immune complex disease or hepatotoxicity were reported (Galun E et al., Clinical evaluation (phase I) of a combination of two human monoclonal antibodies to HBV: safety and antiviral properties, Hepatology 2002 Mar, 35(3):673-9).Similarly, in two studies of chronic HBV patients who received high-dose hepatitis B immune globulin (HBIG) to prevent reinfection after liver transplantation, no adverse events were reported (Reed WD et al., Infusion of hepatitis-B antibody in antigen-positive active chronic hepatitis, Lancet 1973 Dec 15, 2(7842):1347-51, Tsuge M et al., Antiviral effects of anti-HBs immunoglobulin and vaccine on HBs antigen seroclearance for chronic hepatitis B infection, J Gastroenterol 2016 Nov, 51(11):1073-1080).

[0271] Clinical data from the oncology setting suggest that Fc engineering designed to enhance ADCC / ADCP and antigen presentation can improve efficacy without compromising safety or tolerability (Im S-A et al., Long-term responders to single-agent margetuximab, an Fc-modified anti-HER2 monoclonal antibody, in metastatic HER2+ breast cancer patients with prior anti-HER2 therapy, DOI: 10.1158 / 1538-7445.SABCS18-P6-18-11 February 2019). Margetuximab is a modified version of trastuzumab, which is FDA-approved for the treatment of HER2-positive cancers. Margetuximab contains modifications designed to enhance ADCC / ADCP and antigen presentation. Margetuximab was well tolerated in a first-in-human (FIH) phase 1 study in patients with HER-2 positive cancers.General toxicity did not include evidence of cardiotoxicity and was mainly grade 2 or less (Bang YJ et al., First-in-human phase 1 study of margetuximab (MGAH22), an Fc-modified chimeric monoclonal antibody, in patients with HER2-positive advanced solid tumors, Ann Oncol. 2017 Apr 1, 28(4):855-861), which is what has been observed with trastuzumab, the non-Fc-modified parental mAb, in late clinical trials (Ponde NF et al., Twenty years of anti-HER2 therapy-associated cardiotoxicity, ESMO Open. 2016 Jul 21;1(4):e000073, Riccio G et al., Trastuzumab and target-therapy side effects: Is still valid to differentiate anthracycline Type I from Type II cardiomyopathies?, Hum Vaccin Immunother. 2016 May 3, 12(5):1124-31).

[0272] Collectively, these data suggest that mAbs with Fc engineered to extend serum half-life and optimize immune effector cell activity have the potential to improve the efficacy of therapeutic mAbs without compromising safety. AB01 is expected to reduce serum HBsAg, inhibit intrahepatic virus spread, eliminate infected hepatocytes, and stimulate an HBV-specific immune response. Thus, AB01 can achieve functional cure of chronic HBV infection regardless of the presence or absence of other agents such as SIRNA01 and NRTI (tenofovir disoproxil fumarate, "TDF").

[0273] Cohort 1b will evaluate the contribution of AB01 to combination therapy and its potential to achieve functional cure in the absence of any other investigational agents. To assess the response of participants and the potential vaccine effect from AB01, peripheral blood mononuclear cells (PBMCs) will be collected to perform an evaluation of the host cell immune response (e.g., T cells) against HBV antigens. Cohort 1b will evaluate 6 to 12 doses of AB01 administered every 4 weeks for 20 to 44 weeks with NRTI (TDF) in a response induction regimen. Participants who achieve HBsAg loss and HBV DNA below the LLOQ by week 20 may be eligible to discontinue AB01 and TDF. Participants who do not achieve HBsAg loss and HBV DNA below the LLOQ by week 20 will continue to receive AB01 and TDF until week 44.

[0274] SIRNA01 is an siRNA associated with substantial reduction of HBsAg in patients with chronic HBV infection. 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. The use of siRNA provides a new strategy for the treatment of chronic HBV infection. siRNA is a 19- to 21-base pair RNA duplex that enables sequence-specific RNA cleavage and degradation by utilizing the endogenous RNA interference pathway. One siRNA can have multiple antiviral effects, including degradation of pgRNA and thus inhibition of viral replication, and degradation of all viral messenger RNA (mRNA) transcripts, thereby preventing the expression of viral proteins. This can result in the restoration of a functional immune response against HBV, either alone or in combination with other therapies. In contrast, NRTIs act on distinct parts of the viral life cycle and have a different mechanism of action from SIRNA01. NRTIs inhibit HBV polymerase, block the reverse transcription of viral pgRNA into viral DNA, and prevent the production of infectious virions. However, NRTIs do not directly affect the production of viral proteins such as HBsAg. SIRNA01 is a GalNAc-conjugated siRNA that targets all HBV viral RNAs, thereby inhibiting the production and secretion of virions and subviral particles. The reduction of HBsAg-containing non-infectious subviral particles by SIRNA01 is considered an important differentiating factor from currently available treatments.

[0275] New clinical data suggest that a combination of multiple treatment modalities may be required to achieve functional cure in the majority of patients with chronic HBV (Revill PA et al., Meeting the Challenge of Eliminating Chronic Hepatitis B Infection, Genes (Basel) 2019 Apr 1, 10(4):260, Zoulim F et al., Antiviral therapies and prospects for a cure of chronic hepatitis B, Cold Spring Harb Perspect Med. 2015 Apr 1, 5(4):a021501). The excessive secretion of non-virion integrated HBsAg in chronic HBV infection is thought to contribute to T- and B-cell dysfunction and impair the host's ability to eliminate or control the virus (Bertoletti et al., 2016, supra, Burton et al., 2018, supra, Maini et al., 2016, supra). Therefore, the development of functional cure is likely to require the reduction or elimination of HBsAg in conjunction with the induction of host immunity against infection.When administered individually, AB01 and SIRNA01 have been associated with substantial reduction of HBsAg in patients with chronic HBV infection (Gane E et al., Safety and Antiviral Activity of VIR-2218, An X-Targeting RNAi Therapeutic, In Participants With Chronic Hepatitis B Infection: Week 48 Follow-Up Results, Oral Presentation, Presented at the EASL Digital International Liver Conference, June 23-26, 2021, Yuen MF et al., Preliminary Results From a Phase 2 Study Evaluating VIR-2218 Alone and in Combination With Pegylated Interferon Alfa-2a in Participants With Chronic Hepatitis B Infection, Oral Presentation, Presented at AASLD: The Liver Meeting (Virtual), November 12-15, 2021). The combination of AB01 and SIRNA01 can result in a profound and sustained reduction of HBsAg. This is supported by the AAV-HBV mouse model of chronic HBV infection, where combination therapy with AB01 and SIRNA01 has been associated with a significant reduction of HBsAg. Additionally, lower HBsAg levels achieved by pre-treatment or co-treatment with SIRNA01 can enhance the effect of AB01. Reduction of HBsAg can also increase the potential immunological effect of AB01. The Fc modification of AB01 is designed to enhance ADCC / ADCP and antigen presentation, and as a result, to promote the induction of CD4 and CD8 T cell responses. Considering the putative effect of HBsAg on immune function, these potential effects of AB01 can be enhanced in a lower HBsAg environment, which can be achieved by the introduction of SIRNA01 and / or co-treatment with SIRNA01.Administration of AB01 for a longer duration may also enhance these effects.

[0276] The participant population included in this sub - protocol has a low viral load and normal ALT and is not eligible for treatment with NRTIs. However, NRTIs such as TDF help to reduce viral replication and thereby reduce HBV DNA levels. Thus, in addition to AB01 which has potential immunomodulatory effects to ultimately achieve sustained suppression of HBV DNA regardless of the presence or absence of HBsAg loss, they are included in this protocol as part of all regimens to enable these participants with low HBV DNA levels to achieve HBV DNA below the LLOQ.

[0277] To achieve functional cure, a response - induction approach is used to evaluate the timing and duration of AB01 and TDF in combination with SIRNA01. Cohort 2b evaluates 6 - 12 doses of SIRNA01 (200 mg) in a combination regimen containing 6 - 12 doses of AB01 and 20 - 44 weeks of NRTI (TDF). To evaluate the participants' response and the potential vaccine effect from AB01, PBMC will be collected and an assessment of the host cell immune response (e.g., T cells) to HBV antigens will be performed. Participants who achieve HBsAg loss and HBV DNA below the LLOQ by week 20 may be eligible to discontinue AB01, TDF, and SIRNA01 therapy. Participants who do not achieve HBsAg loss and HBV DNA below the LLOQ by week 20 will continue to receive AB01, TDF, and SIRNA01 therapy until week 44.

[0278] Study Design Table 4 shows the treatment groups for the study. Approximately 15 participants will initially be enrolled in each cohort. An additional 30 participants may be added to any of the cohorts. The total duration of the study for all participants is up to 100 weeks, including a maximum 56-day (8-week) screening period, a 20- to 44-week treatment period, and a 48-week follow-up period. During the treatment period, if participants achieve undetectable HBsAg (< 0.05 IU / mL) at two consecutive visits by week 20, all study interventions may be discontinued at week 20 and the participants may transition to the follow-up period. Alternatively, the treatment period may be 44 weeks for all participants with no option to discontinue study interventions at week 20.

[0279]

Table 6

[0280] The overall study scheme is shown in Figures 1A and 1B, and the dosing schemes for all cohorts are shown in Figures 2A and 2B.

[0281] AB01 is provided as a reconstituted lyophilized powder and is administered subcutaneously (SC) at 300 mg every 4 weeks for a total of 6 to 12 doses. SIRNA01 is provided as a liquid and is administered SC at 200 mg every 4 weeks for a total of 6 to 12 doses. Tenofovir disoproxil fumarate (TDF) (Viread®) is provided as a tablet and is administered orally at 300 mg daily for a total of 140 to 308 doses. Tenofovir may be provided outside the United States as tenofovir disoproxil (TD), in which case it is provided as a tablet and is administered orally at 245 mg daily for a total of 140 to 308 doses.

[0282] The cohorts in this study can be registered in parallel. The cohorts can be open cohorts, closed cohorts, or can be terminated. The total duration for participants in Cohorts 1b and 2b is up to 100 weeks, including the screening period, treatment period, and follow-up period. The screening period for all participants is up to 56 days (8 weeks). The treatment period is 20 - 44 weeks for Cohorts 1b and 2b. In particular, the treatment period may be 44 weeks (see Figure 2B). Participants may be eligible to discontinue the study intervention at Week 20 if they achieve undetectable HBsAg (< 0.05 IU / mL) and HBV DNA below the LLOQ at two consecutive visits by Week 20, and may transition to the follow-up period at subsequent visits (see Figure 2A). Alternatively, the treatment period may be 44 weeks for all participants with no option to discontinue the study intervention at Week 20 (see Figure 2B). All other participants will continue the study intervention until the end of the treatment period and will transition to the follow-up period one week after the end of the treatment period. When participants complete the treatment period for each respective cohort, they enter the follow-up period. The maximum duration of the follow-up period is 48 weeks after the last administration of the study intervention. Participants may initiate a commercially available NRTI at any point during the follow-up period if they meet any of the following criteria: within 2 weeks 2log 10An increase in HBV DNA of 100,000 IU / mL or more; an increase in HBV DNA of more than 20,000 IU / mL was confirmed at any follow-up visit (regardless of other biochemical parameters or ALT values), i.e., in two consecutive collections (regardless of other biochemical parameters or ALT values); at the same visit, any of the following criteria: total bilirubin more than twice the ULN and ALT above the ULN were confirmed, (including, but not limited to, an increase in prothrombin time [PT] of 2 or more or an international normalized ratio [INR] of 0.5 or more from baseline, jaundice, ascites, encephalopathy, etc.), any sign of liver decompensation, ALT more than 10 times the ULN, ALT more than twice the ULN persisting for 12 consecutive weeks or more, ALT more than five times the ULN persisting for 4 consecutive weeks or more and HBV DNA more than 2,000 IU / mL; HbeAg seroreversion (i.e., HbeAg positivity after being HbeAg negative) was confirmed; or any other clinically significant event warranting the initiation of NRTI therapy. Participants who meet the criteria for initiating NRTI treatment during the follow-up period will continue to be followed until the end of the study.

[0283] The HBsAg disappearance rate will be evaluated at the end of treatment (EOT) and 24 weeks after EOT. Determine the proportion of participants with HBsAg disappearance (defined as HBsAg below the LLOQ or HBsAg less than 0.05 IU / mL) at EOT. Also determine the proportion of participants who have achieved suppression of HBV DNA (below the LLOQ) with HBsAg disappearance (less than 0.05 IU / mL) at EOT. Determine the proportion of participants with HBsAg disappearance (defined as HBsAg below the LLOQ or HBsAg less than 0.05 IU / mL) 24 weeks after EOT. Additional endpoints to be evaluated are: (i) the proportion of participants who have achieved sustained suppression of HBV DNA below the LLOQ at 24 and 48 weeks after discontinuation of all treatment; (ii) the proportion of participants who have achieved sustained suppression of HBV DNA (below the LLOQ) with HBsAg disappearance (less than 0.05 IU / mL) at 24 and 48 weeks after discontinuation of all treatment; (iii) the mean change in serum HBsAg levels from baseline over multiple time points in the study; (iv) the proportion of participants who have achieved undetectable HBV DNA (below the LLOQ) over multiple time points in the study; (v) the HBV DNA levels and changes from baseline over multiple time points in the study; (vi) the nadir and maximum change in HBV DNA from baseline in the study; (vii) the proportion of participants who meet the criteria for NRTI treatment during the follow-up period; and (viii) the proportion of participants with virological relapse ((1) an increase of 1 log 10 HBV DNA IU / mL or more above the nadir at at least two consecutive visits, or (2) any quantifiable HBV DNA of 1 log10 IU / mL or more above the LLOQ at at least two consecutive visits after being below the LLOQ).

[0284] Pharmacokinetics (PK), immunogenicity, and adverse events will also be evaluated. The concentrations of AB01 and SIRNA01 PK will be quantified using a validated bioanalytical assay. PK parameters are C max 、C last 、T max 、Tlast , AUC inf , AUC last , AUC exp %, t 1 / 2 , λ z , V z / F, and CL / F may be included. Immunogenicity data may include incidence, titer, and neutralization data (e.g., for antibodies against AB01). Additional antiviral activities such as anti-HBs, HBV RNA, and hepatitis B core-related antigen (HbcrAg) are evaluated. The number and percentage of participants showing anti-HBs and anti-HBe seroconversion are evaluated.

[0285] The activity schedules for the cohorts and follow-up periods are provided in FIGS. 3A - 5C. Liver elastography can be performed using FibroScan or equivalent. Additionally, body weight (and BMI) will be determined at screening and at each study visit. Twelve-lead safety electrocardiograms (ECGs) will also be recorded at screening and on day 8 and measured in the supine position after the participant has rested comfortably for approximately 10 minutes. In cohort 2b shown in FIGS. 4A - 4C, PK parameters for AB01 and SIRNA01 can be evaluated at different intervals such as those shown in Table 5 below. During the follow-up period shown in FIGS. 5A - 5C, additional visits may be included at weeks 2, 6, 10, 28, 32, 40, and 44. For example, the activities during the follow-up period may be as shown in FIGS. 5D - 5I.

[0286] [Table 7] AB01 PK sample collection, where applicable, includes samples for free and total PK assays. Pre-dose samples will be collected up to 1 hour before dosing. The AB01 PK post-dose sample on day 2 will be collected between 22 - 28 hours after dosing on D1. AB01 PK post-dose samples may be collected at any time during the D8 visit. The pre-dose sample of SIRNA01 will be collected up to 1 hour before the maximum dose administration. The post-dose samples of SIRNA01 on Day 1, Week 20, and Week 44 will be collected between 2 and 6 hours after dosing. The SIRNA01 PK sample on Day 2 will be collected between 22 and 28 hours after D1 dosing.

[0287] Patient population Participants will have chronic HBV infection, be HbeAg-negative, have low HBV DNA (≤ 2,000 IU / mL) within 1 year before screening, and ALT levels at or below the ULN (or ALT levels < ULN). Chronic HBV infection for the purposes of the study is defined as serum HBsAg, HBV DNA, or HBeAg being positive at least two time points separated by at least 6 months, based on previous or current laboratory evidence (any combination of these tests performed 6 months apart is acceptable). The ULN value for ALT can be, for example, 34 IU / mL for females and 43 IU / mL for males. Participants will be 18 years of age or older (or the legal age of consent, whichever is ≥ 18 years) to < 66 years of age. Participants will also have HBsAg above the lower limit of detection at screening and a body mass index (BMI) of ≥ 18 kg / m 2 and ≤ 35 kg / m 2 2.

[0288] Additional inclusion criteria include the following: · In good health as determined from medical history, with no clinically significant findings from physical examination, vital signs, and laboratory tests. · Female participants must have a negative pregnancy test or confirmation of a postmenopausal state. The postmenopausal state is defined as the absence of menstruation for 12 months without another medical cause. Women of child-bearing potential (WOCBP) must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, must not be lactating, and must intend to use a highly effective method of contraception from 14 days before the administration of the study intervention until 48 weeks after the last dose of AB01 or SIRNA01. Female participants must also agree to refrain from egg donation and in vitro fertilization from the time of administration of the study intervention until 48 weeks after the last dose of AB01 or SIRNA01. · Male participants with female partners of child-bearing potential must agree to meet one of the following contraceptive requirements from the time of administration of the study intervention until 48 weeks after the last dose of AB01 or SIRNA01: vasectomy or proof of azoospermia, or male condom use + use by the partner of one of the contraceptive options listed for contraception of WOCBP. Male participants must also agree not to donate sperm from the time of the first administration of the study intervention until 48 weeks after the last dose of AB01 or SIRNA01. · Be able to understand and comply with the study requirements and be able to provide written informed consent.

[0289] Exclusion criteria include the following: · History of clinically significant liver disease from non-HBV etiology · History or current evidence of liver decompensation, including ascites, hepatic encephalopathy, and / or esophageal or gastric varices · History or current suspicion of a malignant tumor diagnosed or treated within the last 5 years (local treatment of squamous or non-invasive basal cell skin cancer is permitted; in situ cervical cancer is permitted if appropriately treated prior to screening); participants under evaluation for a malignant tumor are not eligible. · History of bone marrow or solid organ transplantation · Known active infections other than chronic HBV infection within 7 days before Day 1, or any clinically significant acute conditions such as fever (above 38°C) or acute respiratory diseases. · Coinfection with human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), hepatitis D virus (HDV), or hepatitis E virus (HEV). · Participants who are HCV antibody or HDV antibody positive but have been proven to be HCV RNA or HDV RNA negative respectively are eligible. Participants who are asymptomatic with positive HAV immunoglobulin M (IgM) or HEV IgM and have positive HAV immunoglobulin G (IgG) or HEV IgG are eligible. · History or clinical evidence of alcohol or drug abuse within 12 months before screening, or positive drug screening at the time of screening (diagnosis and prescription need to be approved by the principal investigator of the clinical trial), unless it can be explained by the prescribed medication. Note: The use of marijuana is permitted. · Received the investigational drug within 90 days before the study intervention administration or within 5 half-lives (if known), whichever is longer, or is active in the follow-up phase of another clinical study including interventional treatment. Participants are also required to consent not to participate in any other interventional studies at any time during their participation in this study, including the follow-up period. · Any clinically significant medical or psychiatric condition that, as determined by the principal investigator of the clinical trial, may interfere with the study intervention, evaluation, or protocol compliance, or render the participant unsuitable for participation in the study. · Defined by either having a liver elastography (FibroScan or equivalent) result of over 8.5 kPa at screening, or having a liver biopsy within one year due to Metavir F3 fibrosis or F4 cirrhosis, significant fibrosis or cirrhosis. · 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 monoclonal antibody, antibody fragment, or any excipient of AB01 · Any previous therapy with NRTI or PEG-IFNα within 24 weeks prior to Day 1 · Use of any of the following systemic medications within 14 days prior to study intervention administration and throughout the study: · Paracetamol (acetaminophen) at 3 g / day or more · Isoniazid · Systemic steroids (prednisone equivalent over 10 mg / day) or other immunosuppressants (Note: Administration of corticosteroids for the treatment of immune-mediated AEs is permitted.) · Administration of HBV-active oligonucleotides (e.g., siRNA, antisense oligonucleotides) within 48 weeks prior to study intervention administration · Administration of AB01 within 24 weeks prior to Day 1 · Participants have the following laboratory parameters at screening by laboratory tests: · ALT above ULN at either screening or 12 months prior to study enrollment · HBeAg positive at either screening or 12 months prior to study enrollment · HBV DNA above 2,000 IU / mL at either screening or 12 months prior to study enrollment · Direct bilirubin or international normalized ratio (INR) above 1.5 times ULN · At screening, calculate by the Cockcroft-Gault formula, with a creatinine clearance (CLcr) of less than 30 mL / min · Clinically significant abnormalities on a 12-lead ECG at screening (determined by the principal investigator of the study)

[0290] Combined therapy · Concurrent therapy was not permitted during the study · Use of NRTI prior to Day 1 is prohibited. During the study, participants will receive TDF according to the protocol. Other NRTIs are not permitted during the treatment period. If a participant requires NRTI therapy during the follow-up period, commercially available NRTIs may be initiated · Use of any of the following systemic medications is prohibited within 14 days before the study intervention administration and throughout the study o Systemic steroids (prednisone equivalent over 10 mg / day) or other immunosuppressants (Note: Administration of corticosteroids for the treatment of immune-mediated AEs is permitted.) o Paracetamol (acetaminophen) over 3 g / day o Isoniazid · Additionally, administration of any potentially hepatotoxic medications during the study should only be considered after careful consideration of the potential risks and benefits to the participant, only if no treatment options can be identified. Medications potentially hepatotoxic or associated with drug-induced liver injury include, but are not limited to, the following (Bjornsson 2016): aspirin over 3 g / day or ibuprofen over 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

[0291] Example 2 Clinical evaluation of combination therapy with antibodies, siRNA, PEG-IFNα, and NRTI inhibitors for the treatment of chronic HBV infection Evaluate the safety and efficacy of anti-HBV siRNA (SIRNA01), PEG-IFNα, and an anti-HBV antibody (AB01) combined with the NRTI tenofovir in a Phase 2 multi-center open-label clinical study in non-cirrhotic human patients with chronic HBV infection who have not previously received NRTI or PEG-IFNα treatment. Background Chronic HBV infection remains an important global public health problem with significant morbidity and mortality (Trepo, 2014, see above). Chronic HBV infection is a dynamic process that depends on the relationship between viral replication and the host immune response. Patients can vary between different stages of the disease, and continuous monitoring of biomarkers such as HBV DNA, aminotransferase (ALT), and HBV antigens helps to determine the natural course of the disease. After infection, the HBV virus can replicate as a stealth virus without a host immune response. Years after viral replication (high HBV DNA), the immune system initiates a response against infected hepatocytes, leading to an increase in ALT in patients positive for hepatitis B e antigen (HBeAg). During this stage of the disease, patients are also characterized by moderate to severe inflammation in the liver and an accelerated progression of fibrosis. Patients infected perinatally may reach this stage of chronic HBV infection 10 to 30 years after infection, while patients infected as adults or in childhood may progress more rapidly or even skip this stage altogether. Also, in this population, the outcomes vary. Among patients positive for HBeAg, some patients rapidly become HBeAg negative, while others take much longer to become HBeAg negative and anti-HBe positive, with or without treatment (EASL, 2017, see above, Fattovich G et al., Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors, J Hepatol. 2008 Feb, 48(2):335-52, Terrault, 2018, see above, Wang G and Duan Z, Guidelines for Prevention and Treatment of Chronic Hepatitis B, J Clin Transl Hepatol. 2021 Oct 28, 9(5):769-791).

[0292] Patients with elevated ALT who are HBeAg-positive and have HBV DNA above 20,000 IU / mL, or HBeAg-negative and have HBV DNA above 2,000 IU / mL, are eligible for treatment with nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and / or peginterferon-alpha-2a (PEG-IFNα) (Liang TJ et al., Present and future therapies of hepatitis B: From discovery to cure, Hepatology 2015 Dec, 62(6):1893-908). NRTIs can suppress HBV DNA in long-term treatment but do not eliminate cccDNA or integrated DNA. In contrast to NRTIs, PEG-IFNα can induce long-term viral control but can only be induced in only a small percentage (less than 10%) of patients and only after 48 weeks of treatment (Konerman MA and Lok AS, Interferon Treatment for Hepatitis B, Clin Liver Dis. 2016 Nov, 20(4):645-665). Therefore, there is an unmet need for better treatment options that can achieve practical cure.

[0293] In patients with chronic HBV infection, 180 μg of PEG-IFNα administered weekly over 48 to 52 weeks generally results in HBsAg loss in approximately 10% or less of the overall patient population (Konerman and Lok, 2016, supra).However, in a subset of patients with baseline HBsAg values of approximately less than 1,000 to 1,500 IU / mL, the HBsAg loss rate after administration of PEG-IFNα with or without NRTI is approximately 20 - 40% (He LT et al., Effect of switching from treatment with nucleos(t)ide analogs to pegylated interferon α-2a on virological and serological responses in chronic hepatitis B patients, World J Gastroenterol. 2016, 22(46):10210-10218, Huang J et al., Switching to PegIFNα-2b leads to HbsAg loss in patients with low HbsAg levels and HBV DNA suppressed by Nas, Sci Rep. 2017, 7(1):13383, Lee et al., 2020, supra, Li et al., 2016, supra, Ning Q et al., Switching from entecavir to PegIFN alfa-2a in patients with HBeAg-positive chronic hepatitis B: a randomized open-label trial (OSST trial), J Hepatol. 2014, 61(4):777-84, Takkenberg B et al., Baseline HbsAg level predict HbsAg loss in chronic hepatitis B patients treated with a combination of peginterferon alfa-2a and adefovir: an interim analysis, EASL International Liver Congress, Copengahen, Denmark, 2009). This suggests that reduction of HBsAg during or before PEG-IFNα therapy can substantially increase the HBsAg loss rate.Therefore, a regimen consisting of both SIRNA01 and AB01, which are associated with substantial reduction of HBsAg, and PEG-IFNα can increase the rate of functional cure beyond that associated with PEG-IFNα monotherapy.

[0294] The combination of SIRNA01 and PEG-IFNα has been associated with substantial reduction of HBsAg beyond that associated with either agent alone. In a study of PEG-IFNα add-on therapy, the mean HBsAg reduction at week 24 was 0.57 log10 IU / mL (Farag MS et al., Addition of Peginterferon Alfa-2a Increases HBsAg Decline in HBeAg-negative Chronic Hepatitis B Patients Treated with Long-Term Nucleos(t)ide Analogue Therapy, Presented at AASLD The Liver Meeting, November 8-12, 2019, Boston, MA.). In contrast, for participants administered both SIRNA01 and PEG-IFNα, the mean HBsAg reduction at week 24 was 2.55 log 10 IU / mL (Yuen et al., 2021, supra). However, the event of HBsAg loss was rarely observed. The addition of AB01 may result in greater reduction of HBsAg, higher rates of HBsAg loss, and functional cure.

[0295] Furthermore, PEG-IFNα has important effects on the immune system and can enhance the potential development of HBV-specific immunity after administration of AB01. Type I interferons regulate antiviral T cells both directly and indirectly through effects on accessory cells such as antigen-presenting cells (Crouse J et al., Regulation of antiviral T cell responses by type I interferons, Nat Rev Immunol. 2015 Apr, 15(4):231-42), and infiltrating or liver-resident immune cells (Dill MT et al., Pegylated IFN-α regulates hepatic gene expression through transient Jak / STAT activation, J Clin Invest. 2014 Apr, 124(4):1568-81). As recently described, liver-resident immune cells such as Kupffer cells play an important role in stimulating a robust HBV-specific T cell response (De Simone G et al., Identification of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming, Immunity 2021 Sep 14, 54(9):2089-2100.e8). These effects may further increase the likelihood of inducing a sustained T cell response (the "vaccine effect") after AB01-containing combination therapy.

[0296] Study design The overall study scheme is shown in FIGS. 6A and 6B, and the dosing schemes for all cohorts are shown in FIGS. 7A and 7B.

[0297] Table 6 shows the treatment groups for the study. Approximately 10 participants will be enrolled in each of cohorts 1a, 2a, and 3a. Approximately 15 participants will be enrolled in each of cohorts 4a and 5a. Additionally, up to 30 floater participants may be added to any cohort. In cohorts 1a, 2a, and 4a, the total study period for all participants is up to 100 weeks, in cohort 3a it is 92 - 96 weeks, and in cohort 5a it is 104 weeks. This includes a screening period of up to 56 days (8 weeks), a treatment period of 44 weeks in cohorts 1a and 2a, 36 or 40 weeks in cohort 3a, 20 - 44 weeks in cohort 4a, and 20 - 48 weeks in cohort 5a, and a follow-up period of up to 48 weeks. Alternatively, the treatment period can be 44 weeks for participants in cohort 4a and 48 weeks for participants in cohort 5a. Participants in cohorts 4a and 5a may be eligible to discontinue study intervention (excluding NRTI) after week 20 and transition to the follow-up period at subsequent visits if they have achieved all of the following at two consecutive visits by week 20: undetectable HBeAg (based on quantitative HBeAg); undetectable HBsAg (< 0.05 IU / mL); and suppression of HBV DNA (below the LLOQ). All other participants will continue study intervention until the end of the treatment period and will transition to the follow-up period one week after the end of the treatment period. Alternatively, there may be no option to discontinue study intervention at week 20, and the treatment period may be 44 or 48 weeks for participants in cohorts 4a and 5a.

[0298] Once participants complete the treatment period for each cohort, they will enter the follow-up period. The maximum duration of the follow-up period is 48 weeks after the last administration of the study intervention. Participants will discontinue NRTI at the F1 or F12 visit during the follow-up period if they meet the criteria for discontinuing NRTI based on the available data. As shown in the activity schedule (Figures 12A - 12C), additional study visits are required for participants who discontinue NRTI during the follow-up period. Liver elastography can be performed using FibroScan or an equivalent. Additionally, weight (and BMI) will be determined at screening and at each study visit. Twelve-lead safety electrocardiogram (ECG) will also be recorded at screening and on day 8, measured in the supine position after the participant has rested comfortably for approximately 10 minutes. Participants who discontinue NRTI at the F1 visit will be required to return to the facility for further visits at F2, F6, and F10, or will be required to return to the facility for further visits at F2, F6, F10, F28, F32, F40, and F44. Participants who discontinue NRTI at the F12 visit will be required to return to the facility for further visits at F14, F18, and F22, or will be required to return to the facility for further visits at F14, F18, F22, F28, F32, F40, and F44. In cohorts 1a, 2a, and 3a, AB01 PK samples will be collected prior to dosing (collected up to 1 hour before dosing).

[0299]

Table 8

[0300] In cohort 4a shown in FIGS. 10A - 10C and cohort 5a shown in FIGS. 11A - 11E, the PK parameters for AB01 and SIRNA01 can be evaluated at different intervals such as those shown in Table 7 below.

[0301]

Table 9

[0302] The follow - up period shown in FIGS. 12A - 12C may include additional visits. For example, the activities during the follow - up period may be as shown in FIGS. 12D - 12I.

[0303] Participants with baseline HBsAg above 3,000 IU / mL may participate in the AB01 PK sub-study. Participants in this sub-study will have up to two additional study visits during the treatment period to collect AB01 PK samples. PK sub-study visit 1 will be conducted 5 to 7 days after any of the 3rd, 4th, or 5th administrations of AB01. PK sub-study visit 2 (optional) will be conducted 5 to 7 days after any of the 7th, 8th, or 9th administrations of AB01. In addition to samples for PK, samples for HBsAg, HBV DNA, and liver function tests will also be collected at the same visit. Participants enrolled in cohort 3a will be excluded from the optional AB01 PK sub-study.

[0304] AB01 is provided as a reconstituted lyophilized powder and administered subcutaneously (SC) at up to 300 mg every 4 to 12 weeks. SIRNA01 is provided as a liquid and administered SC at 200 mg every 4 weeks. PEG-IFNα (Pegasys®) is provided as a liquid and administered SC at 180 mcg weekly. Tenofovir disoproxil fumarate (TDF) (Viread®) is provided as a tablet and administered orally at 300 mg daily. Tenofovir may be provided as tenofovir disoproxil (TD) outside the United States, in which case it is provided as a tablet and administered orally at 245 mg daily.

[0305] Participants in Cohort 1a / 2a will receive AB01 at a maximum of 300 mg every 4 weeks from Day 1 to Week 44, along with 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they become eligible at the later of discontinuation of NRTI or end of the follow-up period. Participants in Cohort 3a will receive AB01 at 300 mg every 8 - 12 weeks from Day 1 to Week 44, along with 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they become eligible at the later of discontinuation of NRTI or end of the follow-up period. Participants in Cohort 4a will receive AB01 at 300 mg and SIRNA01 at 200 mg every 4 weeks from Day 1 to Week 20 or Week 44, along with 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they become eligible at the later of discontinuation of NRTI or end of the follow-up period. Participants in Cohort 5a will receive AB01 at 300 mg and SIRNA01 at 200 mg every 4 weeks from Day 1 to Week 20 or Week 48, PEG-IFNα at 180 mcg weekly from Day 1 to Week 48, and 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they become eligible at the later of discontinuation of NRTI or end of the follow-up period.

[0306] To achieve functional cure, the timing and duration of AB01 and TDF, with or without combination with SIRNA01, are evaluated using a response induction approach. In Cohort 4a, AB01 and SIRNA01 will be administered every 4 weeks starting from Day 1, along with daily administered TDF. Participants who achieve loss of HBsAg and HBeAg with HBV DNA below the LLOQ by Week 20 may be eligible to discontinue AB01 and SIRNA01 therapy. Participants who do not achieve loss of HBsAg and HBeAg with HBV DNA below the LLOQ by Week 20 will continue to receive AB01 and SIRNA01 therapy until Week 44. TDF is administered daily starting from Day 1 and will end when the NRTI discontinuation criteria are met up to a maximum of Week 12 of the follow-up period.

[0307] To achieve functional cure, the timing and duration of AB01 and TDF, with or without combination with SIRNA01 and PEG-IFNα, are evaluated using a response-guided approach. In cohort 5a, AB01 and SIRNA01 will be administered starting on day 1 and every 4 weeks together with daily-administered TDF and weekly-administered PEG-IFNα for 4 weeks. Participants who achieve loss of HBsAg and HBeAg with HBV DNA below the LLOQ by week 20 may be eligible to discontinue AB01, SIRNA01, and PEG-IFNα therapy, while those who do not achieve loss of HBsAg and HBeAg with HBV DNA below the LLOQ by week 20 will continue to receive AB01, SIRNA01, and PEG-IFNα until week 48. TDF will be administered starting on day 1 and daily, and may end if the NRTI discontinuation criteria are met up to week 1 or week 12 at the maximum during the follow-up period.

[0308] Participants may discontinue NRTI at F1 or F12 visit during the follow-up period if they meet all of the following criteria: HBsAg less than 100 IU / mL and a reduction of 1 log10 IU / mL or more from the baseline HBsAg level; suppression of HBV DNA (defined as below the LLOQ); undetectable HBeAg (based on quantitative HBeAg); and ALT less than or equal to 2 times the upper limit of normal (ULN). Participants who meet the criteria for discontinuation of NRTI treatment will continue according to the activity schedule during the follow-up period. Based on the opinion of the principal investigator of the clinical trial, participants who meet the NRTI discontinuation criteria but for other reasons NRTI discontinuation is not appropriate may continue NRTI treatment.

[0309] The principal investigator of the clinical trial will consider retreatment with NRTI therapy during the follow-up period for participants whose HBV DNA has the following within a 2-week period: 2 log 10HBV DNA >100,000 IU / mL at any follow-up visit (regardless of other biochemical parameters or ALT values); confirmed increase in HBV DNA >20,000 IU / mL (i.e., in two consecutive collections, regardless of other biochemical parameters or ALT values); any of the following criteria at the same visit: confirmed total bilirubin >2x ULN and ALT >ULN, confirmed increase in prothrombin time [PT] >2 or international normalized ratio [INR] >0.5 from baseline, any sign of hepatic decompensation (including, but not limited to, jaundice, ascites, encephalopathy, etc.), ALT >10x ULN, ALT >2x ULN for 12 consecutive weeks or more, and HBV >2,000 IU / mL with ALT >5x ULN for 4 consecutive weeks or more confirmed DNA; HBeAg seroreversion (i.e., HBeAg positivity after being HBeAg negative at the time of NRTI cessation); or any other clinically significant event warranting initiation of NRTI therapy.

[0310] The HBsAg disappearance rate will be evaluated at the end of treatment (EOT) and 24 weeks after EOT. Determine the proportion of participants with HBsAg disappearance (defined as HBsAg less than the lower limit of quantification (LLOQ) or less than 0.05 IU / mL) at EOT. Also determine the proportion of participants who have achieved suppression of HBV DNA (less than the LLOQ) accompanied by HBsAg disappearance (less than 0.05 IU / mL) at EOT. Determine the proportion of participants with HBsAg disappearance (defined as HBsAg less than the LLOQ or less than 0.05 IU / mL) at 24 weeks after EOT and at the F48 follow-up visit. For HBeAg-positive participants, evaluate the proportion of participants with HBeAg disappearance (undetectable HBeAg), the time to HBeAg disappearance (undetectable HBeAg), the proportion of participants with anti-HBs and anti-HBe seroconversion, and the time to anti-HBe seroconversion. After discontinuation of all treatments, at week 24 and at the F48 follow-up visit, the proportion of participants who have achieved sustained suppression of HBV DNA (less than the LLOQ) accompanied by HBsAg disappearance (less than 0.05 IU / mL); the mean change in serum HBsAg levels from baseline over multiple time points in the study; the proportion of participants who have achieved HBV DNA less than the LLOQ over multiple time points in the study; the change in HBV DNA levels and from baseline over multiple time points in the study; the change in the nadir and maximum values of HBV DNA from baseline during the study; and the proportion of participants with virological relapse (defined as either (1) an increase of 1 log10 HBV DNA IU / mL or more above the nadir at at least two consecutive visits, or (2) a quantifiable HBV DNA of 1 log10 IU / mL or more above the LLOQ at at least two consecutive visits after being less than the LLOQ).

[0311] Determine the proportion of participants who meet the criteria for NRTI discontinuation or retreatment during the study, and the proportion of participants who have achieved ALT below the upper limit of normal (ULN) over multiple time points during the study. Evaluate the pharmacokinetics (PK), immunogenicity, and adverse events of AB01 and / or SIRNA01. The concentrations of AB01 and SIRNA01 PK will be quantified using a validated bioanalytical assay. The PK 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 be included. The immunogenicity data of AB01 may include the incidence and titer of anti-drug antibodies, as well as neutralization data. Further antiviral activities such as anti-HBs, HBV RNA, and hepatitis B core-related antigen (HBcrAg) are evaluated.

[0312] The activity schedules for the cohorts and follow-up periods are provided in FIGS. 8A-12C.

[0313] Patient population Participants will have chronic HBV infection, be HBeAg positive or negative, and have high HBV DNA (more than 2,000 IU / mL) and ALT levels (above ULN and up to 5 times ULN). Approximately 40% of the participants with a baseline HBsAg of more than 10,000 IU / mL at screening will be enrolled. In addition, approximately 30% of the HBeAg-positive participants will be targeted for enrollment in the cohort. Chronic HBV infection for the purposes of the study is defined as serum HBsAg, HBV DNA, or HBeAg being positive at two time points separated by at least 6 months, based on previous or current laboratory evidence (any combination of these studies performed 6 months apart is allowed). The ULN value of ALT can be, for example, 34 IU / mL for women and 43 IU / mL for men. Participants are 18 years of age or older (or the legal age of consent, whichever is 18 years or older) to less than 66 years of age. Participants will also have an HBsAg of more than 10 IU / mL at screening, and a body mass index (BMI) of 18 kg / m2 or more to 35 kg / m 2 with the following body mass index (BMI).

[0314] Additional inclusion criteria include the following: ·In addition to chronic infection with HBV, the subject must be in good health as determined from the medical history and have no clinically significant findings from physical examinations, vital signs, and laboratory values. ·Female participants must have a negative pregnancy test or confirmation of a postmenopausal status. Postmenopausal status is defined as absence of menstruation for 12 months 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 lactating, and must be willing to use a highly effective contraceptive method from 14 days prior to study intervention dosing until 48 weeks after the last dose of AB01, SIRNA01, or PEG-IFNα. Female participants must also agree to refrain from egg donation and in vitro fertilization from the time of study intervention dosing until 48 weeks after the last dose of AB01, SIRNA01, or PEG-IFNα. ·Male participants with female partners of childbearing potential must agree to meet one of the following contraception requirements from the time of study intervention dosing until 48 weeks after the last dose of AB01, SIRNA01, or PEG-IFNα: vasectomy or proof of azoospermia, or male condom use + use by the partner of one of the contraceptive options listed for contraception of WOCBP. Male participants must also agree not to donate sperm from the time of the first study intervention dosing until 48 weeks after the last dose of AB01, SIRNA01, or PEG-IFNα. ·Be able to understand and comply with the study requirements and be able to provide written informed consent.

[0315] Exclusion criteria include the following: ·History of clinically significant liver disease from non-HBV etiology ·History or current evidence of liver decompensation, including ascites, hepatic encephalopathy, and / or esophageal or gastric varices · History or current suspicion of a malignant tumor diagnosed or treated within the past 5 years (local treatment of squamous or non-invasive basal cell skin cancer is permitted; in situ cervical cancer is permitted if appropriately treated prior to screening); participants under evaluation for a malignant tumor are not eligible. · History of bone marrow or solid organ transplantation · Known active infection other than chronic HBV infection within 7 days prior to Day 1, or any clinically significant acute condition such as fever (> 38°C) or acute respiratory or GI disease · Coinfection with human immunodeficiency virus (HIV), hepatitis A virus (HAV) IgM, hepatitis C virus (HCV), hepatitis D virus (HDV), or hepatitis E virus (HEV) IgM. Participants who are HCV antibody or HDV antibody positive but HCV RNA or HDV RNA negative, respectively, are eligible. · Participants who are asymptomatic with positive HAV immunoglobulin M (IgM) or HEV IgM and have positive HAV immunoglobulin G (IgG) or HEV IgG are eligible. · History or clinical evidence of alcohol or drug abuse within 12 months prior to screening or a positive drug screen at screening, unless otherwise explained by prescribed medications (diagnosis and prescription must be approved by the study's responsible physician). Note: Use of marijuana is permitted. · Received the investigational drug within the longer of 90 days prior to study intervention administration or 5 half-lives (if known) or is active in the follow-up phase of another clinical study that includes an interventional treatment. Participants must also agree not to participate in any other interventional study at any time during their participation in this study, including the follow-up period. · Any clinically significant medical or psychiatric condition that, as determined by the study's responsible physician, may interfere with study intervention, evaluation, or protocol compliance or render the participant unsuitable for participation in the study. Significant fibrosis or cirrhosis, as defined by either a liver elastography (FibroScan or equivalent) result of more than 8.5 kPa at screening or a liver biopsy within 1 year due to Metavir F3 fibrosis or F4 cirrhosis. · 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 monoclonal antibody, antibody fragment, or any excipient of AB01 · Previous NRTI or PEG-IFNα therapy · Use of any of the following systemic medications within 14 days before and throughout the study intervention: Paracetamol (acetaminophen) at 3 g / day or more Isoniazid Systemic steroids (prednisone equivalent more than 10 mg / day) or other immunosuppressants (Note: Administration of corticosteroids for the treatment of immune-mediated AEs is permitted.) Cohort 5a only: Theophylline Cohort 5a only: Methadone · Administration of HBV-active oligonucleotides (e.g., siRNA, antisense oligonucleotides) within 48 weeks before the study intervention · Administration of AB01 within 24 weeks before Day 1 · Participants have the following laboratory parameters at screening by laboratory tests: Direct bilirubin or INR more than 1.5 times the ULN Total bilirubin more than 1.5 times the ULN Platelets less than 150,000 cells / μL Cohort 5a only: Serum amylase or lipase more than 3 times the ULN Cohort 5a only: Thyroid stimulating hormone (TSH) and free T4 more than the ULN or less than the LLN Cohort 5a only: 1,500 cells / mm 3Absolute neutrophil count (ANC) less than · At screening, calculated by the Cockcroft-Gault formula, with creatinine clearance (CLcr) less than 30 mL / min · Cohort 5a only: Known hypersensitivity or contraindication to interferon products · Cohort 5a only: Psychosis, bipolar disorder, schizophrenia, moderate to severe depression, current or previous history of suicidal desire, attempt, or gesture, or high current suicide risk · Cohort 5a only: Current or previous history of clinically significant retinal disease · Cohort 5a only: Current or previous history of chronic uncontrolled hypoglycemia or uncontrolled hyperglycemia / diabetes (defined as HbA1c of 8% or more) at screening · Cohort 5a only: Current or previous history of colitis · Clinically significant abnormalities on 12-lead ECG at screening (determined by the principal investigator of the study)

[0316] Combination therapy · Combination therapy was not permitted during the study · Use of NRTIs before Day 1 was prohibited. During the study, participants were to receive TDF according to the protocol. Other NRTIs were not permitted during the study. · Use of any of the following systemic medications was prohibited within 14 days before the study intervention dosing and throughout the study: o Systemic steroids (prednisone equivalent over 10 mg / day) or other immunosuppressants (Note: Administration of corticosteroids for the treatment of immune-mediated AEs is permitted.) o Paracetamol (acetaminophen) over 3 g / day o Isoniazid o Cohort 5a only: Theophylline o Cohort 5a only: Methadone · Additionally, the administration of any potential hepatotoxic medications under study should only be considered after careful consideration of the potential risks and benefits to participants, only if no treatment options can be identified. Medications that are potentially hepatotoxic or associated with drug-induced liver injury include, but are not limited to, the following (Bjornsson 2016): aspirin at doses of 3 g / day or more or ibuprofen at doses of 1.2 g / day or more; tricyclic antidepressants; valproate; phenytoin; amiodarone; anabolic steroids; allopurinol; amoxicillin-clavulanic acid; minocycline; nitrofurantoin; sulfamethoxazole / trimethoprim; erythromycin; rifampin; azole antifungals; and herbs or natural remedies.

[0317] While specific embodiments have been illustrated and described, it will be readily understood that additional embodiments can be provided by combining the various embodiments described above, and that various changes can be made without departing from the spirit and scope of the invention.

[0318] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein or listed in the application data sheet, including U.S. Provisional Patent Application No. 63 / 343,896, filed May 19, 2022, and U.S. Provisional Patent Application No. 63 / 353,383, filed Jun. 17, 2022, are hereby incorporated by reference in their entirety, unless otherwise indicated. Aspects of the embodiments can be modified if necessary to utilize concepts from various patents, applications, and publications to provide further embodiments.

[0319] These and other modifications can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in this specification and the claims, but rather the claims should be construed to include all possible embodiments together with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

[0320] Array SEQ ID NO:1 (Hepatitis B virus genome - NCBI reference sequence NC_003977.2 (GenBank accession number GI:21326584)) Sequence number 2 (target sequence, nucleotides 1579 - 1597 of NC_003977.2 (GenBank accession number GI:21326584)) GTGTGCACTTCGCTTCAC Sequence number 3 (SIRNA01, sense strand, unmodified) GUGUGCACUUCGCUUCACA Sequence number 4 (SIRNA01, antisense strand, unmodified) UGUGAAGCGAAGUGCACACUU Sequence number 5 (SIRNA01, sense strand, modified) gsusguGfcAfCfUfucgcuucacaL96 Sequence number 6 (SIRNA01, antisense strand, modified) sGfsuga(Agn)gCfGfaaguGfcAfcacsusu Sequence number 7 (SIRNA02, sense strand, modified) gsusguGfcAfCfUfucgcuucacaL96 Sequence number 8 (SIRNA02, antisense strand, modified) usGfsugaAfgCfGfaaguGfcAfcacsusu Sequence number 9 (peptide RFGF containing a membrane translocation sequence) AAVALLPAVLLALLAP Sequence number 10 (peptide RFGF analog containing a membrane translocation sequence) AALLPVLLAAP Sequence number 11 (HIV Tat protein) GRKKRRQRRRPPQ Sequence number 12 (Drosophila Antennapedia protein) RQIKIWFQNRRMKWK Sequence number 13 (HBsAg S domain). MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFF CLWVYI SEQ ID NO: 14 (J02203 (D, ayw3) HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW SEQ ID NO: 15 (FJ899792 (D, adw2) HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW SEQ ID NO: 16 (AM282986 (A) HBsAg antigen loop sequence) QGMLPVCPLIPGTTTTSTGPCKTCTTPAQGNSMFPSCCCTKPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW SEQ ID NO: 17 (D23678 (B1) HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSW SEQ ID NO: 18 (AB117758 (C1) HBsAg antigen loop sequence) QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSW SEQ ID NO: 19 (AB205192 (E) HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTSTGPCRTCTTLAQGTSMFPSCCCSKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLS SEQ ID NO: 20 (X69798 (F4) HBsAg antigen loop sequence) QGMLPVCPLLPGSTTTSTGPCTCTTLAQGTSMFPSCCCSKPSDGNCTCIPIPSSWALGKYLWEWASARFSW SEQ ID NO: 21 (AF160501 (G) HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTSTGPCTCTTPAQGNSMYPSCCCTPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW SEQ ID NO: 22 (AY090454 (H) HBsAg antigen loop sequence) QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFGKYLWEWASARFSW SEQ ID NO: 23 (AF241409 (I) HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCTKPSDGNCTCIPIPSSWAFAKYLWEWASARFSW SEQ ID NO: 24 (AB486012 (J) HBsAg antigen loop sequence) QGMLPVCPLLPGSTTTSTGPCRTCTITAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFAKFLWEWASVRFSW SEQ ID NO: 25 (HBsAg Y100C / P120T HBsAg antigen loop sequence) CQGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGFLWEWASARFSW SEQ ID NO: 26 (HBsAg P120T HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Array number 27 (HBsAg P120T / S143L HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTKPLDGNCTCIPIPSSWAFGKFLWEWASARFSW Array number 28 (HBsAg C121 S HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGPSRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Array number 29 (HBsAg R122D HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGPCDTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Array number 30 (HBsAg R122I HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGPCITCTTPAQGTSMYPSCCCTPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Array number 31 (HBsAg T123N HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGPCRNCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Array number 32 (HBsAg Q129H HBsAg antigen loop sequence) QGMLPVCPLIPGSSTTGTGPCRTCTTPAHGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Array number 33 (HBsA...

Claims

1. A pharmaceutical composition for use in a method of treating hepatitis B virus (HBV) infection or HBV-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, (c) The administration of a nucleoside reverse transcriptase inhibitor (NRTI) A pharmaceutical composition wherein the subject is HBeAg-negative, has an HBV DNA level of 2000 IU / mL or less prior to treatment, is not cirrhotic, has an alanine aminotransferase (ALT) level below the upper limit of normal (ULN), has never been previously administered an NRTI, has not received an NRTI within 24 weeks prior to treatment, has never been previously administered an anti-HBV antibody, and / or has never been previously administered an siRNA targeting HBV mRNA.

2. The pharmaceutical composition according to claim 1, comprising an anti-HBV antibody.

3. The pharmaceutical composition according to claim 1, wherein the subject is HBeAg-negative.

4. The pharmaceutical composition according to claim 1, wherein the subject has an HBV DNA level of 2000 IU / mL or less before treatment.

5. The pharmaceutical composition according to claim 1, wherein the subject has an HBV DNA level of less than 2000 IU / mL before treatment.

6. The pharmaceutical composition according to claim 1, wherein the subject is not cirrhotic and / or has an alanine aminotransferase (ALT) level below the upper limit of normal (ULN).

7. The pharmaceutical composition according to claim 1, wherein the subject is not cirrhotic and / or has an alanine aminotransferase (ALT) level below the upper limit of normal (ULN).

8. The pharmaceutical composition according to claim 1, wherein the subject has never been previously administered NRTI.

9. The pharmaceutical composition according to claim 1, wherein the subject has not received NRTI within 24 weeks prior to treatment.

10. The pharmaceutical composition according to claim 1, wherein the subject has never been previously administered an anti-HBV antibody.

11. The pharmaceutical composition according to claim 1, wherein the subject has never been previously administered siRNA targeting HBV mRNA.

12. A pharmaceutical composition for use in a method of treating hepatitis B virus (HBV) infection or HBV-related disease in a subject requiring treatment, (a) anti-HBV antibody, (b) siRNA targeting HBV mRNA, (c) Interferon-α, or (d) comprising 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, (c) Interferon-α and (d) The administration of a nucleoside reverse transcriptase inhibitor (NRTI) A pharmaceutical composition wherein the subject is HBeAg-negative or HBeAg-positive, has an HBV DNA level greater than 2000 IU / mL prior to treatment, is not cirrhotic, has an alanine aminotransferase (ALT) level greater than the upper limit of normal (ULN) and less than or equal to 5 times the ULN, has not previously received NRTI, has not received NRTI within 24 weeks prior to treatment, has not previously received anti-HBV antibody, has not previously received HCl targeting HBV mRNA, has not previously received interferon-α, is HBsAg-positive prior to treatment, and / or has an HBsAg level greater than 10 IU / mL prior to treatment.

13. The pharmaceutical composition according to claim 12, comprising an anti-HBV antibody.

14. The pharmaceutical composition according to claim 12, wherein the subject is HBeAg-negative.

15. The pharmaceutical composition according to claim 12, wherein the subject is HBeAg positive.

16. The pharmaceutical composition according to claim 12, wherein the subject has an HBV DNA level of more than 2000 IU / mL before treatment.

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

18. The pharmaceutical composition according to claim 12, wherein the subject has never been previously administered NRTI.

19. The pharmaceutical composition according to claim 12, wherein the subject has never been previously administered an anti-HBV antibody.

20. The pharmaceutical composition according to claim 12, wherein the subject has never been previously administered siRNA targeting HBV mRNA.

21. The pharmaceutical composition according to claim 12, wherein the subject has never been previously administered interferon-α.

22. The pharmaceutical composition according to claim 12, wherein the subject is HBsAg positive before treatment.

23. The pharmaceutical composition according to claim 12, wherein the subject has an HBsAg level greater than 10 IU / mL before treatment.

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

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

26. The aforementioned anti-HBV antibody (i) Each of the following: CDRH1, CDRH2, and CDRH3 amino acid sequences described in SEQ ID NOs. 44, 45, or 46, and 47, (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.

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

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

29. 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.

30. The aforementioned anti-HBV antibody (a) Light chain variable domain (V) described in Sequence ID No. 55 Ｌ The pharmaceutical composition according to claim 1, comprising (b) an amino acid sequence and (b) the heavy chain variable domain (VH) amino acid sequence described in SEQ ID NO:

53.

31. 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.

32. The aforementioned anti-HBV antibody The pharmaceutical composition according to claim 1, comprising (a) the light chain amino acid sequence described in Sequence ID No. 59 and (b) the heavy chain amino acid sequence described in Sequence ID No.

57.

33. The pharmaceutical composition according to claim 12, wherein the anti-HBV antibody is a human antibody.

34. The pharmaceutical composition according to claim 12, wherein the antibody is HBC34 or a non-natural variant of HBC34.

35. The aforementioned anti-HBV antibody (i) Each of the following: CDRH1, CDRH2, and CDRH3 amino acid sequences described in SEQ ID NOs. 44, 45, or 46, and 47, (ii) The pharmaceutical composition according to claim 12, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 48, 49, or 50, and 52, respectively.

36. The aforementioned anti-HBV antibody (i) Each of the following: CDRH1, CDRH2, and CDRH3 amino acid sequences described in Sequence ID Nos. 44, 45, and 47, (ii) The pharmaceutical composition according to claim 12, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 48, 49, and 52, respectively.

37. The aforementioned anti-HBV antibody (i) Each of the following: CDRH1, CDRH2, and CDRH3 amino acid sequences described in Sequence ID Nos. 44, 46, and 47, (ii) The pharmaceutical composition according to claim 12, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences described in Sequence ID Nos. 48, 50, and 52, respectively.

38. The aforementioned anti-HBV antibody (a) A light chain variable domain (V) that is at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO:

55. Ｌ ) and (b) a heavy chain variable domain (V) that is at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO:

53. Ｈ The pharmaceutical composition according to claim 12, comprising ) and .

39. The aforementioned anti-HBV antibody (a) Light chain variable domain (V) described in Sequence ID No. 55 Ｌ (b) Amino acid sequence and (b) Heavy chain variable domain (V) described in Sequence ID No. 53 Ｈ The pharmaceutical composition according to claim 12, comprising an amino acid sequence.

40. The aforementioned anti-HBV antibody The pharmaceutical composition according to claim 12, 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.

41. The aforementioned anti-HBV antibody The pharmaceutical composition according to claim 12, comprising (a) the light chain amino acid sequence described in Sequence ID No. 59 and (b) the heavy chain amino acid sequence described in Sequence ID No.

57.

42. 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 replaced by U.

43. 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).

44. The pharmaceutical composition according to claim 43, wherein the sense strand of the siRNA contains or consists of the nucleotide sequence 5'-GUGUGCCACUUCGCCUUCACA-3' (SEQ ID NO: 3).

45. The pharmaceutical composition according to claim 44, wherein at least one strand of the siRNA includes a 3' overhang of at least one nucleotide.

46. The pharmaceutical composition according to claim 44, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs long.

47. The pharmaceutical composition according to claim 44, wherein each chain of the RNAi agent has 15 to 30 nucleotides.

48. 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 44, wherein the sense chain is conjugated to a ligand attached to its 3' end.

49. The pharmaceutical composition according to claim 48, wherein the ligand is one or more GalNAc derivatives linked via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

50. The pharmaceutical composition according to claim 49, wherein the ligand is as follows. 【Chemistry 1】

51. The siRNA is conjugated to the ligand as shown in the following structure, 【Chemistry 2】 The pharmaceutical composition according to claim 50, wherein X is O or S in the formula.

52. The pharmaceutical composition according to claim 51, wherein X is O.

53. The pharmaceutical composition according to claim 44, 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 nucleotide, 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.

54. The pharmaceutical composition according to claim 44, wherein the siRNA includes phosphate backbone modification, 2'-ribose modification, 5'-triphosphate modification, or GalNAc conjugate modification.

55. The pharmaceutical composition according to claim 44, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand of the siRNA are modified nucleotides.

56. 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), In the formula, 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, The pharmaceutical composition according to claim 44, wherein L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol.

57. The L96 is conjugated to the sense chain as shown in the following structure, 【Transformation 3】 The pharmaceutical composition according to claim 56, wherein X is O in the formula.

58. The pharmaceutical composition according to claim 12, 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 replaced by U.

59. The pharmaceutical composition according to claim 12, wherein the antisense strand of the siRNA contains or consists of the nucleotide sequence 5'-UGUGAGCGGAAGUGCAACUCUU-3' (SEQ ID NO: 4).

60. The pharmaceutical composition according to claim 59, wherein the sense strand of the siRNA contains or consists of the nucleotide sequence 5'-GUGUGCCACUUCGCCUUCACA-3' (SEQ ID NO: 3).

61. The pharmaceutical composition according to claim 60, wherein at least one strand of the siRNA includes a 3' overhang of at least one nucleotide.

62. The pharmaceutical composition according to claim 60, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs long.

63. The pharmaceutical composition according to claim 60, wherein each chain of the RNAi agent has 15 to 30 nucleotides.

64. 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 60, wherein the sense chain is conjugated to a ligand bound to its 3' end.

65. The pharmaceutical composition according to claim 64, wherein the ligand is one or more GalNAc derivatives linked via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

66. The pharmaceutical composition according to claim 65, wherein the ligand is as follows. 【Chemistry 4】

67. The siRNA is conjugated to the ligand as shown in the following structure, 【Transformation 5】 The pharmaceutical composition according to claim 66, wherein X is O or S in the formula.

68. The pharmaceutical composition according to claim 67, wherein X is O.

69. The pharmaceutical composition according to claim 60, 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 nucleotide, 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.

70. The pharmaceutical composition according to claim 60, wherein the siRNA comprises a phosphate backbone modification, a 2'-ribose modification, a 5'-triphosphate modification, or a GalNAc conjugate modification.

71. The pharmaceutical composition according to claim 60, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand of the siRNA are modified nucleotides.

72. 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), In the formula, 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, The pharmaceutical composition according to claim 60, wherein L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol.

73. The L96 is conjugated to the sense chain as shown in the following structure, 【Transformation 6】 The pharmaceutical composition according to claim 72, wherein X is O in the formula.

74. The pharmaceutical composition according to claim 1, 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-alpha, and ganciclovir, besifovir (ANA-380 / LB-80380), or tenofovir-exaliades (TLX / CMX157).

75. The pharmaceutical composition according to claim 12, 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-alpha, and ganciclovir, besifovir (ANA-380 / LB-80380), or tenofovir-exaliades (TLX / CMX157).

76. The pharmaceutical composition according to claim 12, wherein the interferon-α is interferon-α-2a.

77. The pharmaceutical composition according to claim 76, wherein the interferon-α is pegylated.

78. The pharmaceutical composition according to claim 77, wherein the interferon-α is peginterferon-α-2a (PEG-IFNα-2a).

79. (i) The anti-HBV antibody is (a) the CDRH1, CDRH2, and CDRH3 amino acid sequences described in SEQ ID NOs. 44, 45, and 47, respectively, and the CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 48, 49, and 52, respectively, and / or (b) A light chain variable domain (V) that is at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO:

55. Ｌ ), and a heavy chain variable domain (VH) that is at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO: 53, and / or (c) 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, (ii) 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), In the formula, 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, The pharmaceutical composition according to claim 1, wherein L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol, and (iii) the NRTI is tenofovir or tenofovir disoproxil fumarate (TDF).

80. (i) The anti-HBV antibody is (a) the CDRH1, CDRH2, and CDRH3 amino acid sequences described in SEQ ID NOs. 44, 45, and 47, respectively, and the CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 48, 49, and 52, respectively, and / or (b) A light chain variable domain (V) that is at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO:

55. Ｌ ), and heavy chain variable domains (V) that are at least 90%, at least 95%, or 100% identical to the amino acid sequence described in SEQ ID NO:

53. Ｈ ), and / or (c) 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, (ii) 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), In the formula, 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 N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol, (iii) The interferon-α is peginterferon-α-2a (PEG-IFNα-2a), (iv) The pharmaceutical composition according to claim 12, wherein the NRTI is tenofovir or tenofovir disoproxil fumarate (TDF).

81. The pharmaceutical composition according to claim 1, wherein the anti-HBV antibody and siRNA are administered every four weeks.

82. The pharmaceutical composition according to claim 12, wherein the anti-HBV antibody and siRNA are administered every four weeks.

83. The pharmaceutical composition according to claim 12, wherein the interferon-α is administered once a week.

84. The pharmaceutical composition according to claim 1 or 12, wherein the NRTI is administered daily.

85. The pharmaceutical composition according to claim 1 or 12, wherein the anti-HBV antibody is administered in a dose of 300 mg.

86. The pharmaceutical composition according to claim 85, wherein the anti-HBV antibody is administered for a treatment period of 44 weeks, 48 ​​weeks, or longer.

87. The pharmaceutical composition according to claim 1 or 12, wherein the siRNA is administered in a dose of 200 mg.

88. The pharmaceutical composition according to claim 87, wherein the siRNA is administered over a treatment period of 44 weeks, 48 ​​weeks, or longer.

89. The pharmaceutical composition according to claim 12, wherein the interferon-α is administered in a dose of 180 mcg.

90. The pharmaceutical composition according to claim 89, wherein the dose of interferon-α is administered over a treatment period of 44 weeks, 48 ​​weeks, or longer.

91. The pharmaceutical composition according to claim 1 or 12, wherein the NRTI is administered in a dose of 300 mg.

92. The pharmaceutical composition according to claim 1 or 12, wherein the NRTI is administered in a dose of 245 mg.

93. The pharmaceutical composition according to claim 1 or 12, wherein the NRTI is administered over a treatment period of 44 weeks, 48 ​​weeks, or longer.

94. The pharmaceutical composition according to claim 1 or 12, wherein the subject is administered the siRNA and the anti-HBV antibody starting on the same day.

95. The pharmaceutical composition according to claim 1 or 12, wherein the subject has chronic HBV.

96. The pharmaceutical composition according to claim 1 or 12, wherein the subject is infected with hepatitis D virus (HDV).

97. The pharmaceutical composition according to claim 1 or 12, wherein the HBV-related disease is chronic HBV.

98. The pharmaceutical composition according to claim 1 or 12, wherein the HBV-related disease is HDV infection.

99. The pharmaceutical composition according to claim 1 or 12, wherein the subject is a human.

100. It's a kit, 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, A kit comprising a pharmaceutical composition containing NRTI and pharmaceutically acceptable excipients.

101. It's a kit, 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, A pharmaceutical composition comprising interferon-α and a pharmaceutically acceptable excipient, A kit comprising a pharmaceutical composition containing NRTI and pharmaceutically acceptable excipients.

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