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

Abstract

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

Description

[0001] Reference to an electronic sequence listing

[0002] The contents of the electronic sequence listing (441WO_SeqListing.xml; size: 70.5KB; and creation date: May 4, 2023) are incorporated herein by reference in their entirety. Background Art

[0003] Hepatitis B virus (HBV) is a DNA virus that infects, replicates and is maintained in human hepatocytes (Protzer U et al., Living in the liver: hepatic infections, Nature Reviews Immunology 2012, 12: 201-213). The small viral genome (3.2 kb) consists of partially double-stranded relaxed circular DNA (rcDNA) and has 4 open reading frames encoding 7 proteins: HBcAg (HBV core antigen, viral capsid protein), HBeAg (hepatitis B e-antigen), HBV Pol / RT (polymerase, reverse transcriptase), PreS1 / PreS2 / HBsAg (large, medium and small surface envelope glycoproteins) and HBx (HBV×antigen, transcriptional regulator required for 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).

[0004] In hepatocytes, rcDNA in the form of HBV nucleic acid introduced by infected virions is converted into covalently closed circular DNA (cccDNA), which is maintained in the host cell nucleus as a free chromatin structure (Allweiss L et al., The Role of cccDNA in HBV Maintenance, Viruses 2017, 9: 156). CccDNA serves as a transcription template for all viral transcripts (Lucifora J et al., Attacking hepatitis B virus cccDNA—The holygrail to hepatitis B cure, Journal of Hepatology 2016, 64 (1): S41-S48). Pregenomic RNA (pgRNA) transcripts are reverse transcribed into new rcDNA of new virions, which are secreted without causing cytotoxicity. In addition to infectious virions, infected hepatocytes also secrete a large number of genome-free subviral particles, the number of which may exceed 10,000 times the number of secreted virions (Seeger et al., 2015, see above). The virus can also randomly integrate into the host genome, which is a mechanism that contributes to hepatocyte transformation (Levrero M et al., Mechanisms of HBV-induced hepatocellular carcinoma, Journal of Hepatology 2016, 64(1): S84-S101). HBV is maintained in hepatocytes in the form of cccDNA and integrated DNA (intDNA).

[0005] Hepatitis B infection is characterized by serum viral markers and antibodies. In acute resolution of infection, the virus is cleared by effective innate and adaptive immune responses, including cytotoxic T cells that cause death of infected hepatocytes, and induction of B cells that produce neutralizing antibodies that prevent viral spread (Bertoletti A, Adaptive immunity in HBV infection, Journal of Hepatology 2016, 64 (1): S71-S83; Maini MK et al., The role of innate immunity in the immunopathology and treatment of HBV infection, Journal of Hepatology 2016, 64 (1): S60-S70; Li Y et al., Genome-wide association study identifies 8p21.3 associated with persistent hepatitis B virus infection among Chinese, Nature Communications 2016, 7: 11664). In contrast, chronic infection is associated with T and B cell dysfunction, mediated by multiple regulatory mechanisms, including presentation of viral epitopes on hepatocytes and secretion of subviral particles (Bertoletti et al., 2016, supra; Maini et al., 2016, supra; Burton AR et al., Dysfunctional surface antigen specific memory B cells accumulate in chronic hepatitis B infection, EASL International Liver Congress, Paris, France 2018). Therefore, the continued expression and secretion of viral proteins due to cccDNA retention in hepatocytes is considered a key step in the host's inability to clear infection.

[0006] Chronic HBV infection remains an important global public health problem due to significant morbidity and mortality (TrepoC, 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 of viral replication and host immune response.

[0007] Patients can be divided into different disease stages based on 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 August, 67(2):370-398; Sarin SK et al., Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update, Hepatol Int. 2016 January, 10(1):1-98; Terrault NA et al., Update on prevention, diagnosis, and treatment of chronic hepatitis B:

[0008] AASLD 2018 hepatitis B guidance, Hepatology 2018 April, 67(4):1560-1599). Of the 300 million patients chronically infected with HBV, those who are HBeAg negative are the largest subgroup. These patients range from inactive carriers to chronic active hepatitis, which can progress to serious 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 for at least 1 year. These patients have a good long-term prognosis, a very low rate of histological progression, a very low risk of cirrhosis or HCC, and a very 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 2020 Sep, 11(9):e00196). Inactive carriers lack indications for currently available treatments targeting HBV DNA suppression, but they are most likely to achieve functional cure, thereby becoming non-infectious, and have a low risk of disease recurrence.

[0009] In non-cirrhotic patients with chronic HBV infection, treatment is currently recommended for subgroups 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 nucleoside (acid) reverse transcriptase inhibitors (NRTIs) and polyethylene glycol interferon α-2a (PEG-IFNα-2a or "PEG-IFNa-2a") (Liang TJ et al., Present and future therapies of hepatitis B: From discovery to cure, Hepatology 201512, 62(6): 1893-908). Long-term therapy NRTI therapy may suppress HBV DNA, but cannot eliminate cccDNA or integrated DNA. In contrast to NRTI, PEG-IFNa can induce long-term viral control, but only in a small percentage of patients (<10%) and after 48 weeks of therapy (Konerman MA et al., Interferon Treatment for Hepatitis B, Clin Liver Dis. November 2016, 20(4): 645-665). Therefore, there is an unmet need for better treatment options that can achieve functional cure.

[0010] Currently available treatments are unable to restore immune control of HBV in the majority of patients. Therefore, there remains a need for effective treatments for HBV infection that can suppress viral replication and restore immune control in the majority of patients. Summary of the invention

[0011] In some aspects, the present disclosure provides a method of treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof, the method comprising: administering to the subject:

[0012] (a) Anti-HBV antibodies;

[0013] (b) siRNA targeting HBV mRNA; and

[0014] (c) nucleoside reverse transcriptase inhibitors (NRTIs); and

[0015] Wherein: the subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤upper limit of normal (ULN), the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks before treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA.

[0016] In some aspects, the present disclosure provides a method of treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof, the method comprising: administering to the subject:

[0017] (a) Anti-HBV antibodies;

[0018] (b) siRNA targeting HBV mRNA;

[0019] (c) interferon-α; and

[0020] (d) nucleoside reverse transcriptase inhibitors (NRTIs); and

[0021] Wherein: the subject is HBeAg negative or HBeAg positive, the subject has an HBVDNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >upper limit of normal (ULN) and ≤5 times of ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks before treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment.

[0022] In some aspects, compositions for use in therapy, compositions for use in the manufacture of a medicament, and kits are provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A to Figure 1B The study protocol used for the clinical study in Example 1 is depicted.

[0024] FIG. 2A to FIG. 2B The cohort dosing regimen used for the clinical study in Example 1 is depicted.

[0025] FIG. 3A to FIG. 3D A timeline of activities for Cohort 1b in the clinical study in Example 1 is depicted.

[0026] 4A to 4D A timeline of activities for Cohort 2b in the clinical study in Example 1 is depicted.

[0027] FIG. 5A to FIG. 5I A timeline of activities for the follow-up period for all cohorts in the clinical study in Example 1 is depicted.

[0028] FIG. 6A to FIG. 6B The study protocol used for the clinical study in Example 2 is depicted.

[0029] FIG. 7A to FIG. 7B The cohort dosing regimen used for the clinical study in Example 2 is depicted.

[0030] FIG. 8A to FIG. 8D A timeline of activities for Cohort 1a / 2a in the clinical study in Example 2 is depicted.

[0031] 9A to 9E A timeline of activities for Cohort 3a in the clinical study in Example 2 is depicted.

[0032] FIG. 10A to FIG. 10E A timeline of activities for Cohort 4a in the clinical study in Example 2 is depicted.

[0033] FIG. 11A to FIG. 11E A timeline of activities for Cohort 5a in the clinical study in Example 2 is depicted.

[0034] FIG. 12A to FIG. 12I A timeline of activities for the follow-up period for all cohorts in the clinical study in Example 2 is depicted. DETAILED DESCRIPTION

[0035] The present disclosure provides methods and compositions for use in treating hepatitis B virus (HBV) infection or HBV-related diseases, wherein one or more of anti-HBV antibodies, anti-HBV siRNA, interferon α and NRTI are administered to a subject, and related kits are provided. In some embodiments, this combination therapy is used to treat chronic hepatitis B. In some embodiments, this combination therapy is used to treat hepatitis D virus (HDV) infection.

[0036] I. Glossary

[0037] The following sections provide a detailed description of the combination therapy for treating HBV infection or HBV-related diseases and a kit related to the combination therapy. Before elaborating on the disclosure in more detail, the definition of certain terms used herein may be helpful in understanding the disclosure. Other definitions are set forth throughout the disclosure.

[0038] In this specification, unless otherwise indicated, the term "about" means +20% of the indicated range, value or structure.

[0039] The term "comprising" (and similar terms such as "including" and "consisting of") means the presence of the features, integers, steps, or components mentioned in the claim, but does not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof. The term "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0040] It should be understood that, as used herein, the term "a / an" refers to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives, and can be used synonymously with "and / or". As used herein, the terms "including" and "having" are used synonymously, and these terms and variations thereof are intended to be interpreted as non-restrictive or open-ended.

[0041] The word "substantially" does not exclude "completely"; for example, a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the word "substantially" may be omitted from the definitions provided herein.

[0042] As used herein, the term "disease" is intended to be generally synonymous and used interchangeably with the terms "disorder" and "condition" (e.g., medical condition), because all reflect an abnormal condition of the human or animal body or one of its parts that impairs normal function, usually manifests itself by obvious signs and symptoms, and reduces the duration or quality of life of the human or animal.

[0043] As used herein, the terms "peptide", "polypeptide" and "protein" and variants of these terms refer to molecules, particularly peptides, oligopeptides, polypeptides or proteins including fusion proteins, which include at least two amino acids joined to each other by a normal peptide bond or such as, for example, by a modified peptide bond in the case of isosteric peptides. For example, a peptide, polypeptide or protein can be composed of an amino acid selected from the 20 kinds of amino acids defined by a genetic code, which are connected to each other by a normal peptide bond ("classical" polypeptide). A peptide, polypeptide or protein can be composed of L-amino acids and / or D-amino acids. In particular, the terms "peptide", "polypeptide" and "protein" also include "peptide mimetics", which are defined as peptide analogs containing non-peptide structural elements, which can simulate or antagonize the biological effects of natural parent peptides. Peptide mimetics lack classical peptide properties such as peptide bonds that are easily enzymatically cleaved. In particular, in addition to these amino acids, a peptide, polypeptide or protein may include amino acids other than the 20 kinds of amino acids defined by the genetic code, or it may be composed of amino acids other than the 20 kinds of amino acids defined by the genetic code. In particular, peptides, polypeptides or proteins in the context of the present disclosure may equally be obtained by methods well known to those skilled in the art such as

[0044] The amino acid composition of the natural process or chemical process of the post-translational maturation process is modified. Such modifications are fully described in the literature. These modifications may occur at any position in the polypeptide: in the peptide backbone, in the amino acid chain or even at the carboxyl or amino terminus. In particular, the peptide or polypeptide may be branched after ubiquitination, or may be cyclic with or without branches. This modification type may be the result of a natural or synthetic post-translational process well known to those skilled in the art. In particular, in the context of the present disclosure, the terms "peptide", "polypeptide" or "protein" also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositols, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of amino acids such as arginylation or ubiquitination. These modifications are fully described in the literature (Proteins Structure and Molecular Properties, 2nd Edition., TECreighton, New York (1993); Post-translational Covalent Modifications of Proteins, BC 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). Therefore, the terms "peptide", "polypeptide" and "protein" include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, etc.

[0045] As used herein, a "(poly)peptide" comprises a single chain of amino acid monomers linked by peptide bonds as explained above. As used herein, a "protein" comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) (poly)peptides, i.e. one or more chains of amino acid monomers linked by peptide bonds as explained above. In certain embodiments, a protein according to the present disclosure comprises 1, 2, 3 or 4 polypeptides.

[0046] As used herein, the term "recombinant" (e.g., recombinant antibodies, recombinant proteins, recombinant nucleic acids, etc.) refers to any molecule (antibody, protein, nucleic acid, siRNA, etc.) that is prepared, expressed, produced, or separated by a recombinant manner and that is not naturally present. 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 a specific embodiment, the nucleic acid molecule is double-stranded RNA.

[0047] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such nomenclature includes progeny. Thus, the words "transformants" and "transformed cells" include primary subject cells and cultures derived therefrom, without regard to the number of transfers. It should also be understood that all progeny may not be identical in terms of DNA content due to intentional or unintentional mutations. Variant progeny having the same function or biological activity as screened in the initially transformed cells are included. Where different nomenclature is desired, it will be apparent from the context.

[0048] As used herein, the term "sequence variant" refers to any sequence with one or more changes compared to a reference sequence, wherein the reference sequence is any sequence listed in the sequence table, i.e., SEQ ID NO: 1 to SEQ ID NO: 61. Therefore, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. For sequence variants in the context of nucleotide sequences, the reference sequence is also a nucleotide sequence, and for sequence variants in the context of amino acid sequences, the reference sequence is also an amino acid sequence. As used herein, a "sequence variant" has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with a reference sequence. Unless otherwise specified, sequence identity is usually calculated relative to the full length of a reference sequence (i.e., a sequence described in this application). The identity percentage as mentioned herein can be determined, for example, using BLAST, using the default parameters specified 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]. "Sequence variants" in the context of nucleic acid (nucleotide) sequences have a changed sequence, wherein one or more nucleotides in the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by standard single-letter names (A, C, G or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence may change or not change the corresponding reference amino acid sequence (i.e., an amino acid "sequence variant"). In certain embodiments, a nucleotide sequence variant is a variant that does not produce an amino acid sequence variant (i.e., a silent mutation). However, nucleotide sequence variants that produce "non-silent" mutations are also within the scope, particularly nucleotide sequence variants that produce an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to a reference amino acid sequence. "Sequence variants" in the context of an amino acid sequence have a changed sequence, wherein one or more amino acids are deleted, substituted or inserted compared to a reference amino acid sequence. As a result of the alteration, such sequence variants have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having no more than 10 alterations (i.e., any combination of deletions, insertions or substitutions) is "at least 90% identical" to the reference sequence for every 100 amino acids of the reference sequence.

[0049] Although it is possible to have non-conservative amino acid substitutions, in certain embodiments, the substitution is a conservative amino acid substitution, in which the substituted amino acid has similar structural or chemical properties as the corresponding amino acid in the reference sequence. By way of example, a conservative amino acid substitution involves the replacement of one aliphatic or hydrophobic amino acid (e.g., alanine, valine, leucine, and isoleucine) by another; one hydroxyl-containing amino acid (e.g., serine and threonine) by another; one acidic residue (e.g., glutamic acid or aspartic acid) by another; one amide-containing residue (e.g., asparagine and glutamine) by another; one aromatic residue (e.g., phenylalanine and tyrosine) by another; one basic residue (e.g., lysine, arginine, and histidine) by another; and a small amino acid (e.g., alanine, serine, threonine, methionine, and glycine) by another.

[0050] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusing the N-terminus or C-terminus of an amino acid sequence to a reporter molecule or enzyme.

[0051] Unless otherwise specified, changes in sequence variants do not necessarily eliminate the functionality of the corresponding reference sequence, for example, in the case of the present invention, the functionality of anti-HBV antibodies or siRNA sequences that are sufficient to neutralize HBV infection or reduce HBV protein expression, respectively. It can be determined by using computer programs known in the art to guide which nucleotides and amino acid residues can be substituted, inserted or deleted without eliminating such functionality.

[0052] As used herein, "derived from" a nucleic acid sequence or amino acid sequence specifying a nucleic acid, peptide, polypeptide or protein refers to the source of a nucleic acid, peptide, polypeptide or protein. In some embodiments, a nucleic acid sequence or amino acid sequence derived from a specific sequence has an amino acid sequence substantially identical to the sequence or a portion thereof derived therefrom, wherein "substantially identical" includes sequence variants as defined above. In certain embodiments, a nucleic acid sequence or amino acid sequence derived from a specific peptide or protein is derived from a corresponding domain in a specific peptide or protein. Thus, "corresponding" refers in particular to the same 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). Therefore, one of ordinary skill in the art can identify the "corresponding" portion of a peptide, protein and nucleic acid. Similarly, a sequence "derived from" another sequence can usually be identified in a sequence by one of ordinary skill in the art as another sequence source.

[0053] In some embodiments, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be identical to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). However, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), and in particular, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a functional sequence variant as described above for the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). For example, in a peptide / protein, one or more amino acid residues may be replaced by other amino acid residues, or one or more amino acid residues may be inserted or deleted.

[0054] As used herein, the term "mutation" refers to a change in a nucleotide sequence and / or an amino acid sequence compared to a reference sequence (e.g., a corresponding genomic sequence). A mutation (e.g., compared to a genomic sequence) can be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation (e.g., induced by an enzyme, a chemical substance, or radiation) or a mutation obtained by site-directed mutagenesis (a molecular biological method for specific and intentional changes in a nucleotide sequence and / or an amino acid sequence). Therefore, the term "mutation" should be understood to also include physical mutations, such as in a nucleotide sequence or an amino acid sequence. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids and inversions of several consecutive nucleotides or amino acids. In order to achieve mutations in an amino acid sequence, mutations can be introduced into the nucleotide sequence encoding the amino acid sequence to express (recombinant) mutant polypeptides. Mutation can be achieved, for example, by changing the codons of a nucleic acid molecule encoding one amino acid to produce codons encoding a different amino acid, for example, by site-directed mutagenesis; or by synthesizing sequence variants, for example, by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide, and by designing the synthesis of a nucleic acid molecule that includes a nucleotide sequence encoding a variant of the polypeptide, without mutating one or more nucleotides of the nucleic acid molecule.

[0055] As used herein, the term "coding sequence" is intended to refer to a polynucleotide molecule that encodes the amino acid sequence of a protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon.

[0056] As used herein, the term "expression" refers to any step involved in the production of a polypeptide including transcription, post-transcriptional modification, translation, post-translational modification, secretion, and the like.

[0057] As used herein, the term "vaccine" is generally understood to provide at least one antigen or immunogen preventive or therapeutic material, including viral vector vaccines, which include nucleic acids encoding antigens or immunogens. Antigens or immunogens can be derived from any material suitable for vaccination. For example, antigens or immunogens can be derived from pathogens, such as from bacterial particles or viral particles, or from tumors or cancerous tissues. Antigens or immunogens stimulate the adaptive immune system of the body to provide an adaptive immune response. In particular, "antigen" or "immunogen" generally refers to a substance that can be recognized by the immune system (e.g., an adaptive immune system) and can trigger an antigen-specific immune response (e.g., by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response). Typically, an antigen can be or can include a peptide or protein that can be presented to a T cell by MHC.

[0058] As used herein, "hepatitis B virus", which can be 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 with four overlapping reading frames (which may be referred to herein as "genes", "open reading frames" or "transcripts"): C, X, P and S. The core protein is encoded by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of pre-core (pre-C) protein. DNA polymerase is encoded by gene P. Gene S is a gene encoding surface antigen (HBsAg). The HBsAg gene is a long open reading frame containing three in-frame "start" (ATG) codons, thereby producing three different sizes of polypeptides, referred to as large, medium and small S antigens: pre-S1+pre-S2+S, pre-S2+S or S. In addition to decorating the envelope of HBV, surface antigens are also part of subviral particles that are produced in large excess compared to virions and play a role in immune tolerance and sequestration of anti-HBsAg antibodies, allowing infectious particles to evade immune detection. The protein encoded by gene X plays a role in transcriptional transactivation and replication and has been associated with the development of liver cancer.

[0059] Nine HBV genotypes named A to I have been identified, and an additional genotype J has been proposed, each with a unique geographical distribution (Velkov S et al., The Global Hepatitis B Virus Genotype Distribution Approximated from Available Genotyping Data, Genes 2018, 9(10): 495). The term "HBV" includes any genotype (A to J) of HBV. The complete coding sequence of the reference sequence of the HBV genome can be found in, for example, GenBank accession numbers GI: 21326584 and GI: 3582357. The amino acid sequences of C, X, P and S proteins can be found, for example, in NCBI accession numbers YP_009173857.1 (C protein); YP_009173867.1 and BAA32912.1 (X protein); YP_009173866.1 and BAA32913.1 (P protein); and YP_009173869.1, YP_009173870.1, YP_009173871.1 and BAA32914.1 (S protein). Additional examples of HBV messenger RNA (mRNA) sequences can be obtained using publicly available databases (e.g., 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, ie, genotypes AJ and their variants.

[0060] In some embodiments, the present disclosure provides a combination therapy to treat HBV including anti-HBV siRNA. siRNA mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) path, thereby achieving the inhibition of gene expression. This process is generally referred to as "RNA interference" (RNAi). Without wishing to be bound by a particular theory, the long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is decomposed into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15: 485). Dicer, i.e., ribonuclease-III-like enzyme, processes dsRNA into 19 to 23 base pair siRNAs using characteristic two base 3' overhangs (Bernstein et al., Nature 2001, 409: 363). Then siRNA is incorporated into RISC, where one or more helicases unwind the siRNA double helix, thereby enabling the complementary antisense strand to guide target recognition (Nykanen et al., Cell 2001, 107: 309). After binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., Genes Dev. 2001, 15:188).

[0061] The terms "silencing", "inhibiting the expression of...", "downregulating the expression of...", etc., as long as they refer to the HBV gene, refer herein to at least a partial reduction in the expression of the HBV gene, as manifested by a reduction in the amount of HBV mRNA, which can be isolated or detected from a first cell or cell group that transcribes the HBV gene, and has been treated with an inhibitor of HBV gene expression, so that the expression of the HBV gene is inhibited compared to a second cell or cell group, which is substantially the same as the first cell or cell group but has not been treated in this way (control cells). The degree of inhibition can be measured, for example, as the difference between the degree of mRNA expression in the control cell minus the degree of mRNA expression in the treated cell. Alternatively, the degree of inhibition can be given according to a reduction in a parameter functionally associated with HBV gene expression (e.g., the amount of protein encoded by the HBV gene, or the number of cells displaying a certain phenotype, such as an HBV-infected phenotype). In principle, HBV gene silencing can be determined in any cell expressing the HBV gene, such as a cell infected with HBV or a cell engineered to express the HBV gene, and by any appropriate assay.

[0062] The level of HBV RNA expressed by a cell or cell group or the level of circulating HBV RNA can be determined using any method known in the art for evaluating mRNA expression, such as the rtPCR method provided in Example 2 of International Application Publication No. WO 2016 / 077321A1 and U.S. Patent Application Publication No. US2017 / 0349900A1, which are incorporated herein by reference. In some embodiments, the expression level of an HBV gene (e.g., total HBV RNA, HBV transcript, e.g., HBV3.5kb transcript) in a sample is determined by detecting a transcribed polynucleotide or a portion thereof, e.g., RNA of an HBV gene. RNA can be extracted using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), RNeasy RNA preparation kit Or PAXgene (PreAnalytix, Switzerland) is extracted from cells. Typical assay forms using ribonucleic acid hybridization include nuclear conjugation assay, RT-PCR, RNase protection assay (Melton DA et al., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nuc. Acids Res. 1984, 12: 7035-56), protein blotting, in situ hybridization and microarray analysis. Circulating HBV mRNA can be detected using the method described in International Application Publication No. WO2012 / 177906A1 and U.S. Patent Application Publication No. US2014 / 0275211A1, which are incorporated herein by reference.

[0063] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during HBV gene transcription, including mRNA as an RNA processing product of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cutting at or near the portion. For example, the length of the target sequence will generally be 9 to 36 nucleotides, for example, 15 to 30 nucleotides in length, including all subranges therebetween. As a non-limiting example, the target sequence 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, 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.

[0064] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0065] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence and to form a double helix structure under certain conditions, as will be understood by the skilled person. Such conditions may, for example, be stringent conditions, wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 ° C or 70 ° C for 12 to 16 hours, followed by washing. Other conditions may be applied, such as physiologically relevant conditions that may be encountered inside an organism. The skilled person will be able to determine the condition set that is most suitable for the complementarity test of the two sequences according to the final application of the hybridized nucleotides.

[0066] As described herein, complementary sequences within siRNA include base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, where a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3 or 2 mismatched base pairs in the case of hybridization producing a duplex of up to 30 base pairs,

[0067] While retaining the ability to hybridize under conditions most relevant to their ultimate application, such as inhibition of gene expression via the RISC pathway. However, in the case where two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered as mismatches relative to the determination of complementarity. For example, an siRNA comprising an oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can still be referred to as "fully complementary" for the purposes described herein.

[0068] As used herein, "complementary" sequences may also include non-Watson-Crick base pairs and / or base pairs formed by non-natural and modified nucleotides, or may be formed entirely by them, as long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0069] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used with respect to base matching between the sense and antisense strands of an siRNA or between the antisense strand of an siRNA agent and a target sequence, as understood from the context of their use.

[0070] As used herein, a polynucleotide that is "substantially complementary" to at least a portion of an mRNA refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding an HBV protein). For example, if the sequence is substantially complementary to an uninterrupted portion of an HBV mRNA, the polynucleotide is complementary to at least a portion of an HBV mRNA.

[0071] As used herein, the term "siRNA" refers to an RNA interference molecule comprising an RNA molecule or a molecular complex having a hybridized duplex region comprising two antiparallel and substantially complementary nucleic acid chains, which will be referred to as having "sense" and "antisense" orientations relative to the target RNA. The duplex region may have any length that allows for specific degradation of the desired target RNA by the RISC pathway, but typically the length range will be 9 to 36 base pairs, for example, 15 to 30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex may have any length within this range, for example 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 15 to 20 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 29 base pairs, 15 to 30 base pairs, 15 to 31 base pairs, 15 to 32 base pairs, 15 to 33 base pairs, 15 to 34 base pairs, 15 to 35 base pairs, 15 to 36 base pairs and any subrange therebetween, including but not limited to 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 20 to 26 base pairs, 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs, and 21 to 22 base pairs. siRNAs generated in cells by treatment with Dicer and similar enzymes are typically in the range of 19 to 22 base pairs in length.

[0072] One strand of the duplex region of siRNA includes a sequence that is substantially complementary to the region of the target RNA. The two strands that form the duplex structure may come from a single RNA molecule having at least one self-complementary region, or may be formed by two or more separate RNA molecules. When the duplex region is formed by two strands of a single molecule, the molecule may have a duplex region separated by a single-stranded nucleotide chain (referred to herein as a "hairpin loop") between the 3' end of one strand and the 5' end of the corresponding other strand that forms the duplex structure. The hairpin loop may include at least one unpaired nucleotide; in some embodiments, the hairpin loop may include 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. In the case where the two substantially complementary strands of siRNA are composed of separate RNA molecules, those molecules do not have to be but may be covalently linked. When the two strands are covalently linked by means other than the hairpin loop, the connecting structure is referred to as a "joint".

[0073] siRNA as described herein can be synthesized by standard methods known in the art, for example by using an automatic DNA synthesizer, such as commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0074] The term "antisense strand" or "guide strand" refers to a strand of the siRNA that includes a region that is substantially complementary to a target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence). In the case where the complementary region is not completely complementary to the target sequence, the mismatch may be in the interior or terminal region of the molecule. Typically, the most tolerated mismatch is in the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends.

[0075] As used herein, the term "sense strand" or "follower strand" refers to the strand of a siRNA that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0076] The term "RNA molecule" or "ribonucleic acid molecule" not only encompasses the RNA molecule expressed or found in nature, but also encompasses analogs and derivatives of RNA comprising one or more ribonucleoside / ribonucleoside analogs or derivatives as described herein or known in the art. Strictly speaking, "ribonucleoside" comprises nucleoside bases and ribose, and "ribonucleotide" is a ribonucleoside with one, two or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleotide" can be considered to be equivalent as used herein. RNA can be modified in a core base structure or in a ribose-phosphate backbone structure, for example, as described in more detail below. However, the siRNA molecule comprising a ribonucleoside analog or a derivative retains the ability to form a duplex. As non-limiting example, RNA molecule can also include at least one modified ribonucleoside, including but not limited to 2'-O-methyl modified nucleoside, including 5' thiophosphate group nucleoside, terminal nucleoside connected with cholesteryl derivative or n-dodecanoic acid bisdecylamide group, locked nucleoside, abasic nucleoside, 2'-deoxy-2'-fluoro modified nucleoside, 2'-amino modified nucleoside, 2'-alkyl modified nucleoside, morpholino nucleoside, phosphoramidate or including nucleoside of non-natural base or their any combination. In another example, RNA molecule can include at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, at most the whole length of siRNA molecule. For each ribonucleoside in this multiple modified ribonucleosides in RNA molecule, modification need not be identical. In some embodiments, modified ribonucleosides include deoxyribonucleosides. For example, the siRNA may include one or more deoxynucleosides, including, for example, a deoxynucleoside overhang or one or more deoxynucleosides within a double-stranded portion of the siRNA. However, as used herein, the term "siRNA" does not include a complete DNA molecule.

[0077] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double helix 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 may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may include or consist of: nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0078] As used herein, the term "blunt / blunt ended" with respect to siRNA refers to the absence of unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs, at a given end of the siRNA. One or both ends of the siRNA may be blunt-ended. When both ends of the siRNA are blunt-ended, the siRNA is referred to as "blunt-ended." Blunt-ended" siRNA is a siRNA with both ends being blunt-ended, i.e., no nucleotide overhangs at either end of the molecule. Often such molecules will be double-stranded over their entire length.

[0079] In some embodiments, the present disclosure provides a combination therapy to treat HBV including an anti-HBV antibody. In certain embodiments, the anti-HBV antibody or its antigen-binding fragment binds to the antigenic loop region of HBsAg and neutralizes hepatitis B virus infection. In certain embodiments, the anti-HBV antibody or its antigen-binding fragment binds to the antigenic loop region of HBsAg and neutralizes hepatitis D virus infection.

[0080] As used herein, the term "antibody" covers various forms of antibodies, including but not limited to complete antibodies, antibody fragments, antigen binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variants or mutant antibodies), as long as the characteristic properties of the antibody are retained. In some embodiments, the antibody shows human antibodies and / or monoclonal antibodies. In specific embodiments, the antibody is a human monoclonal antibody. In certain specific embodiments, the antibody is a recombinant human monoclonal antibody. As used herein, the terms "antigen binding fragment", "fragment" and "antibody fragment" are used interchangeably to refer to any fragment of the antibody of the combination therapy that retains the antigen binding activity of the antibody. The example of an antibody fragment includes but is not limited to single-chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. In addition, as used herein, the term "antibody" includes both antibodies and their antigen binding fragments.

[0081] As used herein, "neutralizing antibodies" are antibodies that neutralize, i.e., prevent, inhibit, reduce, hinder or interfere with the ability of a pathogen to initiate and / or perpetuate an infection in a host. The terms "neutralizing antibody" or "neutralizing antibody" are used interchangeably herein. These antibodies can be used alone or in combination as a prophylactic or therapeutic agent when properly formulated, in conjunction with active vaccination, as a diagnostic tool or as a manufacturing tool as described herein.

[0082] 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, upon immunization, of producing a full repertoire or selection of human antibodies in the absence of endogenous immunoglobulins.

[0083] Transfer of the 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. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner P et al., Immunol. 1991, 147: 86-95). In some embodiments, human monoclonal antibodies are prepared by using modified EBV-B cell immortalization as described in the following literature: Traggiai E et al. (Nat Med. 2004, 10 (8): 871-5). As used herein, the term "human antibody" also includes such antibodies that are modified, for example, in the variable region to produce properties as described herein.

[0084] The antibody of the combination therapy can be any isotype (e.g., IgA, IgG, IgM, i.e., κ, γ or μ heavy chain), but in certain specific embodiments, the antibody is IgG. Within the IgG isotype, the antibody can be an IgG1, IgG2, IgG3 or IgG4 subclass. In a specific embodiment, these antibodies are IgG1. The antibody of the combination therapy may have a κ or λ light chain. Based on the antigen-independent uptake of IgG into hepatocytes through the FcRN-IgG receptor, IgG-type HBsAg-specific antibodies can also advantageously block the release of HBV and HBsAg from infected cells. Therefore, IgG-type HBsAg-specific antibodies can bind intracellularly and thereby block the release of HBV virions and HBsAg.

[0085] As used herein, the term "variable region" (light chain variable region (V L ), heavy chain variable region (V H )) refers to the portion of an antibody light chain (LC) or heavy chain (HC) (usually about 105 to 120 amino-terminal amino acids of a mature antibody heavy chain or light chain) that includes the complementarity determining region ("CDR") and the framework region ("FR") and is directly involved in the binding of the antibody to an antigen. The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-continuous amino acid sequences within the variable region of an antibody that confer antigen specificity and / or binding affinity. Typically, there are three CDRs in each variable region of an immunoglobulin binding protein; for example, for an antibody, V H Area and V L The region generally includes six CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3). Immunoglobulin sequences can be aligned with numbering schemes (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which can allow the annotation of equivalent residue positions and allow the use of Antigen Receptor Numbering And Receptor Classification (ANARCI) software tools (Bioinformatics 2016, 15: 298-300) to compare different molecules. It should be understood that in certain embodiments, the antibodies or antigen-binding fragments of the present disclosure may include all or part of a heavy chain (HC), a light chain (LC), or both. For example, a full-length intact IgG antibody monomer generally includes V H 、CH1、CH2、CH3、V L and CL.

[0086] In certain embodiments, the anti-HBV antibody or antigen-binding fragment thereof according to the combination therapy of the present disclosure is a purified antibody, single-chain antibody, Fab, Fab', F(ab')2, Fv or scFv. Therefore, the antibody of the combination therapy can be a human antibody, a monoclonal antibody, a human monoclonal antibody, a recombinant antibody and / or a purified antibody. The present disclosure also provides fragments of antibodies, in particular fragments that retain the antigen-binding activity of the antibody. Such fragments include, but are not limited to, single-chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Although in some places, the present disclosure may explicitly refer to the antigen-binding fragments, antibody fragments, variants and / or derivatives of antibodies, the terms "antibody" or "antibody for combination therapy" used herein include all categories of antibodies, i.e., antigen-binding fragments, antibody fragments, variants and derivatives of antibodies.

[0087] Fragments of antibodies can be obtained from antibodies by methods including digestion with enzymes such as pepsin or papain and / or by chemical reduction to cleave disulfide bonds. Alternatively, fragments of antibodies can be obtained by cloning and expressing a portion of a heavy chain or light chain sequence. The present disclosure also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of antibodies of the present disclosure. For example, the present disclosure includes scFv, which includes CDRs from antibodies of the present disclosure. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies, such as single chain Fv, in which the heavy chain variable domain and the light chain variable domain are joined by a peptide linker.

[0088] The antibody fragments disclosed herein can confer monovalent or multivalent interactions and are included in various structures as described above. For example, scFv molecules can be synthesized to produce trivalent "trifunctional antibodies" or tetravalent "quadrufunctional antibodies". ScFv molecules can include domains in the Fc region to form bivalent miniantibodies. In addition, the sequence of the antibody / antibody fragment can be a component of a multispecific molecule, wherein the sequence targets an epitope as 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 bifunctional antibodies (Holliger and Hudson, Nature Biotechnology 2005, 9: 1126-36).

[0089] Antibodies according to the present disclosure may be provided in purified form. Typically, the antibodies will be present in a composition that is substantially free of other polypeptides, for example, wherein less than 90% (by weight), typically less than 60% and more typically less than 50% of the composition is constituted by other polypeptides.

[0090] In embodiments, the antibodies and antigen-binding fragments of the present disclosure may be multispecific (e.g., bispecific, trispecific, tetraspecific, etc.) and may be provided in any multispecific format as disclosed herein. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are multispecific antibodies, such as bispecific or trispecific antibodies. The form of bispecific antibodies is disclosed in, for example, Spiess et al. (Mol. Immunol. 2015, 67(2):95) and Brinkmann and Kontermann (mAbs 2017, 9(2):182-212), these bispecific formats and methods for making them are incorporated herein by reference, and include, for example, bispecific T cell engagers (BiTEs), DARTs, knob-in-hole (KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, TandAbs, triplets, TriBi miniantibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCab, intracellular antibodies, CrossMab, bispecific Use Fab (DAF) (two-in-one or four-in-one), DutaMab, DT-IgG, charge pair, Fab arm exchange, SEED body, Triomab, LUZ-Y assembly, Fcab, κλ body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody and DVI-IgG (four-in-one). A bispecific or multispecific antibody may include a HBV and / or HDV-specific binding domain of the present disclosure in combination with another such binding domain of the present disclosure, or with a different binding domain that specifically binds to HBV and / or HDV (e.g., at the same or different epitopes), or with a binding domain that specifically binds to different antigens.

[0091] II. siRNA targeting HBV

[0092] In some embodiments, the present disclosure provides therapeutic methods and related compositions and kits involving the administration of siRNA targeting HBV mRNA.

[0093] In some embodiments, the siRNA targeting HBV mRNA is SIRNA01.

[0094] SIRNA01 is a synthetic chemically modified siRNA targeting HBV RNA with a covalently attached triantennary N-acetyl-galactosamine (GalNAc) ligand that allows specific uptake by hepatocytes. SIRNA01 targets mRNA encoded by a region of the HBV genome that is common to all HBV viral transcripts and is pharmacologically active against HBV genotypes A to J. In preclinical models, SIRNA01 has been shown to inhibit viral replication, translation, and secretion of HBsAg, and may provide or contribute to a functional cure for chronic HBV infection. SIRNA may have a variety of antiviral effects, including degrading pgRNA, thereby inhibiting viral replication and degrading all viral mRNA transcripts, thereby preventing the expression of viral proteins. This may lead to the restoration of a functional immune response to HBV, whether used alone or in combination with other therapies. The ability of SIRNA01 to reduce non-infectious subviral particles containing HBsAg also distinguishes it from currently available treatments.

[0095] SIRNA01 targets and inhibits the expression of mRNA encoded by the HBV genome according to the NCBI reference sequence NC_003977.2 (GenBank accession number GI: 21326584) (SEQ ID NO: 1). More specifically, SIRNA01 targets mRNA encoded by a portion of the HBV genome including the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO: 2), which corresponds to nucleotides 1579-1597 of SEQ ID NO: 1. Because transcription of the HBV genome produces overlapping RNAs of multiple cistrons, SIRNA01 results in significant inhibition of the expression of most or all HBV transcripts. Exemplary methods for synthesizing SIRNA01 and experimental data demonstrating silencing of HBV gene expression are described in International Application Publication No. WO 2020 / 036862A1, and these methods and data are incorporated herein by reference.

[0096] SIRNA01 has a sense strand comprising 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO:3) and an antisense strand comprising 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO:4), wherein these nucleotides include 2'-fluoro (2'F) and 2'-O-methoxy (2'OMe) ribose modifications, phosphorothioate backbone modifications, glycol nucleic acid (GNA) modifications, and is conjugated to a triantennary N-acetyl-galactosamine (GalNAc) ligand at the 3' end of the sense strand to facilitate delivery to hepatocytes through the asialoglycoprotein receptor (ASGPR). Including modifications, the sense strand of SIRNA01 includes 5′-gsusguGfcAfCfUfucgcuucacaL96-3′ (SEQ ID NO: 5), and the antisense strand includes 5′-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3′ (SEQ ID NO: 6), wherein the modifications are abbreviated as shown in Table 1.

[0097] Table 1. Abbreviations for nucleotide monomers used in the expression of modified nucleic acid sequences. It should be understood that unless otherwise indicated, Otherwise these monomers, when present in an oligonucleotide, are linked to each other via 5'-3'-phosphodiester bonds.

[0098] abbreviation Nucleotides A Adenosine-3'-phosphate A 2'-Fluoroadenosine-3'-phosphate

[0099]

[0100]

[0101] 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 includes sequence variants of SIRNA01. In specific embodiments, the HBV transcript portion targeted by the sequence variant of SIRNA01 overlaps with the HBV transcript portion targeted by SIRNA01.

[0102] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein (1) the sense strand comprises SEQ ID NO:3 or SEQ ID NO:5, or a sequence that differs from SEQ ID NO:3 or SEQ ID NO:5 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively; or (2) the antisense strand comprises SEQ ID NO:4 or SEQ ID NO:6, or a sequence that differs from SEQ ID NO:4 or SEQ ID NO:6 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively.

[0103] In some embodiments, a shorter duplex having one of the sequences of SEQ ID NO:4 or SEQ ID NO:6 minus only a few nucleotides on one or both ends is used. Therefore, it is contemplated herein that siRNAs have 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 its ability to inhibit the expression of the HBV gene differs from the siRNA inhibition of the complete sequence by no more than 5%, 10%, 15%, 20%, 25% or 30%. In some embodiments, siRNAs having blunt ends at one or both ends are provided, which are formed by removing nucleotides from one or both ends of SIRNA01.

[0104] In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein (1) the sense strand comprises SEQ ID NO:7, or a sequence that differs from SEQ ID NO:7 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively; or (2) the antisense strand comprises SEQ ID NO:8, or a sequence that differs from SEQ ID NO:8 by no more than 4, no more than 3, no more than 2, or no more than 1 nucleotides, respectively.

[0105] In some embodiments, a shorter duplex having a sequence of SEQ ID NO: 8 minus only a few nucleotides on one or both ends is used. Thus, siRNA having a partial sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from SEQ ID NO: 8 is contemplated herein, and its ability to inhibit the expression of the HBV gene differs by no more than 5%, 10%, 15%, 20%, 25% or 30% from the siRNA inhibition comprising the complete sequence. In some embodiments, siRNA having a blunt end at one or both ends is provided, which is formed by removing nucleotides from one or both ends of SEQ ID NO: 8.

[0106] In some embodiments, siRNA as described herein may include one or more mismatches with the target sequence. In some embodiments, siRNA as described herein includes no more than 3 mismatches. In some embodiments, if the antisense strand of siRNA includes a mismatch with the target sequence, the mismatch region is not located at the center of the complementary region. In a specific embodiment, if the antisense strand includes a mismatch with the target sequence, the mismatch is limited to the last 5 nucleotides from the 5' or 3' end of the complementary region. For example, for 23 nucleotide siRNA chains complementary to the HBV gene region, the RNA chain may not include any mismatch in the central 13 nucleotides. Methods described herein or methods known in the art can be used to determine whether the siRNA comprising a mismatch with the target sequence effectively inhibits the expression of the HBV gene.

[0107] In some embodiments, the siRNA used in the methods, compositions and kits described herein comprises two oligonucleotides, wherein one oligonucleotide is described as a sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described herein and as known in the art, the complementary sequence of the siRNA can also be included as a self-complementary region of a single nucleic acid molecule relative to being located on a separate oligonucleotide.

[0108] In some embodiments, a single-stranded antisense RNA molecule comprising the antisense strand of a siRNA described herein is used in the methods, compositions, and kits described herein.Antisense RNA molecules can have 15 to 30 nucleotides complementary to the target.

[0109] In some embodiments, a single-stranded antisense RNA molecule comprising an antisense strand of SIRNA01 or a sequence variant thereof is used in the methods, compositions, and kits described herein. The antisense RNA molecule may have 15 to 30 nucleotides complementary to the target. For example, the antisense RNA molecule may have a sequence of at least 15, 16, 17, 18, 19, 20, 21 or more consecutive nucleotides from SEQ ID NO: 4 or SEQ ID NO: 6.

[0110] In some embodiments, siRNA includes a sense strand and an antisense strand, wherein the sense strand includes SEQ ID NO:5, and the antisense strand includes SEQ ID NO:6, and also includes additional nucleotides, modifications or conjugates as described herein. For example, in some embodiments, in addition to those indicated in SEQ ID NO:5 and 6, siRNA may include further modifications. This modification can be produced using methods established in the art, such as the methods described in the following document: "Current protocols in nucleic acid chemistry," Beaucage SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Examples of this modification are described in more detail below.

[0111] In one embodiment, substantially all or all nucleotides of the sense strand and substantially all or all nucleotides of the antisense strand of the siRNA are modified nucleotides. The nucleotides may be modified as described herein.

[0112] a. Modified siRNA

[0113] Modifications disclosed herein include, for example, (a) (e.g., at the 2' position or the 4' position) sugar modification or replacement of sugars; (b) backbone modifications, including modification or replacement of phosphodiester bonds; (c) base modifications, for example, replacement of bases with stable bases, destabilizing bases, or bases for base pairing with an expanded partner library, removal of bases (absic nucleotides) or conjugated bases; and (d) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse connection, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse connection, etc.). Some specific examples of modifications that can be incorporated into the siRNA of the present application are shown in Table 1.

[0114] Modifications include substituted sugar moieties. The siRNAs featured herein may include one of the following at the 2' position: F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl; wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2) n NH2, O(CH2) n CH 3、 O(CH2) n ONH2 and O(CH2) n ON[(CH2)n CH3)]2, wherein n and m are 1 to about 10. In some other embodiments, the siRNA includes one of the following at the 2' position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of siRNA or group for improving the pharmacodynamic properties of siRNA and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta 1995, 78: 486-504), i.e., alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2 * -O-dimethylaminoethoxyethyl or 2 * -DMAEOE), i.e., 2 * -O-CH2-O-CH2-N(CH2)2. Other exemplary modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the siRNA, specifically, at the 3' position of the sugar on the 3' terminal nucleotide or in the 5' position of the 2'-5' linked siRNA and the 5' terminal nucleotide. Modifications can also include sugar mimetics, such as cyclobutyl moieties that replace the pentofuranosyl sugar.

[0115] Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,5 No. 76,427; No. 5,591,722; No. 5,597,909; No. 5,610,300; No. 5,627,053; No. 5,639,873; No. 5,646,265; No. 5,658,873; No. 5,670,633; and No. 5,700,920; each of which is incorporated herein by reference for its teachings relating to methods for making such modifications.

[0116] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoamidates), thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' bonds, 2'-5'-linked analogs of these esters, and those with reversed polarity, wherein adjacent pairs of nucleoside units are linked with 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0117] Representative U.S. patents that teach the preparation of the above phosphorus-containing bonds 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 No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. No. 5,571,799; No. 5,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,4 No. 44,423; No. 6,531,590; No. 6,534,639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Pat. No. RE 39464; each of which is incorporated herein by reference for its teachings relating to methods of making such modifications.

[0118] In addition, RNA with modified main chain includes those without phosphorus atom in the main chain. For the purpose of this specification, and as sometimes mentioned in the art, modified RNA without phosphorus atom in its internucleoside main chain can also be regarded as oligonucleoside. The modified RNA main chain not including phosphorus atom has a main chain formed by: short chain alkyl or cycloalkyl nucleoside interbond, mixed heteroatom and alkyl or cycloalkyl nucleoside interbond or one or more short chain heteroatom or heterocyclic nucleoside interbond. These main chains include: those main chains with morpholino bond (partially formed by the sugar part of nucleoside); siloxane main chain; sulfide, sulfoxide and sulfone main chain; acetyl and thioacetyl main chain; methylene acetyl and thioacetyl main chain; olefin-containing main chain; aminosulfonate main chain; methylene imino and methylene hydrazine main chain; sulfonate and sulfonamide main chain; amide main chain; and other main chains with mixed N, O, S and CH2 components.

[0119] Representative U.S. patents that teach 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; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704; No. 5,623,070; No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439; each of which is incorporated herein by reference for its teachings relating to methods of making such modifications.

[0120] In some embodiments, the sugar and internucleoside bond, i.e., the main chain, of the nucleotide unit are replaced with novel groups. The base unit is maintained to hybridize with an appropriate nucleic acid target compound. One such oligomeric compound (i.e., an RNA mimic that has been shown to have excellent hybridization properties) is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing main chain, particularly an aminoethylglycine main chain. The nucleobase is retained and directly or indirectly combined with the aza nitrogen atom of the amide portion of the main chain. Representative U.S. patents for teaching 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 these documents is incorporated herein by reference for teachings related to this preparation method. Additional teachings of PNA compounds can be found in, for example, Nielsen et al. (Science 1991, 254: 1497-1500).

[0121] Some embodiments of the technology described herein include RNAs with thiophosphate backbones and oligonucleotides with heteroatom backbones, and in particular, -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as methylene (methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-] of U.S. Pat. No. 5,489,677 and the amide backbone of U.S. Pat. No. 5,602,240. In some embodiments, the RNAs described herein have the morpholino backbone structure of U.S. Pat. No. 5,034,506.

[0122] Modifications of the siRNA disclosed herein may also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0123] Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, specifically 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional nucleobases include: nucleobases disclosed in U.S. Pat. No. 3,687,808; nucleobases disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine (Herdewijn P, ed., Wiley-VCH, 2008); nucleobases disclosed in The Concise Encyclopedia Of Polymer Science And Engineering (pages 858 to 859, Kroschwitz JL, ed., John Wiley & Sons, 1990); nucleobases disclosed by Englisch et al. (Angewandte Chemie, International Edition, 30, 613, 1991), and nucleobases disclosed by Sanghvi YS (Chapter 15, dsRNA Research and Applications, pages 289 to 302, Crooke ST and Lebleu B ed., CRC Press, 1993). Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured by the technology 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 the stability of nucleic acid duplexes by 0.6°C to 1.2°C (Sanghvi YS et al., eds., dsRNA Research and Applications, CRC Press, Boca Raton,

[0124] 276-278, 1993) and are exemplary base substitutions, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0125] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Patent No. 3,687,808; U.S. Patent No. 4,845,205; 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 its teachings relating to methods for making such modifications.

[0126] siRNA can also be modified to include one or more diol nucleic acids, such as adenosine diol nucleic acids (GNA). A description of adenosine-GNA can be found, for example, in Zhang et al. (JACS 2005, 127 (12): 4174-75), which is incorporated herein by reference for its teachings related to methods for preparing GNA modifications.

[0127] The RNA of siRNA can also be modified to include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety includes an additional bridge connecting 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to improve the stability of siRNA in serum and reduce off-target effects (Elmen J et al., Nucleic Acids Research 2005, 33 (1): 439-47; Mook OR et al., Mol Cane Ther 2007, 6 (3): 833-43; Grunweller A et al., Nucleic Acids Research 2003, 31 (12): 3185-93).

[0128] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845; each of which is incorporated herein by reference for its teachings relating to methods of making such modifications.

[0129] In some embodiments, the siRNA includes modifications involving chemical attachment of one or more ligands, moieties, or conjugates to the RNA that enhance the activity, cellular distribution, or cellular uptake of the siRNA. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA 1989, 86:6553-56), cholic acid (Manoharan et al., Biorg. Med. Chem. Let. 1990, 4:1053-60), thioethers, e.g., beryl-S-triphenylmethanethiol (Manoharan et al., Ann. NY Acad. Sci. 1992, 660:306-9); Manoharan et al., Biorg. Med. Chem. Let. 1993, 3:2765-70), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20:533-38), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J 1991, 10: 1111-18; Kabanov et al., FEBS Lett. 1990, 259: 327-30; Svinarchuk et al., Biochimie 1993, 75: 49-54), phospholipids, for example, hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36: 3651-54; Shea et al., Nucl. Acids Res. 1990, 18: 3777-83), phospholipids, polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides 1995, 14:969-73) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36:3651-54), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264:229-37) or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277:923-37).

[0130] 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 enhanced affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a cell or organ compartment, tissue, organ, or body region), e.g., compared to a species in which such a ligand is not present. In such embodiments, the ligand will not participate in duplex pairing in a duplex nucleic acid.

[0131] The ligand may 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 may also be a recombinant molecule or a synthetic molecule, such as a synthetic polymer, for example a synthetic polyamino acid. Examples of polyamino acids include polyamino acids that are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, and alpha helical peptides.

[0132] The ligand may also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, a lipid or a protein, such as an antibody that binds to a specific cell type, such as a hepatocyte. The targeting group may be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, a surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipid, a cholesterol, a steroid, a bile acid, a folic acid, vitamin B12, vitamin A, biotin or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0 (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1,3-propylene glycol, heptadecanyl, palmitic acid, myristic acid, 03-(oleoyl) lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptide conjugates (e.g., antennapedia mutant peptides, Tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP and AP.

[0133] The ligand can be a protein (e.g., a glycoprotein) or a peptide (e.g., a molecule with a specific affinity for a co-ligand) or an antibody (e.g., an antibody that binds to a specific cell type, such as a hepatocyte). The ligand can also include hormones and hormone receptors. The ligand can also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose and multivalent fucose. The ligand can be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[0134] The ligand can be a substance, such as a drug, which can increase the uptake of siRNA into cells, for example, by disrupting the cytoskeleton of the cell, for example, by disrupting the microtubules, microfilaments and / or intermediate filaments of the cell. The drug can be, for example, a taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine or myoservin.

[0135] In some embodiments, the ligand is a moiety that is taken up by a target cell (e.g., a hepatocyte), such as a vitamin. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins included are B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by a target cell (such as a hepatocyte). HSA and low-density lipoprotein (LDL) are also included.

[0136] In some embodiments, the ligand attached to the siRNA as described herein acts as a pharmacokinetic (PK) modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc.

[0137] Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides that bind to serum proteins are also known to include many phosphorothioate bonds, so short oligonucleotides (e.g., oligonucleotides with about 5 bases, 10 bases, 15 bases, or 20 bases) that include multiple phosphorothioate bonds in the backbone are also suitable for the technology described herein as ligands (e.g., as PK modulators). In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulators in the embodiments described herein.

[0138] (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 into target cells or cell membranes, and / or (c) can be used to modulate binding to serum proteins, such as HSA. Such lipids or lipid-based molecules can bind to serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, neproxin or aspirin can be used.

[0139] Lipid-based ligands can be used to inhibit (e.g., control) the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA will be less likely to be targeted to the kidney and, therefore, less likely to be cleared from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

[0140] In some embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate will be distributed to non-renal tissues. In certain specific embodiments, the HSA-ligand binding is reversible.

[0141] In some embodiments, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate will distribute to the kidney.Other moieties that target to kidney cells may also be used in place of or in addition to the lipid-based ligand.

[0142] (ii) Cell penetrating peptides and agents. On the other hand, the ligand is a cell penetrating agent, such as a helical cell penetrating agent. In some embodiments, the agent is amphipathic. Exemplary agents are peptides, such as tat or antennapedia mutant peptides. If the agent is a peptide, it can be modified, including peptidyl mimetics, 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 specific embodiments, the helical agent has a lipophilic and lipophobic phase.

[0143] "Cell penetrating peptides" are capable of penetrating cells, e.g., microbial cells (such as bacteria or fungal cells) or mammalian cells (such as human cells). Microbial cell penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or CeropinPI), disulfide bond-containing peptides (e.g., α-defensins, β-defensins or bacteriocins) or peptides containing only one or two main amino acids (e.g., PR-39 or indolicidin).

[0144] The part can be a peptide or a peptide mimetic. Peptide mimics (also referred to as oligopeptide mimics in this article) are molecules that can be folded into a defined three-dimensional structure similar to a natural peptide. The attachment of peptides and peptide mimics to siRNA may affect the pharmacokinetic distribution of RNAi such as by enhancing cell recognition and absorption. The length of peptide or peptide mimics part can be about 5 to 50 amino acids, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0145] The peptide or peptide mimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide portion can be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting portion. The peptide portion 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 mutant protein (RQIKIWFQNRRMKWK (SEQ ID NO: 12)) have been found to function as delivery peptides. The peptide or peptide mimetic may be encoded by a random sequence of DNA, such as peptides identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 1991, 354: 82-84).

[0146] The cell penetrating peptide may also include a nuclear localization signal (NLS). For example, the cell penetrating peptide may be a bipartite amphipathic peptide such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 1993, 31: 2717-24).

[0147] (iii) Carbohydrate conjugates. In some embodiments, the siRNA oligonucleotides described herein also include carbohydrate conjugates. Carbohydrate conjugates may be advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate composed of one or more monosaccharide units, each of which has at least 6 carbon atoms (which may be linear, branched or cyclic), each carbon atom being bound to an oxygen, nitrogen or sulfur atom; or a compound having a carbohydrate portion as a part thereof, the carbohydrate portion being composed of one or more monosaccharide units, each of which has at least six carbon atoms (which may be linear, branched or cyclic), each carbon atom being bound to an oxygen, nitrogen or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides comprising about 4 to 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharide units include C5 and above (in some embodiments, C5-C8) sugars; and disaccharides and trisaccharides include sugars having two or three monosaccharide units (in some embodiments, C5-C8).

[0148] In some embodiments, the carbohydrate conjugate is selected from the group consisting of:

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] Another representative carbohydrate conjugate for use in the embodiments described herein includes

[0155]

[0156] (Formula XXII), wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0157] In some embodiments, the carbohydrate conjugate further includes another ligand, such as, but not limited to, a PK modulator, an endosomolytic ligand, or a cell penetrating peptide.

[0158] (iv) Linkers. In some embodiments, the conjugates described herein can be attached to siRNA oligonucleotides using a variety of linkers, which can be cleavable or non-cleavable.

[0159] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound. Linkers typically include a direct bond or an atom such as oxygen or sulfur; a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH; or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aralkyl, aralkenyl, aralkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaralkyl, alkylaralkenyl, alkylaralkynyl, alkenylaralkyl, alkenylaralkenyl, alkenylaralkynyl, alkynylaralkyl, alkynylaralkenyl, alkynylaralkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenyl heteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocycloalkyl, alkylheterocycloalkenyl, alkylheterocycloalkynyl, alkenylheterocycloalkyl, alkenylheterocycloalkenyl, alkenylheterocycloalkynyl, alkynylheterocycloalkyl, alkynylheterocycloalkenyl, alkynylheterocycloalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl and alkynylheteroaryl, the one or more methylene groups being interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heterocyclyl; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between 1 and 24 atoms, between 4 and 24 atoms, between 6 and 18 atoms, between 8 and 18 atoms, or between 8 and 16 atoms.

[0160] A cleavable linking group is a linking group that is sufficiently stable outside of a cell, but which is cleaved after entering a target cell to release the two parts of the linker that are held together. In certain embodiments, the cleavage of a cleavable linking group in a target cell or under a first reference condition (which may, for example, be selected to simulate or represent intracellular conditions) is at least 10 times or at least 100 times greater than the cleavage in a subject's blood or under a second reference condition (which may, for example, be selected to simulate or represent conditions found in blood or serum).

[0161] The cleavable linking group is susceptible to cleavage agents (e.g., pH, redox potential, or the presence of degrading molecules). Typically, cleavage agents are more prevalent inside cells than in serum or blood, or are found at higher levels or activities. Examples of such degrading agents include: redox agents that are selective for a particular substrate or that are not substrate specific, including, for example, oxidative or reductases or reducing agents such as thiols present in cells that can

[0162] Degradation of the redox cleavable linking group by reduction; esterases; endosomes or agents that can produce an acidic environment, such as those that make the pH five or lower; enzymes, peptidases (which can be substrate specific) and phosphatases that can hydrolyze or degrade acid-cleavable linking groups by acting as a universal acid. Cleavable bond groups, such as disulfide bonds, may be sensitive to pH. The pH of human serum is 7.4, while the average value of intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, about 5.0. Some linkers will have a cleavable linking group that is cleaved at a specific pH, thereby releasing the cationic lipid from the ligand inside the cell, or releasing it into the desired compartment of the cell.

[0163] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the targeted cell. For example, a liver targeting ligand can be linked to a cationic lipid via a linker that includes an ester group. Hepatocytes are rich in esterases, and thus the linker will be more efficiently cleaved in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells in the lung, renal cortex, and testis.

[0164] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0165] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradation agent (or condition) to cut a candidate linking group. It may also be desirable to test the ability of a candidate cleavable linking group to resist cutting in blood or when contacting with other non-target tissues. Therefore, the relative susceptibility to cutting between the first condition and the second condition can be determined, wherein the first condition is selected to indicate the cutting in the target cell, and the second condition is selected to indicate the cutting in other tissues or biofluids, such as blood or serum. The evaluation can be performed in a cell-free system, in a cell, in a cell culture, in an organ or tissue culture, or in a whole animal. An initial evaluation is performed under cell-free or culture conditions and it can be useful to confirm by further evaluating in a whole animal. In certain embodiments, the cutting of a useful candidate compound in a cell (or under in vitro conditions selected to simulate intracellular conditions) is at least 2 times, at least 4 times, at least 10 times, or at least 100 times of the cutting in blood or serum (or under in vitro conditions selected to simulate extracellular conditions).

[0166] A class of cleavable linking groups is a redox cleavable linking group that is cleaved under reduction or oxidation. The example of a reductive cleavable linking group is a disulfide linking group (-SS-). In order to determine whether a candidate cleavable linking group is a suitable "reductive cleavable linking group", or whether it is suitable for use with a specific RNAi part and a specific targeting agent, reference can be made to the method described herein. For example, a candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which simulates the cleavage rate observed in cells (e.g., target cells). Candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In some embodiments, candidate compounds are cut up to 10% in blood. In certain embodiments, the degradation of useful candidate compounds in cells (or under in vitro conditions selected to simulate intracellular conditions) is at least 2 times, at least 4 times, at least 10 times or at least 100 times of the degradation in blood (or under in vitro conditions selected to simulate extracellular conditions). The cleavage rate of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0167] Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. Examples of agents that cleave the phosphate group in a cell are enzymes in the cell, such as phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In certain embodiments, the phosphate-based linking group is selected from: -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -O-P(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In specific embodiments, the phosphate linking group is -OP(O)(OH)-O-. Methods similar to those described above can be used to evaluate these candidates.

[0168] Acid cleavable linking groups are linking groups that are cut under acidic conditions. In some embodiments, acid cleavable linking groups are cut in an acidic environment of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower) or by agents that can act as broad acids, such as enzymes. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid cleavable linking groups. Examples of acid cleavable linking groups include, but are not limited to, esters of hydrazones, esters, and amino acids. Acid cleavable groups may have the general formula -C=N-, C(O)O, or -OC(O). In some embodiments, the carbon (alkoxy) attached to the oxygen of the ester is an aryl, a substituted alkyl or a tertiary alkyl, such as a dimethylpentyl or tert-butyl group. These candidates can be evaluated using methods similar to those described above.

[0169] Ester-based cleavable linking groups are cleaved by enzymes in cells, such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene. 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.

[0170] The cleavable linking group based on peptide is cut by enzymes in cells, such as peptidases and proteases. The cleavable linking group based on peptide is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The cleavable group based on peptide does not include amide groups (-C (O) NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. The peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The cleavable group based on peptide is usually limited to the peptide bond (i.e., amide bond) formed between the amino acids producing peptides and proteins and does not include the entire amide functional group. The cleavable linking group based on peptide has the general formula -NHCHRAC (O) NHCHRBC (O) -, wherein RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to the method described above.

[0171] Representative carbohydrate conjugates with linkers include, but are not limited to:

[0172]

[0173]

[0174]

[0175] wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0176] In certain embodiments of the compositions and methods described herein, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker. For example, in some embodiments, the siRNA is conjugated to a GalNAc ligand as shown in the following schematic: wherein X is O or S. In some of these embodiments, X is O.

[0177] In some embodiments, the combination therapy comprises an siRNA conjugated to a bivalent or trivalent branched linker selected from the group of structures represented by any one of Formulae (XXXI)-(XXXIV):

[0178]

[0179] in:

[0180] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0 to 20 at each occurrence, and wherein the repeating units may be the same or different;

[0181] 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 absent occurrence is independently CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;

[0182] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B and Q 5C is independently alkylene or substituted alkylene at each occurrence, wherein one or more methylene groups may be interrupted or terminated by one or more of: O, S, S(O), SO2, N(R N ), C(R')=C(R"),C≡C or C(O);

[0183] R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B and R 5C Each time it does not occur, it is independently NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-

[0184] NH-,CO,CH=NO, or a heterocyclic group;

[0185] L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, independently at each occurrence is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a Is H or an amino acid side chain. The trivalent conjugated GalNAc derivative is particularly suitable for use with siRNA to inhibit the expression of a target gene, such as the expression of formula (XXXV):

[0186]

[0187] Where L 5A , L 5B and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0188] Examples of suitable divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures of Formula I, Formula VI, Formula X, Formula IX, and Formula XII mentioned above.

[0189] Representative U.S. patents that teach the preparation of RNA conjugates include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 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; 5,567,81 No. 0; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928 and No. 5,688,941; No. 6,294,664; No. 6,320,017; No. 6,576,752; No. 6,783,931; No. 6,900,297; and No. 7,037,646; each of which is incorporated herein by reference for its teachings relating to this method of preparation.

[0190] In some cases, the RNA of the siRNA can be modified with non-ligand groups. Several non-ligand molecules have been conjugated to siRNA 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 have included 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), bile acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4:1053), thioethers, e.g., hexyl-S-triphenylmethanethiol (Manoharan et al., Ann. NY Acad. Sci. 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20: 533), aliphatic chains, for example, 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, for example, hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36: 3651; Shea et al., Nucl. Acids Res. 1990, 18: 3777), phospholipids, polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides 1995, 14:969) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277:923).

[0191] Typical conjugation protocols involve synthesizing RNA with 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 agent or activating agent. The conjugation reaction can be performed in the solution phase while the RNA is still bound to a solid support or after RNA cleavage. Purification of RNA conjugates by HPLC generally produces pure conjugates.

[0192] b. Pharmaceutical compositions and delivery of siRNA

[0193] In some embodiments, a pharmaceutical composition comprising siRNA as described herein and a pharmaceutically acceptable carrier or excipient is provided. The pharmaceutical composition comprising siRNA can be used to treat HBV infection. This pharmaceutical composition is usually formulated based on the delivery mode. For example, the composition can be formulated for delivery via parenteral, for example, by subcutaneous (SC) delivery for systemic administration.

[0194] A "pharmaceutically acceptable carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more agents (such as nucleic acids) to an animal. Excipients can be liquid or solid and are selected with consideration of the intended mode of administration so as to provide a desired volume, consistency, etc. when combined with the agent (e.g., nucleic acid) and other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers or excipients include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0195] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not react adversely with nucleic acids can also be used to prepare siRNA compositions. Suitable pharmaceutically acceptable carriers for use in non-parenteral delivery include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, straight-chain starch, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, etc.

[0196] Preparations for topical administration of nucleic acids may 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 bases. These solutions may also include buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not adversely react with nucleic acids that are suitable for non-parenteral administration may be used.

[0197] In some embodiments, administration of the pharmaceutical compositions and formulations described herein can be topical (e.g., by a transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by a nebulizer); intratracheal; intranasal; epidermal and transdermal; oral; or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subcutaneous administration (e.g., via an implanted device); or intracranial administration (e.g., by intraparenchymal, intrathecal, or intraventricular administration).

[0198] In some embodiments, the pharmaceutical composition comprises a sterile solution of siRNA (e.g., SIRNA01) prepared in water for subcutaneous injection. In some embodiments, the pharmaceutical composition comprises a sterile solution of SIRNA01 prepared in water for subcutaneous injection at a free acid concentration of 200 mg / mL.

[0199] In some embodiments, the pharmaceutical composition comprising siRNA described herein is administered with a dosage sufficient to inhibit HBV gene expression. In some embodiments, the dosage of siRNA is within the range of 0.001 milligrams per kilogram of body weight to 200.0 milligrams per kilogram of body weight recipients per day, or within the range of 1 milligram per kilogram of body weight to 50 milligrams per kilogram of body weight per day. For example, siRNA can be administered per single dose 0.01mg / kg, 0.05mg / kg, 0.5mg / kg, 1mg / kg, 1.5mg / kg, 2mg / kg, 3mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg or 50mg / kg. The pharmaceutical composition can be administered once a day, or the pharmaceutical composition can be administered with two, three or more sub-doses at appropriate intervals throughout the day, or even using continuous infusion or delivered by controlled release formulations. In this case, the siRNA contained in each sub-dose must be correspondingly smaller to reach a daily total dose. Dosage units can also be mixed for delivery in a few days, for example, using conventional sustained-release formulations that provide sustained release of siRNA over a period of several days. Sustained-release formulations are well known in the art and can be used specifically for delivering medicaments at specific sites, such as those that can be used together with the medicaments of the technology described herein. In this embodiment, the dosage unit comprises a corresponding multiple of the daily dose.

[0200] In some embodiments, a pharmaceutical composition comprising an siRNA targeting HBV mRNA described herein (eg, SIRNA01) comprises the siRNA at a dose of 0.8 mg / kg, 1.7 mg / kg, 3.3 mg / kg, 6.7 mg / kg, or 15 mg / kg.

[0201] In some embodiments, the pharmaceutical composition including siRNA described herein (e.g., SIRNA01) includes a dosage of 20mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg or 900mg siRNA. In some embodiments, the pharmaceutical composition including siRNA described herein (e.g., SIRNA01) includes a dosage of 20mg to 900mg siRNA. In some embodiments, the pharmaceutical composition including siRNA described herein (e.g., SIRNA01) includes a dosage of 100mg to 300mg siRNA.

[0202] In some embodiments, a pharmaceutical composition comprising a siRNA described herein (eg, SIRNA01) comprises a dose of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg of siRNA.

[0203] In some embodiments, a pharmaceutical composition comprising an siRNA described herein (eg, SIRNA01) comprises a dose of 200 mg of the siRNA.

[0204] III. Anti-HBV Antibodies

[0205] The present disclosure also provides anti-HBV antibodies for use in combination therapy for treating HBV or HBV-related diseases.

[0206] a. Antibodies that bind to HBV proteins

[0207] In some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy binds to the antigenic loop region of HBsAg. The envelope of the hepatitis B virus contains three "HBV envelope proteins" (also known as "HBsAg", "hepatitis B surface antigen"): S protein (for "small", also known as S-HBsAg), M protein (for "middle", also known as M-HBsAg) and L protein (for "large", also known as L-HBsAg). S-HBsAg, M-HBsAg and L-HBsAg share the same C-terminus (also known as "S domain", 226 amino acids), which corresponds to the S protein (S-HBsAg) and is involved in viral assembly and infectivity. S-HBsAg, M-HBsAg and L-HBsAg are synthesized, assembled and secreted in the form of particles through the Golgi apparatus in the endoplasmic reticulum (ER). The S domain includes four predicted transmembrane (TM) domains, whereby both the N-terminus and C-terminus of the S domain are exposed to the lumen. Transmembrane domains TM1 and TM2 are both necessary for the integration of co-translational proteins into the ER membrane, and transmembrane domains TM3 and TM4 are located at the C-terminal third of the S domain. The "antigenic loop region" of HBsAg is located between the predicted TM3 and TM4 transmembrane domains of the S domain of HBsAg, whereby the antigenic loop region includes amino acids 101-172 of the S domain (Salisse J and Sureau C, Journal of Virology 2009, 83: 9321-8). The determinant cluster of infectivity lies in the antigenic loop region of the HBV envelope protein. In particular, the residues between 119 and 125 of HBsAg contain the CXXC motif, which has been shown to be the most important sequence required for HBV infectivity (Jaoude GA and Sureau C, Journal of Virology 2005, 79: 10460-6).

[0208] As used herein, the S domain of HBsAg refers to the amino acid sequence as set forth in SEQ ID NO: 13 (shown below) or a natural or artificial sequence variant thereof.

[0209] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTA QGTSMYPSCCCTKPSD GNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI (SEQ ID NO: 13; amino acids 101-172 are underlined)

[0210] For example, the expression "amino acids 101-172 of the S domain" refers to the amino acid residues at positions 101-172 of the polypeptide according to SEQ ID NO: 13. However, it will be understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions, such as HBsAg of different genotypes or different HBsAg mutants described herein) may occur naturally in the amino acid sequence of the S domain of HBsAg or be artificially introduced into the amino acid sequence of the S domain of HBsAg without affecting its biological properties. Therefore, the term "S domain of HBsAg" includes all such polypeptides, including, for example, polypeptides according to SEQ ID NO: 13 and natural or artificial mutants thereof. 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 natural or artificial variants thereof. For example, the expression "amino acid residues from positions 101-172 of the S domain of HBsAg" encompasses amino acid residues from positions 101-172 of SEQ ID NO: 13 and corresponding fragments of mutants (natural or artificial mutants) thereof.

[0211] As used herein, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at the same position of the sequence when the sequence is subjected to optimized alignment, i.e., the sequence is aligned to obtain the highest identity percentage. The M protein (M-HBsAg) corresponds to the S protein extended by the N-terminal domain of 55 amino acids called "pre-S2". The L protein (L-HBsAg) corresponds to the M protein extended by the N-terminal domain of 108 amino acids called "pre-S1" (genotype D). The pre-S1 and pre-S2 domains of the L protein may be present at the inner surface of the virion (on the cytoplasmic side of the ER), playing a vital role in the viral assembly, or present on the outer surface (on the lumen side of the ER), which can be used to interact with target cells and is necessary for viral infectivity. In addition, the HBV surface protein (HBsAg) is not only incorporated into the virion envelope, but also spontaneously buds from the ER-Golgi intermediate compartment membrane to form an empty "subviral particle (SVP)" released from the cell by secretion.

[0212] Because all three HBV envelope proteins S-HBsAg, M-HBsAg and L-HBsAg include the S domain, all three HBV envelope proteins S-HBsAg, M-HBsAg and L-HBsAg also include an "antigenic loop region." Therefore, an antibody or an antigen-binding fragment thereof that binds to the HBsAg antigenic loop region binds to all three HBV envelope proteins: S-HBsAg, M-HBsAg and L-HBsAg.

[0213] In addition, in some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy neutralizes infection of hepatitis B virus. In other words, the antibody or antigen-binding fragment thereof can reduce the viral infectivity of hepatitis B virus. In some embodiments, the anti-HBV antibody or antigen-binding fragment thereof of the combination therapy neutralizes infection of 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 explanation of hepatitis D virus as a dedicated satellite of hepatitis B virus).

[0214] In order to study and quantify viral infectivity (or "neutralization") in the laboratory, those skilled in the art are aware of various standard "neutralization assays". For neutralization assays, animal viruses are typically propagated in cells and / or cell lines. In the context of the present disclosure, for neutralization assays, cultured cells can be incubated with a fixed amount of HBV in the presence (or absence) of the antibody to be tested. As a readout, the level of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant can be used and / or HBcAg staining can be assessed. In one embodiment of the HBV neutralization assay, cultured cells (e.g., HepaRG cells, in particular differentiated HepaRG cells) are incubated with a fixed amount of HBV in the presence or absence of the antibody to be tested, for example at 37°C for 16 hours. The incubation can be carried out in a culture medium (e.g., supplemented with 4% PEG 8000). After incubation, the cells can be washed and further cultured. To measure viral infectivity, the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the culture supernatant can be determined by enzyme-linked immunosorbent assay (ELISA), for example, on days 7 to 11 after infection. In addition, HBcAg staining can be assessed in an immunofluorescence assay.

[0215] In some embodiments, antibodies and antigen-binding fragments have high and high efficacy. The concentration of the antibody of the present disclosure required for neutralizing 50% of the hepatitis B virus (HBV) is, for example, about 10pg / ml or less. In certain embodiments, the concentration of the antibody of the present disclosure required for neutralizing 50% of HBV is about 5pg / ml, about 1pg / ml, or about 750ng / ml. In certain embodiments, the concentration of the antibody of the present disclosure required for neutralizing 50% of HBV is 500ng / ml or less, for example, 450ng / ml, 400ng / ml, 350ng / ml, 300ng / ml, 250ng / ml, 200ng / ml, 175ng / ml, 150ng / ml, 125ng / ml, 100ng / ml, 90ng / ml, 80ng / ml, 70ng / ml, 60ng / ml, or about 50ng / ml or less. This means that only low concentrations of antibodies are needed to neutralize 50% of HBV. Specificity and potency can be measured using standard assays known to those skilled in the art.

[0216] In some embodiments, anti-HBV antibodies can be used as a component of a combination therapy to prevent and / or treat hepatitis B or hepatitis B-related diseases.

[0217] In some embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure promote the removal of HBsAg and HBV. In particular, antibodies or antigen-binding fragments thereof according to the present disclosure can promote the removal of both subviral particles of hepatitis B virus and HBV. The removal of HBsAg or subviral particles can be assessed by measuring, for example, the level of HBsAg in a blood sample (e.g., from a hepatitis B patient). Similarly, the removal of HBV can be assessed by measuring, for example, the level of HBV in a blood sample (e.g., from a hepatitis B patient).

[0218] In the serum of patients infected with HBV, in addition to infectious particles (HBV), there is usually an excess (usually 1,000 to 100,000 times) of empty subviral particles (SVPs) in the form of relatively small spheres and variable length filaments consisting only of HBV envelope proteins (HBsAg). 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). This is also important in the context of the infectivity of serum containing HBV, because infectivity depends not only on the amount of virus, but also on the amount of SVPs (Bruns et al., 1998, supra).

[0219] In addition, excessive subviral particles can act as bait by absorbing neutralizing antibodies and thus delay the removal of infection. Generally, achieving hepatitis B surface antigen (HBsAg) loss is therefore considered to be the ideal endpoint of treatment and the closest result of curing chronic hepatitis B (CHB). Therefore, in some embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure (which promote the removal of HBsAg, and in particular the removal of subviral particles of hepatitis B virus and HBV) can improve the treatment of hepatitis B, especially in the case of chronic hepatitis B. Therefore, antibodies or antigen-binding fragments thereof according to the present disclosure can effectively neutralize HBV because less antibodies are absorbed by SVP acting as bait. In addition, in certain embodiments, antibodies or antigen-binding fragments thereof according to the present disclosure promote the removal of subviral particles of hepatitis B virus and reduce the infectivity of HBV in serum.

[0220] Based on the genome sequence, HBV is differentiated into many genotypes. To date, eight well-known genotypes (AH) of the HBV genome have been defined.

[0221] In addition, two new genotypes I and J have been identified (Sunbul M, World J Gastroenterol 2014, 20 (18): 5427-34). It is known that genotype affects disease progression, and the differences in response to antiviral therapy have been determined for each genotype. For example, genotype A has a tendency to chronicity, while genotype C frequently undergoes viral mutations. Both chronicity and mutation frequency are common in genotype D. In addition, HBV genotypes are distributed differently around the world (Sunbul, 2014, see above). In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure binds to at least 6, at least 8, or all 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of HBsAg genotypes A, B, C, D, E, F, G, H, I and J. Examples of different genotypes of HBsAg include the following: GenBank Accession No. J02203 (HBV-D, ayw3), GenBank Accession No. FJ899792.1 (HBV-D, adw2), GenBank Accession No. AM282986 (HBV-A), GenBank Accession No. D23678 (HBV-B1 Japan), GenBank Accession No. AB117758 (HBV-C1 Cambodia), GenBank Accession No. AB205192 (HBV-E Ghana), GenBank Accession No. X69798 (HBV-F4 Brazil), GenBank Accession No. AF160501 (HBV-G United States), GenBank Accession No. AY090454 (HBV-H Nicaragua), GenBank Accession No. AF241409 (HBV-I Vietnam) and GenBank Accession No. AB486012 (HBV-J Borneo). The amino acid sequences of the antigenic loop region of the S domain of HBsAg of different genotypes are shown in Table 2 (SEQ ID NO: 14-42).

[0222] Table 2. Antigenic loop sequences from various HBV genotypes.

[0223]

[0224]

[0225]

[0226] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the HBsAg mutants having mutations in the antigenic loop region: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121 S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141 E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R and HBsAg N146A. These mutants are naturally occurring mutants of the S domain based on HBsAg genotype D (SEQ ID NO: 43), Genbank Accession No. FJ899792 (wherein the mutated amino acid residue is indicated in the name).

[0227] MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSD GNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI (SEQ ID NO: 43) (the antigenic loop region, ie, amino acids 101-172, is underlined).

[0228] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to at least 12, at least 15, or all 18 of the HBsAg mutants having mutations in the antigenic loop regions: HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121 S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141 E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A.

[0229] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to an epitope comprising at least one amino acid, at least two amino acids, at least three amino acids, or at least four amino acids of the antigenic loop region of HBsAg, wherein the at least two amino acids, at least three amino acids, or at least four amino acids are selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. Note: The positions of the amino acids (e.g., 115-133, 120-133, 120-130) refer to the S domain of HBsAg as described above, which is present in all three HBV envelope proteins S-HBsAg, M-HBsAg, and L-HBsAg.

[0230] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to an epitope in the antigenic loop region of HBsAg, wherein the epitope is formed by one or more amino acids located at amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the HBsAg S domain.

[0231] As used herein in the context of epitopes, the term "formed by..." means that the epitope bound by the antibody or antigen-binding fragment thereof of the present disclosure may be linear (continuous) or conformational (discontinuous). A linear or sequential epitope is an epitope recognized by an antibody through its linear sequence or primary structure of amino acids. In contrast, a conformational epitope has a specific three-dimensional shape and protein structure. Therefore, if the epitope is a linear epitope and includes more than one amino acid located at amino acid positions 115-133 or amino acid positions 120-133 selected from the S domain of HBsAg, the amino acids included in the epitope may be located at adjacent positions of the primary structure (i.e., continuous amino acids in the amino acid sequence). In contrast, in the case of a conformational epitope (3D structure), the amino acid sequence generally forms a 3D structure as an epitope, and thus the amino acids forming the epitope (or the amino acids "included" by the epitope) may or may not be located at adjacent positions of the primary structure (i.e., may or may not be continuous amino acids in the amino acid sequence). In certain embodiments, the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind is formed only by amino acids selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. In specific embodiments, (additional) amino acids, which are located outside positions 115-133, positions 120-133, or positions 120-130, are not required to form the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind.

[0232] In certain embodiments, the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind in the antigenic loop region of HBsAg is formed by two or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. In certain embodiments, the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind in the antigenic loop region of HBsAg is formed by three or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. In some embodiments, the epitope to which the antibodies or antigen-binding fragments thereof of the present disclosure bind in the antigenic loop region of HBsAg is formed by four or more amino acids located at positions selected from amino acid positions 115-133, amino acid positions 120-133, or amino acid positions 120-130 of the S domain of HBsAg. Therefore, the antibodies or antigen-binding fragments thereof according to the present disclosure may bind to at least one amino acid, at least two amino acids, at least three amino acids, or at least four amino acids of the antigenic loop region of HBsAg, which 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. In a specific embodiment, the antibodies or antigen-binding fragments thereof according to the present disclosure bind to an epitope comprising at least two amino acids, at least three amino acids, or at least four amino acids of the antigenic loop region of HBsAg, wherein the at least two amino acids, at least three amino acids, or at least four amino acids are selected from amino acids 120-133 or amino acids 120-130 of the S domain of HBsAg, and wherein the at least two amino acids, at least three amino acids, or at least four amino acids are located in adjacent positions (i.e., consecutive amino acids in the amino acid sequence / primary structure).

[0233] In certain embodiments, the epitope to which the antibody or antigen-binding fragment thereof according to the present disclosure binds is a conformational epitope. Therefore, the antibody or antigen-binding fragment thereof according to the present disclosure may bind to an epitope comprising at least two amino acids, at least three amino acids, or at least four amino acids in the antigenic loop region of HBsAg, wherein at least two amino acids, at least three amino acids, or at least four amino acids are selected from amino acids 120-133 or amino acids 120-130 of the S domain of HBsAg, and wherein at least two amino acids, or at least three amino acids, or at least four amino acids are not located in adjacent positions (of the primary structure).

[0234] In certain specific embodiments, the antibodies of the present disclosure are bispecific antibodies having a first specificity for HBsAg and a second specificity for stimulating immune effector cells (e.g., by targeting T cell surface proteins, such as, for example, the extracellular portion of the CD3 protein). The second specificity may cause, for example, a cytotoxic effect or a vaccine effect.

[0235] b. Fc part

[0236] In some embodiments, the binding protein (e.g., an antibody or antigen-binding fragment thereof) includes an Fc portion. In certain embodiments, the Fc portion may be derived from a human source, such as from human IgG1, IgG2, IgG3, and / or IgG4. In specific embodiments, the antibody or antigen-binding fragment may include an Fc portion derived from human IgG1.

[0237] As used herein, the term "Fc moiety" refers to a sequence including or derived from a part of an immunoglobulin heavy chain, which starts from the hinge region upstream of the papain cleavage site (e.g., residue 216 in native IgG, considering that the first residue of the heavy chain constant region is 114) and ends at the C-terminal of an immunoglobulin heavy chain. Therefore, the Fc moiety can be a complete Fc moiety or a part thereof (e.g., domain). In certain embodiments, the complete Fc moiety includes a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid position 216-446). Additional lysine residues (K) are sometimes present in the extreme C-terminal of the Fc moiety, but are often cleaved from mature antibodies. The amino acid positions in the Fc moiety have been numbered according to the EU numbering system of Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983 and 1987).

[0238] The amino acid positions of the Fc portion may also be numbered according to the IMGT numbering system (including unique numbering of the C domain and exon numbering) and the Kabat numbering system.

[0239] In some embodiments, the Fc portion includes at least one of the following: a hinge (e.g., an upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion or fragment thereof. In some embodiments, the Fc portion includes at least a hinge domain, a CH2 domain, or a CH3 domain. In other embodiments, the Fc portion is a complete Fc portion. An amino acid sequence of an exemplary Fc portion of a human IgG1 isotype is provided in SEQ ID NO: 60. Relative to a naturally occurring Fc portion, the Fc portion may also include one or more amino acid insertions, deletions, or substitutions. For example, at least one of a hinge domain, a CH2 domain, or a CH3 domain, or a portion thereof may be missing. 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.

[0240] The Fc portion of the present disclosure can be modified so that it differs from the complete Fc portion of a naturally occurring immunoglobulin molecule in amino acid sequence while retaining (or enhancing) at least one desired function conferred by the naturally occurring Fc portion. Such functions include, for example, Fc receptor (FcR) binding, antibody half-life regulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Portions of the naturally occurring Fc portion that are involved in such functions have been described in the art.

[0241] For example, to activate the complement cascade, when an immunoglobulin molecule is attached to an antigen target, the C1q protein complex can bind to at least two IgG1 molecules or one IgM molecule (Ward ES and Ghetie V, Ther. Immunol. 1995, 277-94). The heavy chain region including amino acid residues 318 to 337 is involved in complement fixation (Burton DR, Mol. Immunol. 1985, 22: 161-206). Duncan AR and Winter G. (Nature 1988, 332: 738-40), using site-directed mutagenesis, reported that Glu318, Lys320 and Lys322 form a binding site for C1q. The role of Glu318, Lys320 and Lys322 residues in C1q binding was confirmed by the ability of short synthetic peptides including these residues to inhibit complement-mediated lysis.

[0242] For example, FcR binding can be mediated by the interaction of the Fc portion (of an antibody) with an Fc receptor (FcR), which is a specialized cell surface receptor on cells including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes and by antibody-dependent cell-mediated 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; the Fc receptors for IgG antibodies are called FcγRs, the Fc receptors for IgE antibodies are called FcεRs, the Fc receptors for IgA antibodies are called FcαRs, etc., and the neonatal Fc receptor is called FcRn. Fc receptor binding is described, for example, in Ravetch JV and Kinet JP, Annu. Rev. Immunol. 1991, 9:457-92; Capel PJ et al., Immunomethods 1994, 4:25-34; de Haas M et al., J Lab. Clin. Med. 1995, 126:330-41; and Gessner JE et al., Ann. Hematol. 1998, 76:231-48.

[0243] Cross-linking of the Fc domain of a natural IgG antibody (FcγR) to the receptor triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. The present invention contemplates an Fc portion that provides cross-linking of a receptor (e.g., FcγR). In humans, the following three classes of FcγRs have been characterized: (i) FcγRI (CD64), which binds to monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) FcγRII (CD32), which binds to complexed IgG with moderate to low affinity, is widely expressed, especially on leukocytes, is considered to be a central player in antibody-mediated immunity, and can be divided into FcγRIIA, FcγRIIB, and FcγRIIC, which Perform different functions in the immune system, but bind to IgG-Fc with similar low affinity, and the extracellular domains of these receptors are highly homologous; and (iii) FcγRIII (CD16), which binds to IgG with moderate to low affinity and has been found in two forms: FcγRIIIA, which has been found 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.

[0244] FcγRIIA is found on many cells (e.g., macrophages, monocytes, neutrophils) involved in killing and seems to be able to activate the killing process. FcγRIIB seems to play a role in the inhibition process and is found on B cells, macrophages and mast cells and eosinophils. It has been shown that 75% of all FcγRIIB are found in the liver (Ganesan LP et al., Journal of Immunology 2012, 189: 4981-8). FcγRIIB is expressed in large quantities in Kupffer cells on the liver sinusoidal endothelium known as LSEC and in the liver, and LSEC is the main site for clearing small immune complexes (Ganesan et al., 2012, see above).

[0245] In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein include an Fc portion, particularly an Fc region, for binding to FcγRIIb, such as, for example, an IgG-type antibody. In addition, the Fc portion can be engineered by introducing mutations S267E and L328F to enhance FcγRIIB binding, as described by Chu SY et al. (Molecular Immunology 2008, 45: 3926-33). Thus, the clearance of immune complexes can be enhanced (Chu S et al., Am J Respir Crit, AmericanThoracic Society International Conference Abstracts 2014). In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein include an engineered Fc portion with mutations S267E and L328F, particularly as described by Chu SY et al. (2008, supra).

[0246] On B cells, FcγRIIB appears to act to inhibit further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB is thought to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the b-form may help inhibit the activation of these cells by binding of IgE to its independent receptors.

[0247] Regarding FcγRI binding, modification in native IgG of at least one of E233-G236, P238, D265, N297, A327, and P329 reduced binding to FcγRI. IgG2 residues at positions 233-236 substituted to the corresponding positions in IgG1 and IgG4 reduced binding of IgG1 and IgG4 to FcγRI by 10 3The cytotoxicity of cytokines in human monocytes was folded and eliminated the response of human monocytes to antibody-sensitized erythrocytes (Armour KL et al., Eur. J. Immunol. 1999, 29: 2613-2624).

[0248] With respect to FcγRII binding, reduced binding to FcγRIIA was found, for example, for IgG mutations of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

[0249] With respect to FcγRIII binding, reduced binding to FcγRIIIA was found, for example, for mutations in at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376.

[0250] Mapping of the Fc receptor binding site on human IgG1, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields RL et al. (J. Biol. Chem. 2001, 276:6591-6604).

[0251] With regard to binding to FcγRII, two regions of native IgG Fc appear to be involved in the interaction between FcγRII and IgG, namely (i) the lower hinge site of IgG Fc, in particular amino acid residues L, L, G and G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, in particular the loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example in the region of P331 (Wines BD et al., J. Immunol. 2000, 164: 5313-8). In addition, FcγRI appears to bind to the same site on IgG Fc, while FcRn and protein A bind to different sites on IgG Fc, which appear to be located at the CH2-CH3 interface (Wines BD et al., 2000, supra).

[0252] Mutations that increase the binding affinity of the Fc portion of the present disclosure to (i.e., one or more) Fcγ receptors (e.g., compared to a reference Fc portion or antibody that does not include the mutation) are also contemplated. See, e.g., Delillo and Ravetch, Cell 2015, 161(5):1035-45 and Ahmed et al., J. Struc. Biol. 2016, 194(1):78, the Fc mutations and techniques of which are incorporated herein by reference. In any of the embodiments disclosed herein, the binding protein may include an Fc portion comprising a mutation selected from the group consisting of: G236A; S239D; A330L; and I332E; or a combination thereof; for example, S239D / I332E; S239D / A330L / I332E; G236A / S239D / I332E; G236A / A330L / I332E; and G236A / S239D / A330L / I332E.

[0253] In certain embodiments, the Fc portion may include at least a portion of or be composed of the Fc portion of the binding involved in binding to FcRn. In certain embodiments, the Fc portion includes one or more amino acid modifications that improve the binding affinity to FcRn, and in some embodiments, the in vivo half-life of the molecule including the Fc portion is extended (e.g., compared to a reference Fc portion or antibody that does not include modification). In certain embodiments, the Fc portion includes or is derived from IgG Fc, and the half-life extension mutation includes any one or more of the following: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; and E380A (EU numbering). In certain embodiments, the half-life extension mutation includes M428L / N434S. In certain embodiments, the half-life extension mutation includes M252Y / S254T / T256E. In certain embodiments, the half-life extension mutation includes T250Q / M428L. In certain embodiments, the half-life extension mutation includes P257I / Q311I. In certain embodiments, the half-life extension mutation includes P257I / N434H. In certain embodiments, the half-life extension mutation includes D376V / N434H. In certain embodiments, the half-life extension mutation includes T307A / E380A / N434A.

[0254] 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.

[0255] In certain embodiments, the binding protein comprises an Fc portion comprising the substitution mutations: G236A / A330L / I332E. In certain embodiments, the antibody or antigen binding fragment comprises an Fc portion comprising the substitution mutations: G236A / S239D / A330L / I332E.

[0256] Alternatively or additionally, the Fc portion of the binding protein of the present disclosure may include at least a portion required for protein A binding known in the art; and / or the Fc portion of the antibody of the present disclosure includes at least a portion of the Fc molecule required for protein G binding known in the art. In some embodiments, the retained function includes clearance of HBsAg and HBVg. Therefore, in certain embodiments, the Fc portion includes at least a portion required for FcγR binding known in the art. As described above, the Fc portion may therefore include at least: (i) the lower hinge site of native IgG Fc, in particular amino acid residues L, L, G and G (234-237, EU numbering); and (ii) an adjacent region of the CH2 domain of native IgG Fc, in particular loops and chains in the upper CH2 domain adjacent to the lower hinge region, for example in the P331 region, for example a region of at least 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids in the upper CH2 domain of native IgG Fc around P331, for example between amino acids 320 and 340 (EU numbering) of native IgG Fc.

[0257] In some embodiments, the binding proteins according to the present disclosure include an Fc region. As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by two or more Fc portions of an antibody heavy chain. For example, the Fc region can be a monomer or a "single-chain" Fc region (i.e., a scFc region). A single-chain Fc region includes an Fc portion connected within a single polypeptide chain (e.g., encoded in a single continuous nucleic acid sequence).

[0258] Exemplary scFc regions are disclosed in WO 2008 / 143954A2, and are incorporated herein by reference. The Fc region may be or include a dimeric Fc region. "Dimeric Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two independent immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc portions (e.g., the Fc region of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc portions (e.g., one Fc monomer of the dimeric Fc region includes at least one amino acid modification (e.g., substitution, deletion, insertion or chemical modification) not present in another Fc monomer, or one Fc monomer may be truncated compared to the other).

[0259] The Fc part of the present disclosure may include Fc sequences or regions of the same or different classes and / or subclasses. For example, the Fc part may be derived from an immunoglobulin (e.g., human immunoglobulin) of an IgG1, IgG2, IgG3 or IgG4 subclass or from any combination thereof. In certain embodiments, the Fc part of the Fc region belongs to the same class and subclass. However, the Fc region (or one or more Fc parts of the Fc region) may also be chimeric, whereby the chimeric Fc region may include Fc parts derived from different immunoglobulin classes and / or subclasses.

[0260] For example, at least two of the Fc parts of a dimer or single-chain Fc region may be from different immunoglobulin classes and / or subclasses. In certain embodiments, the dimer Fc region may include sequences from two or more different isotypes or subclasses; for example, SEED bodies ("chain exchange engineered domains") (see Davis et al., Protein Eng. Des. Sel. 2010, 23 (4): 195).

[0261] Additionally or alternatively, the chimeric Fc region may include one or more chimeric Fc portions. For example, a chimeric Fc region or portion may include one or more portions of an immunoglobulin derived from a first subclass (e.g., IgG1, IgG2, or IgG3 subclass), while the remainder of the Fc region or portion belongs to a different subclass. For example, the Fc region or portion of an Fc polypeptide may include a CH2 and / or CH3 domain derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG4 subclass) and a hinge region of an immunoglobulin from a second subclass (e.g., IgG3 subclass). For example, the Fc region or portion may include a hinge and / or CH2 domain derived from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and a CH3 domain from an immunoglobulin of a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). For example, a chimeric Fc region may include an Fc portion (e.g., a complete Fc portion) of an immunoglobulin from a first subclass (e.g., an IgG4 subclass) and an Fc portion of an immunoglobulin from a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, an Fc region or portion may include a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, an Fc region or portion may include a CH1 domain and a CH2 domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. For example, an Fc region or portion may include a portion of a CH2 domain from a specific antibody subclass, such as EU positions 292-340 of a CH2 domain. For example, an Fc region or portion may include amino acid positions 292-340 of a CH2 derived from an IgG4 portion and the remainder of a CH2 derived from an IgG1 portion (alternatively, 292-340 of CH2 may be derived from an IgG1 portion and the remainder of CH2 is derived from an IgG4 portion).

[0262] In addition, the Fc region or portion may (additionally or alternatively) for example include a chimeric hinge region. For example, a chimeric hinge may be, for example, partially derived from an IgG1, IgG2 or IgG4 molecule (e.g., upper and lower middle hinge sequences) and partially derived from an IgG3 molecule (e.g., middle hinge sequence). In another example, the Fc region or portion may include a chimeric hinge partially derived from an IgG1 molecule and partially derived from an IgG4 molecule. In another example, a chimeric hinge may include upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge may be obtained, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the middle hinge domain of the IgG4 hinge region. In some other embodiments, the chimeric hinge may include amino acids at EU positions 233-236 from an IgG2 antibody and / or a Ser228Pro mutation, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g., a chimeric hinge of sequence ESKYGPPCPPCPAPPVAGP (SEQ ID NO: 61)). Additional chimeric hinges that can be used in the Fc portion of an antibody according to the present disclosure are described in U.S. Patent Application Publication No. US2005 / 0163783A1.

[0263] In some embodiments, the Fc portion or Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., derived from an Fc region or Fc portion from 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 murine amino acids may be present in the Fc portion or Fc region.

[0264] c.HBC34 antibody

[0265] 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 antigenic loop of HBsAg with high affinity (in the pM range), recognizes all 10 HBV genotypes and 18 mutants, and binds to spherical SVPs at a low stoichiometric ratio. The activity of HBC34 as determined by immunoassay diagnostics is 5000 IU / mg. In comparison, the activity of HBIG is about 1 IU / mg.

[0266] As referred to herein, unless otherwise specified, the terms "HBC34 antibody" and "HBC antibody" may include wild-type HBC34 antibody or engineered variants thereof (eg, HBC34 and HBC34 variants described in Table 3).

[0267] Table 3 shows the amino acid sequences of the CDRs, the heavy chain variable region (V H ) and the light chain variable region (V L ) and their 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.

[0268] Table 3. Sequence of the HBC34 antibody.

[0269]

[0270]

[0271]

[0272] In certain embodiments, the anti-HBV antibody has one or more amino acid sequences as set forth in Table 3. In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure comprises a CDR sequence, a V H Sequence, V L The present invention relates to an antibody or antigen-binding fragment of an antigen-binding fragment having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a CDR, VL, or LC sequence. In any of the presently disclosed embodiments, the antibody or antigen-binding fragment may include a CDR, VL, or LC sequence as set forth in Table 3. H 、V L , HC and / or LC sequences. Exemplary methods for synthesizing antibodies having the sequences shown in Table 3 and experimental data demonstrating binding and neutralization by AB01 are described in International Application Publication No. WO 2020 / 132091A2, which are incorporated herein by reference.

[0273] In some embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise: (i) CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 or 46 and 47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 or 50 and 51 or 52, respectively.

[0274] Thus, in some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 44, 45, and 47, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 44, 46, and 47, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 48, 49, and 52, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 are according to SEQ ID NOs: 48, 50, and 52, respectively.

[0275] It should be understood that the antibodies or antigen-binding fragments of the present disclosure may include any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 44-50 and 52.

[0276] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure include:

[0277] The CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

[0278] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure include: (a) a light chain variable domain (V L ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO: 55; and (b) a heavy chain variable domain (V H ), which comprises or consists of an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:53.

[0279] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure comprise (a) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:59, and (b) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:57.

[0280] d. Pharmaceutical Compositions

[0281] In some embodiments, the antibody or antigen-binding fragment thereof of the combination therapy is provided as a pharmaceutical composition comprising an anti-HBV antibody and an optional pharmaceutically acceptable carrier. In some embodiments, the composition may comprise an anti-HBV antibody, wherein the antibody may account for at least 50% by weight of the total protein in the composition (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more). In such a composition, the antibody may be in a purified form.

[0282] Pharmaceutical compositions of anti-HBV antibodies may include an antimicrobial agent, particularly when packaged in multidose form. These pharmaceutical compositions may include a detergent, e.g., Tween (polysorbate), such as Tween 80. When present, the detergent is typically present at low levels, e.g., less than 0.01%. The composition may also include a sodium salt (e.g., sodium chloride) to provide tonicity. For example, in some embodiments, the pharmaceutical composition includes NaCl at a concentration of 10 ± 2 mg / ml.

[0283] In addition, the pharmaceutical composition may comprise, for example, a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) at about 15 mg / ml to 30 mg / ml (e.g., 25 mg / ml), particularly where they are to be lyophilized or where they comprise materials that have been reconstituted from lyophilized materials. The pH of the lyophilized composition may be adjusted to between 5 and 8 or between 5.5 and 7 or to about 6.1 prior to lyophilization.

[0284] The antibody compositions of the present disclosure may also include one or more immunomodulators. In some embodiments, one or more of the immunomodulators include an adjuvant.

[0285] The method for preparing a pharmaceutical composition of an anti-HBV antibody may include the following steps: (i) preparing an antibody; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

[0286] In some embodiments, the pharmaceutical composition comprising the anti-HBV antibody described herein (e.g., AB01) comprises an antibody at a dose of 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg. In some embodiments, the pharmaceutical composition comprising the anti-HBV antibody described herein (e.g., AB01) comprises an antibody at a dose of 100 mg to 300 mg. In some embodiments, the pharmaceutical composition comprising the anti-HBV antibody described herein (e.g., AB01) comprises an antibody at a dose of 100 mg to 200 mg.

[0287] In some embodiments, a pharmaceutical composition comprising an anti-HBV antibody described herein (eg, AB01) comprises a dose of 200 mg of the antibody. IV. Methods of Treatment Using Combination Therapies

[0288] In some embodiments, the present disclosure provides methods for treating HBV infection or HBV-related disease in a subject.

[0289] In some embodiments, a method for treating HBV infection or HBV-related disease in a subject is provided, wherein the method comprises: administering to the subject siRNA and antibodies as described herein. In some embodiments, SIRNA01 and ABO1 are administered to the subject.

[0290] In some embodiments, a method for treating HBV infection or HBV-related diseases in a subject is provided, wherein the method comprises: administering to the subject siRNA and antibodies as described herein, and also administering to the subject a nucleoside / nucleotide reverse transcriptase inhibitor. As used herein, "nucleoside / nucleotide reverse transcriptase inhibitor" or "nucleoside (acid) reverse transcriptase inhibitor" (NRTI) refers to a DNA replication inhibitor 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), clavudine, ritonavir, fovir dipivoxil, lobucavir, famciclovir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-α, ganciclovir, besifovir (ANA-380 / LB-80380) and tenofovir-exaliades (TLX / CMX157). In some embodiments, NRTI is tenofovir. In some embodiments, NRTI is tenofovir disoproxil fumarate (TDF). In some embodiments, NRTI is an antagonist is tenofovir 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 these methods, the subject is HBeAg negative; has an HBV DNA level of ≤2000 IU / mL before treatment; has an HBV DNA level of <2000 IU / mL before treatment; and / or is non-cirrhotic, and / or alanine aminotransferase (ALT) level ≤ upper limit of normal (ULN), and / or alanine aminotransferase (ALT) level < upper limit of normal (ULN). In some embodiments, the ALT ULN value is 34 IU / mL for women and 43 IU / mL for men. In some embodiments of these methods, the subject has not previously been administered one or more elements of the combination therapy (e.g., the subject has not previously been administered NRTI; the subject has not previously been administered anti-HIV antibodies; and / or the subject has not previously been administered siRNA targeting HBV mRNA). In some embodiments of these methods, the subject has not been previously administered, has not previously received, or has not received an NRTI within 24 weeks prior to treatment with the combination therapy.

[0291] In some embodiments, a method for treating HBV infection or HBV-related disease in a subject is provided, wherein the method comprises: administering to the subject siRNA and antibodies as described herein, and also administering to the subject a nucleoside / nucleotide reverse transcriptase inhibitor and interferon alpha (e.g., PEG-IFNα). In some embodiments of these methods, the subject is HBeAg negative or HBeAg positive; has an HBV DNA level of >2000 IU / mL before treatment; and / or an alanine aminotransferase (ALT) level>upper limit of normal (ULN) and ≤5 times the ULN. In some embodiments of these methods, the subject has not previously been administered one or more elements of the combination therapy (e.g., the subject has not previously been administered NRTI; the subject has not previously been administered interferon alpha; the subject has not previously been administered anti-HIV antibodies; and / or the subject has not previously been administered siRNA targeting HBV mRNA). In some embodiments, the ALT ULN value is 34 IU / mL for women and 43 IU / mL for men.

[0292] As used herein, "subject" is an animal, such as a mammal, including any mammal that can be infected with HBV, for example, a primate (such as a human, a non-human primate, such as a monkey or a chimpanzee), or an animal, HBV-AAV mouse model (see, for example, Yang et al., Cell and Mol Immunol 2014, 11: 71) or HBV 1.3xfs transgenic mouse model (Guidotti et al., J. Virol. 1995, 69: 6158) that is considered to be an acceptable HBV infection clinical model. In some embodiments, the subject suffers from hepatitis B virus (HBV) infection. In some other embodiments, the subject suffers from hepatitis B virus (HBV) infection and hepatitis D virus (HDV) infection at the same time. In some other embodiments, the subject is a human, such as a human with HBV infection, especially chronic hepatitis B virus (CHBV) infection.

[0293] As used herein, the term "treat" refers to a beneficial or desired result, including but not limited to alleviating or ameliorating one or more signs or symptoms associated with undesirable HBV gene expression or HBV replication, such as the presence of serum or liver HBV cccDNA, the presence of serum HBV DNA, presence of serum or liver HBV antigens, e.g., HBsAg or HBeAg, elevated ALT, elevated AST (normal range is generally considered to be about 10 U / L to 34 U / L), absence or low levels of anti-HBV antibodies; liver damage; cirrhosis; hepatitis D; acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, low-grade fever; myalgia; easy fatigability; taste acuity and smell disturbances (aversion to food and cigarettes); or right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; lethargy; disturbed sleep pattern; confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (mildly enlarged, soft liver); splenomegaly; palmar erythema; spider angiomas; muscle atrophy; spider angiomas; vasculitis; varicose bleeding; peripheral edema; male Gynaecomastia; testicular atrophy; abdominal collateral veins (capitulum medullaris); ALT levels higher than AST levels; elevated gamma-glutamyl transpeptidase (GGT) (normal range is generally considered to be about 8 U / L to 65 U / L) and alkaline phosphatase (ALP) levels (normal range is generally considered to be about 44 IU / L to 147 IU / L (international units per liter), not more than 3 times the ULN); slightly low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; elevated erythrocyte sedimentation rate (ESR); shortened red blood cell survival; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); presence of serum or liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HB), hepatitis B e antibody (anti-HBe), or HBV DNA; elevated bilirubin levels; hyperglobulinemia; the presence of tissue nonspecific antibodies, such as anti-smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10% to 20%); the presence of tissue specific antibodies, such as antibodies to the thyroid gland (10% to 20%); elevated levels of rheumatoid factor (RF); low platelet and leukocyte counts; lobular, with degenerative and regenerative hepatocyte changes, and with inflammation; and significant centrilobular necrosis, whether detectable or undetectable. For example, when an individual with one or more risk factors for liver fibrosis (e.g., chronic hepatitis B infection) fails to develop liver fibrosis or develops less severe liver fibrosis relative to a population with the same risk factors and not receiving treatment as described herein, the likelihood of developing, for example, liver fibrosis is reduced. "Treatment" can also mean prolonging survival compared to the expected survival without treatment.

[0294] As used herein, the term "prevent" refers to the failure to develop a disease, disorder, or condition, or to reduce the development of signs or symptoms associated with such a disease, disorder, or condition (e.g., by reducing a clinically relevant amount), or to delay the manifestation of signs or delayed symptoms (e.g., by days, weeks, months, or years). Prevention may require administration of more than one dose.

[0295] Doses are often expressed in relation to body weight. Thus, a dose expressed as [g, mg or other unit] / kg (or g, mg, etc.) usually means [g, mg or other unit] "per kg (or g, mg, etc.) body weight", even if the term "body weight" is not explicitly mentioned.

[0296] In some embodiments, the treatment of HBV infection leads to a "functional cure" for hepatitis B. As used herein, functional cure is understood to be the removal of circulating HBsAg, and is a transformation that may be accompanied by a state in which HBsAg antibodies become detectable using clinically relevant assays. For example, detectable antibodies may include a signal higher than 10mIU / ml as measured by chemiluminescent microparticle immunoassay (CMIA) or any other immunoassay. Functional cure does not require the removal of all replicated forms of HBV (e.g., cccDNA from the liver). Anti-HB seroconversion occurs spontaneously in approximately 0.2% to 1% of chronically infected patients each year. However, even after anti-HB seroconversion, low levels of HBV are often observed to persist for decades, indicating that functional cure rather than complete cure has occurred. Without being constrained by a specific mechanism, the immune system may be able to limit HBV under conditions where functional cure has been achieved. Functional cure allows any treatment for HBV infection to be stopped. However, it should be understood that "functional cure" of HBV infection may not be sufficient to prevent or treat diseases or conditions caused by HBV infection, such as liver fibrosis, HCC or cirrhosis. In some specific embodiments, "functional cure" may refer to a sustained decrease in serum HBsAg, such as <1 IU / mL, for at least 3 months, at least 6 months, or at least 1 year after the start of a treatment regimen or completion of a treatment regimen.

[0297] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" is a disease or condition caused by or associated with HBV infection or replication. The term "HBV-related disease" includes diseases, disorders, or conditions that would benefit from a reduction in HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, hepatitis D, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0298] In some embodiments, the HBV-related disease is chronic hepatitis B.

[0299] Chronic hepatitis B is defined by one of the following criteria: (1) two positive serum HBsAg, HBV DNA, or HBeAg tests at least 6 months apart (any combination of these tests performed 6 months apart is acceptable); or (2) negative immunoglobulin M (IgM) antibodies against HBV core antigen (IgM anti-HBc) and a positive result on one of the following tests: HBsAg, HBeAg, or HBV DNA. Chronic HBV generally involves inflammation of the liver that persists for more than six months. Subjects with chronic HBV are HBsAg positive and have high viremia (≥10 4 HBV-DNA copies / mL blood) or low viremia (<10 3 HBV-DNA copies / ml blood). In certain embodiments, the subject has been infected with HBV for at least five years. In certain embodiments, the subject has been infected with HBV for at least ten years. In certain embodiments, the subject is infected with HBV at birth. Subjects with chronic hepatitis B disease may have immune tolerance, or suffer from inactive chronic infection in the absence of any evidence of active disease, and they are also asymptomatic. Chronic active hepatitis patients (especially during the replication state) may have symptoms similar to acute hepatitis. Subjects with chronic hepatitis B disease may have active chronic infection with necrotic inflammatory liver disease, increased liver cell renewal rate in the absence of detectable necrotic inflammation, or suffer from inactive chronic infection in the absence of any evidence of active disease, and they are also asymptomatic. The persistence of HBV infection in chronic HBV subjects is the result of cccHBV DNA. In some embodiments, subjects with chronic HBV are HBeAg positive. In some other embodiments, subjects with chronic HBV are HBeAg negative. Subjects with chronic HBV have serum HBV DNA levels less than 105 and persistent elevations of transaminases (eg, ALT, AST, and gamma-glutamyl transferase). Subjects with chronic HBV may have a liver biopsy score (eg, necroinflammatory score) less than 4.

[0300] In some embodiments, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or HDV (HDV) is a human pathogen. However, the virus is defective and relies on mandatory helper functions provided by HBV for transmission; in fact, HDV requires an associated or pre-existing HBV infection to be infectious and reproduce, in particular a viral envelope containing the hepatitis B surface antigen. HDV can cause severe acute and chronic liver diseases associated with HBV. Hepatitis D infection or hepatitis D is very prevalent in several African countries, the Amazon River Basin and the Middle East, while the prevalence is very low in industrialized countries outside the Mediterranean region.

[0301] Transmission of HDV can occur either via co-infection with HBV (coinfection) or superimposed on chronic hepatitis B or hepatitis B carrier status (superinfection). Both dual infection and coinfection with HDV generally result in more severe complications than infection with HBV alone. These complications include a greater likelihood of developing liver failure in acute infection and 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 of all hepatitis infections at 20%.

[0302] In some embodiments, the HBV-related disease is acute hepatitis B. Acute hepatitis B includes inflammation of the liver that lasts for less than 6 months. Typical symptoms of acute hepatitis B are fatigue, anorexia, nausea and vomiting. Very high transaminase values ​​(>1000U / L) and hyperbilirubinemia are often observed.

[0303] Severe cases of acute hepatitis B may rapidly progress to acute liver failure, which is marked by poor 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 pre-existing liver disease. Acute hepatitis B may evolve into chronic hepatitis B.

[0304] In some embodiments, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B have symptoms of acute hepatitis and additional symptoms of confusion or coma (due to the liver's failure to detoxify chemicals) and bruising or bleeding (due to lack of clotting factors).

[0305] Subjects with HBV infection (e.g., chronic HBV) may develop liver fibrosis. Thus, in some embodiments, the HBV-related disease is liver fibrosis. Liver fibrosis or cirrhosis is histologically defined as a diffuse liver process characterized by fibrosis (excess fibrous connective tissue) and transformation of normal liver architecture into structurally abnormal nodules.

[0306] Subjects with HBV infection (e.g., chronic HBV) may develop end-stage liver disease. Therefore, in some embodiments, HBV-related diseases are end-stage liver disease. For example, liver fibrosis may develop to the extent that the body may no longer be able to compensate for, for example, reduced liver function due to liver fibrosis (i.e., decompensated liver), and lead to, for example, psychiatric and neurological symptoms and liver failure.

[0307] Subjects with HBV infection (e.g., chronic HBV) may develop hepatocellular carcinoma (HCC), also known as malignant liver cancer. Therefore, in some embodiments, the HBV-related disease is HCC. HCC usually occurs in subjects with CHB and may be fibrolamellar cells, pseudoglandular cells (adenoid cells), pleomorphic cells (giant cells), or clear cells.

[0308] An "HDV-associated disorder" or "hepatitis D virus-associated disorder" is a disease or disorder associated with HDV expression. Exemplary HDV-associated disorders include hepatitis B virus infection, acute hepatitis B, acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0309] As used herein, a "therapeutically effective amount" is intended to include an amount of siRNA, anti-HBV antibodies, or other active agents (e.g., PEG-IFNα, tenofovir) that is sufficient to affect the treatment of the disease (e.g., by reducing or maintaining one or more symptoms of an existing disease or disease) when administered to a patient to treat a subject with HBV infection or HBV-related disease. A "therapeutically effective amount" may vary according to the following: active agent, mode of administration, disease and its severity and medical history, age, weight, family history, genetic composition, stage of pathological process mediated by HBV gene expression, type of previous or concomitant treatment (if any), and other individual characteristics of the patient to be treated. A therapeutically effective amount may require administration of more than one dose.

[0310] "Therapeutically effective amount" also includes the amount of siRNA, anti-HBV antibody or other active agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutic agent (e.g., siRNA, anti-HBV antibody) used in the methods of the present disclosure can be administered in sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0311] As used herein, the term "sample" includes a collection of similar fluids, cells or tissues separated from a subject, and fluids, cells and tissues present in a subject. The example of a biological fluid includes blood, serum and serosal fluids, plasma, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs or local areas. For example, a sample may be derived from a specific organ, a part of an organ or a fluid or cell in these organs. In certain embodiments, a sample may be derived from liver (for example, whole liver or some parts of liver or some types of cells in liver, such as, for example, hepatocytes). In certain embodiments, "sample derived from subject" refers to blood or plasma or serum obtained from the blood extracted from a subject. In other embodiments, "sample derived from subject" refers to liver tissue (or its subcomponents) or blood tissue (or its subcomponents, for example, serum) derived from a subject.

[0312] 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 4 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 anti-HBV antibody is administered to the subject for a period of 44 weeks. In some embodiments, the anti-HBV antibody is administered to the subject for a period of 48 weeks. In some embodiments, the anti-HBV antibody is administered to the subject for a period of at least 44 weeks, 44 weeks, 44 weeks or longer, at least 48 weeks, 48 ​​weeks, or 48 weeks or longer. In some embodiments, the anti-HBV antibody is administered to the subject for a period of 20 weeks, 40 weeks, 44 weeks, or 48 weeks. In some embodiments, the anti-HBV antibody is administered to the subject 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.

[0313] In some embodiments of the methods described herein, siRNA is administered subcutaneously. In some embodiments, siRNA is administered every 4 weeks. In some embodiments, siRNA is administered to a subject for a period of 20 weeks, 44 weeks, or 48 weeks. In some embodiments, siRNA is administered to a subject for a period of 44 weeks. In some embodiments, siRNA is administered to a subject for a period of 48 weeks. In some embodiments, siRNA is administered to a subject for a period of at least 44 weeks, 44 weeks, 44 weeks, or longer, at least 48 weeks, 48 ​​weeks, or 48 weeks or longer. In some embodiments, siRNA is administered at a dosage of 20 mg to 900 mg. In some embodiments, siRNA is administered at a dosage of 100 mg to 300 mg. In some embodiments, siRNA is administered at a dosage of 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 450 mg. In some embodiments, siRNA is administered at a dosage of 200 mg.

[0314] 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, interferon-α is administered to a subject for a period of 44 weeks. In some embodiments, interferon-α is administered to a subject for a period of 48 weeks. In some embodiments, interferon-α is administered to a subject for a period of at least 44 weeks, 44 weeks, 44 weeks or longer, at least 48 weeks, 48 ​​weeks or 48 weeks or longer. In some embodiments, interferon-α is administered at a dose of at least 120mcg. In some embodiments, interferon-α is administered at a dose of 120mcg to 180mcg. In some embodiments, interferon-α is administered at a dose of 180mcg.

[0315] In some embodiments of the methods described herein, the NRTI is administered orally. In some embodiments, the NRTI is administered daily. In some embodiments, the NRTI is administered to the subject for a period of 44 weeks. In some embodiments, the NRTI is administered to the subject for a period of 48 weeks. In some embodiments, the NRTI is administered to the subject for a period of at least 44 weeks, 44 weeks, 44 weeks or longer, at least 48 weeks, 48 ​​weeks or 48 weeks or longer. 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.

[0316] In some embodiments of the methods described herein, administration of the siRNA and the anti-HBV antibody to the subject begins on the same day.

[0317] In some embodiments of the methods described herein, the siRNA and the anti-HBV antibody are administered to the subject starting on the same day for a period of 20 weeks, 44 weeks, or 48 weeks.

[0318] In some embodiments of the methods described herein, the subject is HBsAg negative after treatment continues for example 24 weeks, 48 ​​weeks or later. In some embodiments of the methods described herein, the subject achieves functional cure, i.e., after treatment continues for example 24 weeks, 48 ​​weeks or later, there is undetectable HBsAg (being HBsAg negative) and continues to suppress HBV DNA (HBV DNA is not detected) . In some embodiments of the methods described herein, the subject has undetectable HBsAg and / or achieves anti-HBe seroconversion after treatment continues for example 24 weeks, 48 ​​weeks or later. In some embodiments of the methods described herein, the subject has normal alanine aminotransferase (ALT) levels after treatment continues for example 24 weeks, 48 ​​weeks or later.

[0319] The present disclosure also provides antibodies, siRNA, NRTI and / or interferon described herein, and pharmaceutical compositions thereof, for use in the above methods. Also provided is the use of antibodies, siRNA, NRTI and / or interferon described herein in the manufacture of a medicament for use in the above methods.

[0320] V. Kits for Combination Therapy

[0321] Also provided herein are kits including therapy components for treating HBV infection or HBV-related diseases. These kits may include siRNA (e.g., SIRNA01), anti-HBV antibodies (e.g., AB01) and NRTI (e.g., tenofovir disoproxil fumarate, tenofovir disoproxil). These kits may include siRNA (e.g., SIRNA01), anti-HBV antibodies (e.g., AB01), PEG-IFNα and NRTI (e.g., tenofovir disoproxil fumarate, tenofovir disoproxil). The kit may additionally include instructions for preparing and / or administering components of HBV combination therapy.

[0322] VI. Example Implementation

[0323] In some embodiments, the present disclosure provides:

[0324] 1. A method for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof, the method comprising: administering to the subject:

[0325] (a) Anti-HBV antibodies;

[0326] (b) siRNA targeting HBV mRNA; and

[0327] (c) Nucleoside reverse transcriptase inhibitors (NRTIs).

[0328] 2. The method of embodiment 1, wherein the subject is HBeAg negative.

[0329] 3. The method according to embodiment 1 or 2, wherein the subject has an HBV DNA level of ≤2000 IU / mL before treatment.

[0330] 4. The method according to embodiment 1 or 2, wherein the subject has an HBV DNA level of <2000 IU / mL before treatment.

[0331] 5. The method of any one of embodiments 1 to 4, wherein the subject is non-cirrhotic and / or has an alanine aminotransferase (ALT) level ≤ upper limit of normal (ULN).

[0332] 6. A method according to any one of embodiments 1 to 4, wherein the subject is non-cirrhotic and / or has an alanine aminotransferase (ALT) level < the upper limit of normal (ULN).

[0333] 7. The method of any one of embodiments 1 to 6, wherein the subject has not been previously administered an NRTI.

[0334] 8. The method of any one of embodiments 1 to 6, wherein the subject has not received an NRTI within 24 weeks prior to treatment.

[0335] 9. The method according to any one of embodiments 1 to 8, wherein the subject has not been previously administered anti-HIV antibodies.

[0336] 10. The method of any one of embodiments 1 to 9, wherein the subject has not been previously administered siRNA targeting HBV mRNA.

[0337] 11. A method for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof, the method comprising: administering to the subject:

[0338] (a) Anti-HBV antibodies;

[0339] (b) siRNA targeting HBV mRNA;

[0340] (c) interferon-α; and

[0341] (d) Nucleoside reverse transcriptase inhibitors (NRTIs).

[0342] 12. The method of embodiment 11, wherein the subject is HBeAg negative.

[0343] 13. The method of embodiment 11, wherein the subject is HBeAg positive.

[0344] 14. The method of any one of embodiments 11 to 13, wherein the subject has an HBV DNA level of >2000 IU / mL before treatment.

[0345] 15. The method of any one of embodiments 11 to 14, wherein the subject has an alanine aminotransferase (ALT) level > the upper limit of normal (ULN) and ≤ 5 times the ULN.

[0346] 16. The method of any one of embodiments 11 to 15, wherein the subject has not been previously administered an NRTI.

[0347] 17. The method of any one of embodiments 11 to 16, wherein the subject has not been previously administered anti-HIV antibodies.

[0348] 18. The method of any one of embodiments 11 to 17, wherein the subject has not been previously administered siRNA targeting HBV mRNA.

[0349] 19. The method of any one of embodiments 11 to 18, wherein the subject has not been previously administered interferon-α.

[0350] 20. The method of any one of embodiments 1 to 19, wherein the subject is HBsAg positive prior to treatment.

[0351] 21. The method of any one of embodiments 11 to 20, wherein the subject has an HBsAg level of >10 IU / mL prior to treatment.

[0352] 22. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody recognizes HBV genotypes A, B, C, D, E, F, G, H, I and J.

[0353] 23. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is a human antibody.

[0354] 24. The method according to any one of the preceding embodiments, wherein the antibody is HBC34 or a non-natural variant of HBC34.

[0355] 25. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0356] (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NO: 44, 45 or 46 and 47, respectively; and

[0357] (ii) the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NO: 48, 49 or 50 and 52, respectively.

[0358] 26. The method of embodiment 25, wherein the anti-HBV antibody comprises:

[0359] (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and

[0360] (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

[0361] 27. The method of embodiment 25, wherein the anti-HBV antibody comprises:

[0362] (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 46 and 47, respectively; and

[0363] (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 50 and 52, respectively.

[0364] 28. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0365] (a) a light chain variable domain (V L) having at least 90%, at least 95% or 100% identity to the amino acid sequence according to SEQ ID NO: 55 L );and

[0366] (b) a heavy chain variable domain (V) having at least 90%, at least 95% or 100% identity to the amino acid sequence according to SEQ ID NO: 53 H ).

[0367] 29. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0368] (a) a light chain variable domain (V L ) amino acid sequence; and

[0369] (b) the heavy chain variable domain (V H ) amino acid sequence.

[0370] 30. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0371] (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

[0372] (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:57.

[0373] 31. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody comprises:

[0374] (a) a light chain amino acid sequence according to SEQ ID NO: 59, and

[0375] (b) The heavy chain amino acid sequence according to SEQ ID NO:57.

[0376] 32. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is a monoclonal antibody.

[0377] 33. A method according to any of the preceding embodiments, wherein the anti-HBV antibody is a bispecific antibody having a first specificity for HBsAg and a second specificity for stimulating immune effectors.

[0378] 34. A method according to embodiment 33, wherein the second specificity stimulates cytotoxicity or a vaccine effect.

[0379] 35. The method of any one of the preceding embodiments, wherein the subject is a human and a therapeutically effective amount of the anti-HBV antibody is administered; wherein the therapeutically effective amount is about 3 mg / kg to about 30 mg / kg.

[0380] 36. The method according to any one of the preceding embodiments, wherein the siRNA inhibits the expression of HBV transcripts encoding HBsAg protein, HBcAg protein and HBx protein or HBV DNA polymerase protein.

[0381] 37. A method according to any one of the preceding embodiments, wherein the siRNA comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from nucleotides 1579-1597 of SEQ ID NO: 1 by no more than 3 nucleotides, wherein T is replaced by U.

[0382] 38. The method according to any one of the preceding embodiments, wherein the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotides 1579-1597 of SEQ ID NO: 1, wherein T is replaced by U.

[0383] 39. A method according to any of the preceding embodiments, wherein the siRNA binds to at least 15 consecutive nucleotides of a target encoded by: the P gene, nucleotides 2309-3182 and 1-1625 of NC_003977.2; the S gene (encoding L, M and S proteins), nucleotides 2850-3182 and 1-837 of NC_003977.2; HBx, nucleotides 1376-1840 of NC_003977.2; or the C gene, nucleotides 1816-2454 of NC_003977.2.

[0384] 40. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA comprises at least 15 consecutive nucleotides of the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0385] 41. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA comprises at least 19 consecutive nucleotides of the nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0386] 42. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA comprises a nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0387] 43. The method according to any one of the preceding embodiments, wherein the antisense strand of the siRNA consists of a nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

[0388] 44. The method according to any one of the preceding embodiments, wherein the sense strand of the siRNA comprises a nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

[0389] 45. The method according to any one of the preceding embodiments, wherein the sense strand of the siRNA consists of a nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

[0390] 46. ​​The method according to any one of the preceding embodiments, wherein at least one strand of the siRNA comprises a 3' overhang of at least 1 nucleotide.

[0391] 47. The method according to any one of the preceding embodiments, wherein at least one strand of the siRNA comprises a 3' overhang of at least 2 nucleotides.

[0392] 48. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs in length.

[0393] 49. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 17 to 23 nucleotide pairs in length.

[0394] 50. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 17 to 25 nucleotide pairs in length.

[0395] 51. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 23 to 27 nucleotide pairs in length.

[0396] 52. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 19 to 21 nucleotide pairs in length.

[0397] 53. The method according to any one of the preceding embodiments, wherein the double-stranded region of the siRNA is 21 to 23 nucleotide pairs in length.

[0398] 54. The method of any one of embodiments 1 to 40, wherein each strand of the RNAi agent has 15 to 30 nucleotides.

[0399] 55. The method of any one of the preceding embodiments, wherein each strand of the RNAi agent has 19 to 30 nucleotides.

[0400] 56. A method according to any of the preceding embodiments, wherein substantially all nucleotides of the sense strand of the siRNA and substantially all nucleotides of the antisense strand of the siRNA are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end.

[0401] 57. A 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.

[0402] 58. The method according to embodiment 56 or 57, wherein the ligand is

[0403]

[0404] 59. The method of embodiment 58, wherein the siRNA is conjugated to the ligand as shown in the following structure:

[0405]

[0406] Wherein X is O or S.

[0407] 60. The method according to embodiment 59, wherein X is O.

[0408] 61. A method according to any one of the preceding embodiments, 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 conformationally restricted nucleotide, a constrained 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 phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a thiophosphate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate ester, an adenosine-ethylene glycol nucleic acid, or a nucleotide comprising a 5'-phosphate mimetic.

[0409] 62. The method according to any one of the preceding embodiments, wherein the siRNA comprises a phosphate backbone modification, a 2' ribose modification, a 5' triphosphate modification, or a GalNAc conjugation modification.

[0410] 63. A method according to any of the preceding embodiments, wherein the phosphate backbone modification comprises a phosphorothioate bond.

[0411] 64. The method according to any one of the preceding embodiments, wherein the siRNA comprises a 2'-fluoro or 2'-O-methyl substitution.

[0412] 65. The method according to any one of the preceding embodiments, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand of the siRNA are modified nucleotides.

[0413] 66. The method of any one of the preceding embodiments, wherein the siRNA comprises a sense strand and an antisense strand, the sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), the antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0414] wherein 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;

[0415] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0416] (Agn) is adenosine-glycol nucleic acid (GNA);

[0417] s is a phosphorothioate bond; and

[0418] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0419] 67. The method of any one of the preceding embodiments, wherein the siRNA comprises a sense strand and an antisense strand, the sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 7), the antisense strand comprising 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 8), wherein 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;

[0420] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0421] s is a phosphorothioate bond; and

[0422] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0423] 68. The method of embodiment 66 or 67, wherein the L96 is conjugated to the sense strand as shown in the following structure:

[0424]

[0425] Where X is O.

[0426] 69. A method according to any one of the preceding embodiments, wherein the subject is a human, and a therapeutically effective amount of siRNA is administered to the subject; and wherein the effective amount of the siRNA is about 1 mg / kg to about 8 mg / kg.

[0427] 70. The method of any one of the preceding embodiments, wherein the siRNA is administered to the subject twice daily, once daily, once every two days, once every three days, twice a week, once a week, once every other week, once every four weeks, or once a month.

[0428] 71. The method of any one of the preceding embodiments, wherein the siRNA is administered to the subject once every four weeks.

[0429] 72. The method of any one of embodiments 11 to 71, wherein the interferon-α is interferon-α-2a.

[0430] 73. The method of any one of embodiments 11 to 72, wherein the interferon-α is pegylated.

[0431] 74. The method of any one of embodiments 11 to 73, wherein the interferon-α is polyethylene glycol interferon-α-2a (PEG-IFNα-2a).

[0432] 75. The method according to any one of the preceding embodiments, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil (TD), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, fovir dipivoxil, lobcavir, famciclovir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-alpha and ganciclovir, besifovir (ANA-380 / LB-80380) or tenofovir-exaliades (TLX / CMX157).

[0433] 76. The method according to any one of the preceding embodiments, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), or tenofovir disoproxil (TD).

[0434] 77. The method according to any one of the preceding embodiments, wherein the NRTI is tenofovir disoproxil fumarate (TDF).

[0435] 78. The method according to any one of the preceding embodiments, wherein:

[0436] (a) the anti-HBV antibody comprises: a light chain amino acid sequence according to SEQ ID NO: 59, and a heavy chain amino acid sequence according to SEQ ID NO: 57;

[0437] (b) the siRNA comprises: a sense strand and an antisense strand, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), the antisense strand comprises 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0438] wherein 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;

[0439] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0440] (Agn) is adenosine-glycol nucleic acid (GNA);

[0441] s is a phosphorothioate bond; and

[0442] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; and

[0443] (c) The NRTI is tenofovir disoproxil fumarate (TDF).

[0444] 79. The method of any one of embodiments 11 to 78, wherein:

[0445] (a) the anti-HBV antibody comprises: a light chain amino acid sequence according to SEQ ID NO: 59, and a heavy chain amino acid sequence according to SEQ ID NO: 57;

[0446] (b) the siRNA comprises: a sense strand and an antisense strand, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), the antisense strand comprises 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

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

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

[0449] (Agn) is adenosine-glycol nucleic acid (GNA);

[0450] s is a phosphorothioate bond; and

[0451] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol;

[0452] (c) the interferon-α is PEG-IFNα-2a; and

[0453] (d) The NRTI is tenofovir disoproxil fumarate (TDF).

[0454] 80. The method according to any one of the preceding embodiments, wherein:

[0455] (a) the anti-HBV antibody consists of: a light chain amino acid sequence according to SEQ ID NO: 59, and a heavy chain amino acid sequence according to SEQ ID NO: 57;

[0456] (b) the siRNA consists of a sense strand and an antisense strand, wherein the sense strand consists of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and the antisense strand consists of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0457] wherein 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;

[0458] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0459] (Agn) is adenosine-glycol nucleic acid (GNA);

[0460] s is a phosphorothioate bond; and

[0461] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; and

[0462] (c) The NRTI is tenofovir disoproxil fumarate (TDF).

[0463] 81. The method of any one of embodiments 11 to 78, wherein:

[0464] (a) the anti-HBV antibody consists of: a light chain amino acid sequence according to SEQ ID NO: 59, and a heavy chain amino acid sequence according to SEQ ID NO: 57;

[0465] (b) the siRNA consists of a sense strand and an antisense strand, wherein the sense strand consists of 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and the antisense strand consists of 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6),

[0466] wherein 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;

[0467] Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively;

[0468] (Agn) is adenosine-glycol nucleic acid (GNA);

[0469] s is a phosphorothioate bond; and

[0470] L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol;

[0471] (c) the interferon-α is PEG-IFNα-2a; and

[0472] (d) The NRTI is tenofovir disoproxil fumarate (TDF).

[0473] 82. The method according to any one of the preceding embodiments, wherein Figure 1A , Figure 1B , Figure 2A , Figure 2B or FIG. 3A to FIG. 4D The anti-HBV antibody, the siRNA and the NRTI are administered to the subject according to the procedures described in .

[0474] 83. The method according to any one of the preceding embodiments, wherein Fig. 6A , Figure 6B , Fig. 7A , Figure 7B or FIG. 8A to FIG. 11E The anti-HBV antibody, the siRNA, the interferon-α, and the NRTI are administered to the subject according to the procedure described in .

[0475] 84. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered subcutaneously.

[0476] 85. The method according to any one of the preceding embodiments, wherein the siRNA is administered subcutaneously.

[0477] 86. The method of any one of embodiments 11 to 85, wherein the interferon-α is administered subcutaneously.

[0478] 87. The method of any one of the preceding embodiments, wherein the NRTI is administered orally.

[0479] 88. The method according to any one of the preceding embodiments, wherein the anti-HBV antibody is administered every 4 weeks.

[0480] 89. The method of any one of embodiments 1 to 87, wherein the anti-HBV antibody is administered every 8 to 12 weeks.

[0481] 90. The method according to any one of the preceding embodiments, wherein the siRNA is administered every 4 weeks.

[0482] 91. The method of any one of embodiments 11 to 90, wherein the interferon-α is administered once a week.

[0483] 92. The method of any one of the preceding embodiments, wherein the NRTI is administered daily.

[0484] 93. A method according to any of the preceding embodiments, wherein the anti-HBV antibody is administered at a dose of 300 mg.

[0485] 94. The method of embodiment 93, wherein 6 to 13 doses of the anti-HBV antibody are administered.

[0486] 95. The method of embodiment 93, wherein 12 doses of the anti-HBV antibody are administered.

[0487] 96. The method of embodiment 93, wherein 13 doses of the anti-HBV antibody are administered.

[0488] 97. The method of any one of embodiments 93 to 97, wherein the dose of the anti-HBV antibody is administered over a period of 44 weeks.

[0489] 98. The method of any one of embodiments 93 to 97, wherein the dose of the anti-HBV antibody is administered over a period of 44 weeks or longer.

[0490] 99. The method of any one of embodiments 93 to 97, wherein the dose of the anti-HBV antibody is administered over a period of 48 weeks.

[0491] 100. The method of any one of embodiments 93 to 97, wherein the dose of the anti-HBV antibody is administered over a period of 48 weeks or longer.

[0492] 101. The method according to any one of the preceding embodiments, wherein the siRNA is administered at a dose of 200 mg.

[0493] 102. The method of embodiment 94, wherein 6 to 13 doses of the siRNA are administered.

[0494] 103. The method of embodiment 94, wherein 12 doses of the siRNA are administered.

[0495] 104. The method of embodiment 94, wherein 13 doses of the siRNA are administered.

[0496] 105. The method of any one of embodiments 100 to 104, wherein the dose of the siRNA is administered over a period of 44 weeks.

[0497] 106. The method of any one of embodiments 100 to 104, wherein the dose of the siRNA is administered over a period of 44 weeks or longer.

[0498] 107. The method of any one of embodiments 100 to 104, wherein the dose of the siRNA is administered over a period of 48 weeks.

[0499] 108. The method of any one of embodiments 100 to 104, wherein the dose of the siRNA is administered over a period of 48 weeks or longer.

[0500] 109. The method of any one of embodiments 11 to 108, wherein the interferon-α is administered at a dose of 180 mcg.

[0501] 110. The method of embodiment 109, wherein at least 308 doses of the interferon-α are administered.

[0502] 111. The method of any one of embodiments 109 to 110, wherein said dose of said interferon-α is administered over a period of 44 weeks.

[0503] 112. The method of any one of embodiments 109 to 110, wherein said dose of said interferon-α is administered over a period of 44 weeks or longer.

[0504] 113. The method of any one of embodiments 109 to 110, wherein said dose of said interferon-α is administered over a period of 48 weeks.

[0505] 114. The method of any one of embodiments 109 to 110, wherein said dose of said interferon-α is administered over a period of 48 weeks or longer.

[0506] 115. The method according to any of the preceding embodiments, wherein the NRTI is administered at a dose of 300 mg.

[0507] 116. The method of any one of Embodiments 195, wherein the NRTI is administered at a dose of 245 mg.

[0508] 117. The method of embodiment 96 or 97, wherein 140 to 308 doses of the NRTI are administered.

[0509] 118. The method of embodiment 96 or 97, wherein 308 doses of the NRTI are administered.

[0510] 119. The method of any one of embodiments 116 to 118, wherein said dose of said NRTI is administered over a period of 44 weeks.

[0511] 120. The method of any one of embodiments 116 to 118, wherein said dose of said NRTI is administered over a period of 44 weeks or longer.

[0512] 121. The method of any one of embodiments 116 to 118, wherein said dose of said NRTI is administered over a period of 48 weeks.

[0513] 122. The method of any one of embodiments 116 to 118, wherein said dose of said NRTI is administered over a period of 48 weeks or longer.

[0514] 123. The method of any one of the preceding embodiments, wherein administration of the siRNA and the anti-HBV antibody to the subject begins on the same day.

[0515] 124. A method according to any of the preceding embodiments, wherein the subject has chronic HBV.

[0516] 125. A method according to any of the preceding embodiments, wherein the subject has hepatitis delta virus (HDV) infection.

[0517] 126. A method according to any of the preceding embodiments, wherein the HBV-related disease is chronic HBV.

[0518] 127. A method according to any of the preceding embodiments, wherein the HBV-related disease is HDV infection.

[0519] 128. A method according to any of the preceding embodiments, wherein the subject is human.

[0520] 129. An anti-HBV antibody; an siRNA targeting HBV mRNA; and an NRTI; for use in the method of any one of embodiments 1 to 128.

[0521] 130. An anti-HBV antibody; an siRNA targeting HBV mRNA; and a NRTI; for use in the manufacture of a medicament for use in a method according to any one of embodiments 1 to 128.

[0522] 131. Use of an anti-HBV antibody in the manufacture of a first drug; use of an siRNA targeting HBV mRNA in the manufacture of a second drug; and use of an NRTI in the manufacture of a third drug; wherein the first drug, the second drug and the third drug are to be used in a combination therapy according to any one of embodiments 1 to 128.

[0523] 132. An anti-HBV antibody for use in a method of treating a hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein:

[0524] The subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL prior to treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤ the upper limit of normal (ULN), the subject has not been previously administered NRTIs, the subject has not received NRTIs within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA; and

[0525] The subject was also administered siRNA targeting HBV mRNA and a nucleoside reverse transcriptase inhibitor (NRTI).

[0526] 133. An anti-HBV antibody; an siRNA targeting HBV mRNA; and a nucleoside(acid) reverse transcriptase inhibitor (NRTI) for use in a method for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: the subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤the upper limit of normal (ULN), the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA.

[0527] 134. An anti-HBV antibody for use in the manufacture of a medicament for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein:

[0528] The subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL prior to treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤ the upper limit of normal (ULN), the subject has not been previously administered NRTIs, the subject has not received NRTIs within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA; and

[0529] The subject was also administered siRNA targeting HBV mRNA and a nucleoside reverse transcriptase inhibitor (NRTI).

[0530] 135. An anti-HBV antibody for use in the manufacture of a first drug; an siRNA targeting HBV mRNA for use in the manufacture of a second drug; and a nucleoside(acid) reverse transcriptase inhibitor (NRTI) for use in the manufacture of a third drug, wherein the first drug, the second drug and the third drug are to be used in a combination therapy for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: the subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤the upper limit of normal (ULN), the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA.

[0531] 136. An anti-HBV antibody; an siRNA targeting HBV mRNA; interferon-α; and an NRTI; for use in the method of any one of embodiments 11 to 128.

[0532] 137. An anti-HBV antibody; an siRNA targeting HBV mRNA; an interferon-α; and an NRTI; for use in the manufacture of a medicament for use in a method according to any one of embodiments 11 to 128.

[0533] 138. Use of an anti-HBV antibody in the manufacture of a first drug; use of an siRNA targeting HBV mRNA in the manufacture of a second drug; use of interferon-α in the manufacture of a third drug; and use of NRTI in the manufacture of a fourth drug; wherein the first drug, the second drug, the third drug and the fourth drug are to be used in a combination therapy according to the method described in any one of Embodiments 11 to 128.

[0534] 139. An anti-HBV antibody for use in a method of treating a hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein:

[0535] The subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment; and

[0536] The subject was also administered siRNA targeting HBV mRNA, interferon-α, and nucleos(t)ide reverse transcriptase inhibitors (NRTIs).

[0537] 140. An anti-HBV antibody; an siRNA targeting HBV mRNA; an interferon-α; and a nucleoside(a) reverse transcriptase inhibitor (NRTI) for use in a method of treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: the subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment.

[0538] 141. An anti-HBV antibody for use in the manufacture of a medicament for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein:

[0539] The subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment; and

[0540] The subject was also administered siRNA targeting HBV mRNA, interferon-α, and nucleos(t)ide reverse transcriptase inhibitors (NRTIs).

[0541] 142. An anti-HBV antibody for use in the manufacture of a first drug, an siRNA targeting HBV mRNA for use in the manufacture of a second drug, an interferon-α for use in the manufacture of a third drug, and a nucleoside (acid) reverse transcriptase inhibitor (NRTI) for use in the manufacture of a fourth drug, wherein the first drug, the second drug, the third drug and the fourth drug are used to treat hepatitis B virus (HBV) infection or HBV-related diseases in subjects in need thereof; wherein: the subject is HBeAg negative or HBeAg positive, and the subject has HBV> 2000 IU / mL before treatment DNA level, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level > the upper limit of normal (ULN) and ≤ 5 times the ULN, the subject has not been previously administered NRTIs, the subject has not received NRTIs within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered siRNA targeting HBV mRNA, the subject has not been previously administered interferon-α, the subject is HBsAg positive prior to treatment, and / or the subject has an HBsAg level > 10 IU / mL prior to treatment.

[0542] 143. A kit, comprising:

[0543] A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient;

[0544] A pharmaceutical composition comprising siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient; and

[0545] A pharmaceutical composition comprising a NRTI and a pharmaceutically acceptable excipient.

[0546] 144. The kit of embodiment 143, further comprising instructions for performing the method of any one of embodiments 1 to 128.

[0547] 145. A kit, comprising:

[0548] A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient;

[0549] A pharmaceutical composition comprising siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient;

[0550] A pharmaceutical composition comprising interferon-α and a pharmaceutically acceptable excipient; and

[0551] A pharmaceutical composition comprising a NRTI and a pharmaceutically acceptable excipient.

[0552] 146. The kit of embodiment 145, further comprising instructions for performing the method of any one of embodiments 11 to 128.

[0553] Example

[0554] Example 1

[0555] Clinical trial of combination therapy for the treatment of HBeAg-negative participants with chronic HBV infection with low viral load evaluate

[0556] In a phase 2, multicenter, open-label clinical study, the safety and efficacy of anti-HBV antibodies, optionally in combination with anti-HBV siRNA and / or the nucleos(t)ide reverse transcriptase inhibitor tenofovir, were evaluated in HBeAg-negative, non-cirrhotic human patients with low-load chronic HBV infection.

[0557] background

[0558] Chronic HBV infection remains an important global public health problem due to significant morbidity and mortality (TrepoC, 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 of viral replication and host immune response.

[0559] Patients can be divided into different disease stages based on levels of hepatitis B e antigen (HBeAg), hepatitis B virus (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 August, 67(2):370-398; Sarin SK et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update, Hepatol Int. 2016 January, 10(1):1-98; Terrault NA et al., Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance, Hepatology 2018 April, 67(4):1560-1599).

[0560] Chronic HBV infection is a dynamic process and depends on the relationship between viral replication and the host immune response. Patients may fluctuate between different stages of the disease, and serial monitoring of biomarkers such as HBV DNA, ALT, and HBV antigens helps determine the natural history of the disease. Of the 300 million patients chronically infected with HBV, those who are HBeAg-negative are the largest subgroup. These patients range from being immune-inactive (also known as "inactive carriers" or "inactive" hepatitis) to having chronic active hepatitis that may progress to serious 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 for at least 1 year. These patients have a favorable long-term prognosis, with a low rate of histological progression, 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 indications for currently available treatments targeting HBV DNA suppression, but they are most likely to achieve a functional cure, thereby becoming non-infectious, and have a low risk of disease relapse.

[0561] AB01 is a monoclonal antibody (mAb) targeting hepatitis B surface antigen (HBsAg) with multiple potential mechanisms of action, including strong neutralization activity and enhanced immune activity due to Fc domain engineering. AB01 (HBC34v35-MLNS-GAALIE) includes a light chain amino acid sequence of SEQ ID NO: 59 and a heavy chain amino acid sequence of SEQ ID NO: 57.

[0562] In patients with chronic HBV infection, previous studies have shown that restoring HBV-specific CD8+T cell function by PD-1 blockade in inactive carrier patients is linked to T cell differentiation (Bengsch B et al., Restoration of HBV-specific CD8+T cell function by PD-1blockade in inactive carrier patients is linked to T cell differentiation, J Hepatol. December 2014, 61(6):1212-9). The report of this study suggests that the potential of restoring HBV-specific T cells in an immune-inactive population may be feasible using the proposed mechanism of AB01 as a vaccine mAb with T cell engagement activity. AB01 neutralizes HBV virus and subviral particles by targeting HBsAg and inhibiting viral entry into hepatocytes, thereby providing a new strategy for treating chronic HBV infection. In addition, the Fc region of AB01 is engineered to increase affinity with neonatal Fc receptors (FcRn) and promote the binding properties of Fc-γ receptors (FcγR) to activating receptors. These modifications can prolong serum half-life, increase 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 between FcRn-mediated endocytosis and recycling and endosomal degradation. To produce a product with sustained activity against HBV, well-characterized LS modifications (M428L and N434S) (Gaudinski MR et al., Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01 LS: 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) were included in the Fc region of AB01.LS modifications increase IgG1 binding to FcRn only at the acidic pH of the endosomal compartment, thereby increasing IgG recycling back into the circulation. Monoclonal antibodies containing LS mutations have been previously studied in humans (Gaudinski et al., 2018, supra). For example, VRC01LS is a mAb directed against the CD4 binding site of the HIV-1 glycoprotein that was found to be safe and well tolerated in healthy volunteers at doses of 5 mg / kg to 40 mg / kg IV and 5 mg / kg SC. No anti-drug antibodies (ADA) were detected over 48 weeks. Similarly, AB01 is expected to have an extended half-life in humans, resulting in a prolonged duration of exposure. In vitro data for AB01 indicate that LS mutations are not expected to interfere with the additional Fc modifications described below. The Fc region of AB01 has also been engineered to include modifications that modulate binding to human FcγRs by enhancing binding to the activating receptors FcγRIIa and FcγRIIIa while reducing binding to the inhibitory receptor FcγRIIb. The Fc modification is designed to enhance ADCP and antigen presentation of HBsAg and HBV virions, and thus promotes the guidance of T cell responses ("vaccine effect"). Similar modifications have been studied in FcγR humanized lymphoma mouse models. In this model, anti-CD20 antibodies promote direct killing of tumor cells via the participation of FcγRIIIa on macrophages and monocytes. In addition, anti-CD20 immune complexes induce CD8+T cell responses via FcγRIIa-dependent presentation of tumor antigens by dendritic cells. In general, anti-CD20 antibodies are Fc engineered to increase FcγRIIa and FcγRIIIa binding (GASDALIE mutation) with excellent therapeutic activity (DiLillo DJ et al., Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect, Cell May 21, 2015, 161 (5): 1035-1045). A parallel reduction in binding to inhibitory FcγRIIb has the potential to further enhance the efficacy of this vaccine (DiLillo et al., 2015, supra).

[0563] Available data on the use of antibodies against HBsAg in the treatment of patients with chronic HBV infection indicate that these molecules have the potential to reduce HBsAg levels while maintaining acceptable safety and tolerability. As a fully human anti-HBsAg mAb being developed for the treatment of chronic HBV infection and prevention of HBV recurrence after liver transplantation, GC1102 effectively reduces HBsAg by 2log10IU / mL to 3log10 IU / mL and is well tolerated in a phase 1 study in patients with chronic HBV infection without 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) as two human anti-HBsAg mAbs was administered to 27 patients with different HBsAg levels ranging from approximately 20 IU / mL to 85,000 IU / mL. HBV-ABXTL was found to have favorable safety and tolerability at doses up to 80 mg for 4 doses per week, 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 March, 35(3): 673-9).Similarly, no adverse events were reported in two studies of chronic HBV patients receiving high-dose hepatitis B immune globulin (HBIG) to prevent reinfection after liver transplantation (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).

[0564] Clinical data from the oncology setting show that Fc engineering designed to enhance ADCC / ADCP and antigen presentation can improve efficacy without compromising safety or tolerability (Im SA 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 approved by the FDA for the treatment of HER2-positive cancer. Margetuximab contains modifications designed to enhance ADCC / ADCP and antigen presentation. Margetuximab was well tolerated in the first human (FIH) phase 1 study in patients with HER-2-positive cancer. Common toxicities were mainly ≤ grade 2, including no evidence of cardiotoxicity (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 has been observed in late-stage clinical trials with the non-Fc-modified parental mAb (trastuzumab) (Pondé 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 it still valid to differentiate anthracycline Type I from Type II cardiomyopathies?, Hum J Med. Vaccin Immunother. 2016 May 3, 12(5):1124-31).

[0565] In summary, these data suggest that mAbs engineered with Fc to extend serum half-life and optimize immune effector cell activity have the potential to enhance the efficacy of therapeutic mAbs without compromising safety. AB01 is expected to reduce serum HBsAg, inhibit intrahepatic virus spread, eliminate infected hepatocytes, and stimulate HBV-specific immune responses. Thus, AB01 may achieve functional cure of chronic HBV infection with or without other agents such as SIRNA01 and NRTI (tenofovir disoproxil fumarate, "TDF").

[0566] Cohort 1b will evaluate the contribution of AB01 to combination therapy and its potential to achieve functional cure in the absence of any other study agents. To assess participant responses and potential vaccine effects from AB01, peripheral blood mononuclear cells (PBMCs) will be collected to evaluate host cell immune responses (e.g., T cells) against HBV antigens. Cohort 1b will evaluate 6 to 12 doses of AB01 administered every 4 weeks, and NRTI (TDF) for 20 to 44 weeks, in a response-guided manner. Participants who achieve HBsAg loss and HBV DNA <LLOQ before week 20 may be eligible to discontinue AB01 and TDF. Participants who do not achieve HBsAg loss and HBV DNA <LLOQ before week 20 will continue to receive AB01 and TDF until week 44.

[0567] SIRNA01 is an siRNA associated with a substantial reduction in HBsAg in patients with chronic HBV infection. SIRNA01 has a positive strand comprising a nucleotide sequence of SEQ ID NO:5 and an antisense strand comprising a nucleotide sequence of SEQ ID NO:6. The use of siRNA provides a new strategy for treating chronic HBV infection. siRNA is an RNA duplex of 19 to 21 base pairs that utilizes an endogenous RNA interference pathway to achieve sequence-specific RNA cleavage and degradation. One siRNA can have a variety of antiviral effects, including degradation of pgRNA, thereby inhibiting viral replication and degrading all viral messenger RNA (mRNA) transcripts, thereby preventing the expression of viral proteins. This may lead to the restoration of a functional immune response to HBV, whether used alone or in combination with other therapies. In contrast, NRTIs act at different parts of the viral life cycle and have a different mechanism of action from SIRNA01. NRTIs inhibit HBV polymerase, block reverse transcription of viral pgRNA into viral DNA, and prevent the production of infectious viral particles. 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 non-infectious subviral particles containing HBsAg by SIRNA01 is considered an important distinction from currently available treatments.

[0568] Emerging clinical data suggest that a combination of multiple treatment modalities may be necessary to achieve a functional cure in most 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). Excessive secretion of non-virion-incorporated HBsAg in chronic HBV infection is thought to contribute to T and B cell dysfunction and impair the host's ability to clear or control the virus (Bertoletti et al., 2016, supra; Burton et al., 2018, supra; Maini et al., 2016, supra). Therefore, the development of a functional cure may require the reduction or elimination of HBsAg in combination with the induction of host immunity to infection. When administered alone, AB01 and SIRNA01 were associated with substantial reductions in 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 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 may lead to a substantial and sustained decline in HBsAg. This is supported by the AAV-HBV mouse model of chronic HBV infection, in which treatment with the combination of AB01 and SIRNA01 is associated with a significant reduction in HBsAg. Additionally, lower HBsAg levels achieved by prior treatment or treatment concomitant with SIRNA01 may enhance the effect of AB01. The reduction in HBsAg may also enhance the potential immunological effects of AB01. The Fc modification of AB01 is designed to enhance ADCC / ADCP and antigen presentation and thus facilitate the induction of CD4 and CD8 T cell responses. Considering the putative effect of HBsAg on immune function, these potential effects of AB01 may be enhanced in the context of lower HBsAg, which can be achieved using lead-in treatment and / or treatment concomitant with SIRNA01. Longer-duration administration of AB01 may also enhance these effects.

[0569] The participant group included in this subprotocol had low viral load and normal ALT and was not eligible for treatment with NRTIs. However, NRTIs such as TDF help reduce viral replication and thus lower HBV DNA levels. Therefore, in addition to AB01, which has potential immunomodulatory effects, they are included as part of all protocols to allow these participants with low HBV DNA levels to achieve HBV DNA <LLOQ and ultimately achieve sustained suppression of HBV DNA, with or without HBsAg loss.

[0570] A response-guided approach is used to evaluate the timing and duration of functional cure achieved with AB01 and TDF in combination with SIRNA01. Cohort 2b will evaluate 6 to 12 doses of SIRNA01 (200 mg) in combination with a regimen that includes 6 to 12 doses of AB01 and 20 to 44 weeks of NRTI (TDF). To evaluate participant response and the potential vaccine effect from AB01, PBMC will be collected to assess the host cellular immune response (e.g., T cells) against HBV antigens. Participants who achieve HBsAg loss and HBV DNA <LLOQ before week 20 may be eligible to discontinue AB01, TDF, and SIRNA01 therapy. Participants who do not achieve HBsAg loss and HBV DNA <LLOQ before week 20 will continue AB01, TDF, and SIRNA01 therapy until week 44.

[0571] Study Design

[0572] Table 4 shows the treatment groups of the study. Each cohort will initially include approximately 15 participants. An additional 30 participants may be added to any cohort. The total duration of the study for all participants is up to 100 weeks - including a screening period of up to 56 days (8 weeks), a treatment period of 20 to 44 weeks, and a follow-up period of 48 weeks. During the treatment period, if the participant achieves undetectable HBsAg (<0.05IU / mL) at 2 consecutive visits before the 20th week, they can stop all study interventions at the 20th week and enter the follow-up period. Alternatively, the treatment period for all participants can be 44 weeks, with no option to stop the study intervention at the 20th week.

[0573] Table 4. Treatment groups used in clinical studies.

[0574]

[0575] a The dosage of tenofovir disoproxil fumarate (TDF) will be 300 mg per label. Supply outside the United States may be tenofovir disoproxil (TD), and the corresponding dosage will be 245 mg per label.

[0576] The overall research plan is Figure 1A and Figure 1B The dosing schedule for all cohorts is shown in Figure 2A and Figure 2B Shown in.

[0577] AB01 is provided as a reconstituted lyophilized powder that is administered subcutaneously (SC) at 300 mg every 4 weeks for 6 to 12 doses. SIRNA01 is provided as a liquid that is administered SC at 200 mg every 4 weeks for 6 to 12 doses. Tenofovir disoproxil fumarate (TDF) It is provided in the form of a tablet that is taken orally at 300 mg daily for a total of 140 to 308 doses. Tenofovir is available outside the United States as tenofovir disoproxil (TD), in the United States, tenofovir is provided in the form of a tablet that is taken orally at 245 mg daily for a total of 140 to 308 doses.

[0578] Cohorts can be included in the study in parallel. Cohorts can be open, closed or stopped. The total duration for participants in Cohorts 1b and 2b will be up to 100 weeks, including screening, treatment and follow-up periods. The screening period for all participants will be up to 56 days (8 weeks). The treatment period for Cohorts 1b and 2b will be between 20 weeks and 44 weeks. In particular, the treatment period can be 44 weeks (see Figure 2B)。If participants achieve undetectable HBsAg (<0.05 IU / mL) and HBV DNA <LLOQ at two consecutive visits before week 20, they may be eligible to stop the study intervention at week 20 and enter the follow-up period at subsequent visits (see Figure 2A ). Alternatively, the treatment period for all participants may be 44 weeks, with no option to stop 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 enter the follow-up period 1 week after the end of the treatment period. Once participants complete the treatment period of their respective cohort, they will enter the follow-up period. The maximum duration of the follow-up period is 48 weeks after the last dose of the study intervention. If participants meet any of the following criteria, they may start commercial NRTI at any time during the follow-up period: HBV DNA increases by ≥2 log10 IU / mL over a 2-week period; HBV 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); HBV DNA >2,000 IU / mL, in combination with any of the following criteria, at the same visit: confirmed total bilirubin >2x ULN and ALT >ULN, any signs of liver decompensation (including but not limited to confirmed increase in prothrombin time [PT] ≥2 or increase in international normalized ratio [INR] ≥0.5, jaundice, ascites, encephalopathy, etc.) from baseline, ALT >10x ULN, ALT >2x ULN for ≥12 consecutive weeks, ALT >5x ULN for ≥4 consecutive weeks; confirmed HbeAg seroconversion (i.e., positive HbeAg after negative HbeAg); or any other clinically significant event, to initiate NRTI therapy. Participants who meet the criteria for starting NRTI treatment during the follow-up period will continue to be followed until the end of the study.

[0579] The HBsAg loss rate will be evaluated at the end of treatment (EOT) and 24 weeks after EOT. The proportion of participants with HBsAg loss (defined as HBsAg < LLOQ or HBsAg < 0.05 IU / mL) at EOT was determined. The proportion of participants who achieved suppression of HBV DNA (< LLOQ) and had HBsAg loss (0.05 < IU / mL) at EOT was also determined. The proportion of participants with HBsAg loss (defined as HBsAg < LLOQ or HBsAg < 0.05 IU / mL) at 24 weeks after EOT was also determined. The additional endpoints evaluated included: (i) the proportion of participants who achieved sustained suppression of HBV DNA < LLOQ at 24 and 48 weeks after stopping all treatment; (ii) the proportion of participants who achieved sustained suppression of HBV DNA (< LLOQ) and had HBsAg loss (< 0.05 IU / mL) at 24 and 48 weeks after stopping all treatment; (iii) the mean change in serum HBsAg levels relative to baseline across time points in the study; (iv) the proportion of participants who achieved undetectable HBV DNA (< LLOQ) across time points in the study; (v) the HBV DNA levels and changes relative to baseline across time points in the study; (vi) the nadir and maximum change in HBV DNA relative to baseline in the study; (vii) the proportion of participants who met the NRTI treatment criteria during the follow-up period; and (viii) the proportion of participants with virological relapse (defined as: (1) an increase in HBV DNA ≥ 1 log 10 10 HBV DNA IU / mL higher than the nadir at least at two consecutive visits, or (2) quantifiable HBV DNA ≥ 1 log10 IU / mL higher than LLOQ at least at two consecutive visits after < LLOQ).

[0580] Pharmacokinetics (PK), immunogenicity, and adverse events were also evaluated. Concentrations of AB01 and SIRNA01 PK will be quantified using validated bioanalytical assays. PK parameters may include Cmax, Clast, Tmax, Tlast, AUCinf, AUClast, %AUC exp 、t 1 / 2 、λ z 、V z / F, and CL / F. Immunogenicity data may include incidence, titers, and neutralization data (e.g., antibodies to AB01). Additional antiviral activities such as anti-HB, HBV RNA, and hepatitis B core-related antigen (HbcrAg) were evaluated. The number and percentage of participants who exhibited anti-HB and anti-HBe seroconversion were evaluated.

[0581] FIG. 3A to FIG. 5CThe activity timeline of the cohort and follow-up period is provided in Liver elasticity imaging can be performed using FibroScan or equivalent.

[0582] In addition, weight (and BMI) will be determined at Screening and at each study visit. A 12-lead safety electrocardiogram (ECG) will also be recorded at Screening and on D8, with the participant resting comfortably for approximately 10 minutes in the supine position. FIG. 4A to FIG. 4C As shown, the PK parameters of AB01 and SIRNA01 can be evaluated at different time intervals, such as shown in Table 5 below. FIG. 5A to FIG. 5C During the follow-up period shown, additional visits may be made at weeks 2, 6, 10, 28, 32, 40, and 44. For example, activities during the follow-up period may include: FIG. 5D to FIG. 5I shown.

[0583] Table 5. Timing of PK assessments for Cohort 2b.

[0584]

[0585] AB01 PK sample collection includes samples for free PK assays and total PK assays, as applicable. Pre-dose samples will be collected up to 1 hour prior to dosing.

[0586] Day 2 AB01 PK post-dose samples will be collected between 22 hours and 28 hours after D1 dosing.

[0587] AB01 PK post-dose samples may be collected at any time during the D8 visit.

[0588] SIRNA01PK pre-dose samples will be collected up to 1 hour prior to dosing.

[0589] Day 1, Week 20, and Week 44 SIRNA01 PK post-dose samples will be collected between 2 hours and 6 hours after dosing.

[0590] Day 2 SIRNA01 PK samples will be collected between 22 hours and 28 hours after D1 dosing.

[0591] Patient population

[0592] Participants will have chronic HBV infection and be HbeAg negative within 1 year prior to screening, have low HBV DNA (≤2000 IU / mL) and ALT levels less than or equal to (≤) ULN (or ALT levels less than (<) ULN). For the purposes of the study, chronic HBV infection is defined as 2 serum HBsAg, HBV DNA, or HBeAg positive tests at least 6 months apart (any combination of these tests 6 months apart is acceptable) based on previous or current laboratory documentation. The ULN value of ALT can be, for example, 34 IU / mL for women and 43 IU / mL for men. Participants will be aged ≥18 (or legal age of consent, whichever is greater) to <66 years. Participants will also have HBsAg>lower limit of detection at screening, and a body mass index (BMI) ≥18 kg / m 2 To ≤35kg / m 2 .

[0593] Additional inclusion criteria included the following:

[0594] Must be in good health, as determined by medical history, with no clinically significant findings on physical examination, vital signs, and laboratory values

[0595] Female participants must have a negative pregnancy test or confirmed postmenopausal status. Postmenopausal status is defined as the absence of menstruation for 12 months without an alternative medical reason. Women of childbearing potential (WOCBP) must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, cannot breastfeed, and must be willing to use highly effective contraception from 14 days before 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 start of the study intervention until 48 weeks after the last dose of AB01 or SIRNA01.

[0596] Male participants with female partners of reproductive potential must agree to meet 1 of the following contraceptive requirements from the start of the study intervention until 48 weeks after the last dose of AB01 or SIRNA01: documented vasectomy or azoospermia, or use of male condoms plus partner use of 1 of the contraceptive options listed for WOCBP contraception. Male participants must also agree not to donate sperm from the start of the first study intervention until 48 weeks after the last dose of AB01 or SIRNA01.

[0597] ·Able to understand and comply with study requirements and able to provide written informed consent.

[0598] Exclusion criteria included the following:

[0599] History of clinically significant liver disease due to non-HBV etiology

[0600] History or current evidence of signs of liver decompensation, including ascites, hepatic encephalopathy, and / or esophageal or gastric varices

[0601] History or current suspicion of malignancy diagnosed or treated within 5 years (topically treated squamous or noninvasive basal cell skin cancer was allowed; in situ cervical cancer was allowed if appropriately treated prior to screening); participants undergoing evaluation for malignancy were not eligible.

[0602] History of bone marrow or solid organ transplantation

[0603] Known active infection other than chronic HBV viral infection or any clinically significant acute condition (such as fever (>38°C) or acute respiratory illness) within 7 days before Day 1

[0604] Co-infection with human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

[0605] Participants who were positive for HCV antibody or HDV antibody but had documented negative HCV RNA or HDV RNA, respectively, were eligible. Participants who were positive for HAV immunoglobulin M (IgM) or HEV IgM but were asymptomatic and positive for HAV immunoglobulin G (IgG) or HEV IgG were eligible.

[0606] · History or clinical evidence of alcohol or drug abuse within 12 months prior to screening, or a positive drug screen at screening, unless explained by prescription medication (diagnosis and prescription must be approved by the investigator). Note: Marijuana use is permitted

[0607] · Received study medication within 90 days or 5 half-lives (if known), whichever is longer, prior to the study intervention, or is active during the follow-up period of another clinical study involving the 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.

[0608] Any clinically significant medical or psychiatric condition that, as determined by the investigator, may interfere with study interventions, assessments, or compliance with the protocol or otherwise render the participant unsuitable for study participation.

[0609] Significant fibrosis or cirrhosis, as defined by liver elastography (FibroScan or equivalent) results >8.5 kPa at screening or Metavir F3 fibrosis or F4 cirrhosis on liver biopsy within 1 year.

[0610] History of immune complex disease

[0611] History of autoimmune disease

[0612] History of HBV-related extrahepatic disease, including but not limited to HBV-related rash, arthritis, or glomerulonephritis

[0613] History of anaphylaxis, hypersensitivity, or intolerance to the monoclonal antibodies, antibody fragments, or any excipients of AB01

[0614] Prior NRTI therapy within 24 weeks prior to Day 1, or any prior therapy with PEG-IFNα

[0615] Use of any of the following systemic medications within 14 days prior to study intervention and throughout the study period:

[0616] Acetaminophen (paracetamol) ≥ 3 g / day

[0617] Isoniazid

[0618] Systemic steroids (prednisone equivalents >10 mg / day) or other immunosuppressants (Note: corticosteroid administration is permitted for the treatment of immune-mediated AEs.)

[0619] Receipt of oligonucleotides with anti-HBV activity (e.g., siRNA, antisense oligonucleotides) within 48 weeks prior to study intervention

[0620] Received AB01 within 24 weeks prior to Day 1

[0621] Participants had the following laboratory tests at the time of screening

[0622] parameter:

[0623] ALT > ULN at screening and at any time within 12 months prior to study entry

[0624] HBeAg positive at screening and at any time within 12 months prior to study entry

[0625] HBV DNA > 2,000 IU / mL at screening and at any time within 12 months prior to study entry

[0626] Direct bilirubin or international normalized ratio (INR) >1.5ULN

[0627] Creatinine clearance (CLcr) < 30 mL / min, as calculated by the Cockcroft-Gault formula at screening

[0628] Clinically significant abnormalities on 12-lead ECG at screening (as determined by the investigator) Concomitant therapy

[0629] No concomitant treatment was allowed during the study

[0630] NRTI use prior to Day 1 is prohibited. During the study, participants will receive TDF as per protocol. No other NRTIs are allowed during the treatment period. If a participant requires NRTI treatment during the follow-up period, a commercial NRTI may be started.

[0631] The use of any of the following systemic medications within 14 days prior to the study intervention and throughout the study period is prohibited:

[0632] ○ Systemic steroids (prednisone equivalents >10 mg / day) or other immunosuppressants (Note: corticosteroid administration is permitted for the treatment of immune-mediated AEs.)

[0633] ○ Acetaminophen (paracetamol) ≥ 3 g / day

[0634] ○ Isoniazid

[0635] In addition, the use of any potentially hepatotoxic medication during the study should only be considered if no therapeutic alternatives can be identified and after careful consideration of the potential risks and benefits to the participants. Medications that are potentially hepatotoxic or have been associated with drug-induced liver injury include, but are not limited to, the following ( 2016): aspirin > 3 g / day or ibuprofen ≥ 1.2 g / day; tricyclic antidepressants; valproic acid; phenytoin; amiodarone; anabolic steroids; allopurinol; amoxicillin-clavulanate; minocycline; nitrofurantoin;

[0636] Sulfadiazine / trimethoprim; erythromycin; rifampin; azole antifungals; and herbal or natural remedies.

[0637] Example 2

[0638] Clinical trials of combination therapy of antibodies, siRNA, PEG-IFNα and NRTI inhibitors for the treatment of chronic HBV infection evaluate

[0639] In a phase 2, multicenter, open-label study, the safety and efficacy of an anti-HBV antibody (AB01) combined with anti-HBV siRNA (SIRNA01), PEG-IFNα, and the NRTI tenofovir were evaluated in non-cirrhotic human patients with chronic HBV infection who had not received prior NRTI or PEG-IFNα treatment.

[0640] background

[0641] Chronic HBV infection remains a major global public health problem due to significant morbidity and mortality (Trepo, 2014, supra). Chronic HBV infection is a dynamic process and depends on the relationship between viral replication and the host immune response. Patients may fluctuate between different stages of the disease, and serial monitoring of biomarkers such as HBV DNA, alanine aminotransferase (ALT), and HBV antigens helps determine the natural history of the disease. Following infection, the HBV virus may replicate as a stealth virus in the absence of a host immune response. After years of viral replication (high HBV DNA), the immune system responds to infected hepatocytes, resulting in elevated ALT in patients who are positive for hepatitis B e antigen (HBeAg). At this stage of the disease, patients are also characterized by moderate to severe liver inflammation and accelerated fibrosis progression. Patients infected perinatally may reach this stage of chronic HBV infection 10 to 30 years after infection, while patients infected during adulthood or childhood progress more rapidly or may even skip this stage entirely. The outcomes for this group are also variable. Among HBeAg-positive patients, some become HBeAg-negative rapidly, while others take longer to become HBeAg-negative and anti-HBe-positive with or without treatment (EASL, 2017, supra; 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, supra; 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).

[0642] Patients with elevated ALT (HBeAg positive and HBV DNA>20,000 IU / mL or HBeAg negative and HBV DNA>2,000 IU / mL) are eligible for treatment with nucleoside (acid) reverse transcriptase inhibitors (NRTIs) and / or polyethylene glycol interferon-α-2a (PEG-IFNα) (Liang TJ et al., Present and future therapies of hepatitis B: From discovery to cure, Hepatology 2015 December, 62(6): 1893-908). NRTIs can suppress HBV DNA through long-term therapy, but cannot eliminate cccDNA or integrated DNA. In contrast to NRTIs, PEG-IFNα can induce long-term viral control, but only in a small percentage of patients (<10%) and after 48 weeks of therapy (Konerman MA and Lok AS, Interferon Treatment for Hepatitis B, Clin Liver Dis. 2016 November, 20(4):645-665). Therefore, there is an unmet need for better treatment options that can achieve functional cure.

[0643] In patients with chronic HBV infection, weekly administration of 180 μg of PEG-IFNα for 48 to 52 weeks generally results in total HBsAg loss in approximately ≤ 10% of patients (Konerman and Lok, 2016, supra). However, in the subset of patients with baseline HBsAg values ​​less than approximately 1,000 IU / mL to 1,500 IU / mL, the rate of HBsAg loss after receiving PEG-IFNα (with or without NRTI) was approximately 20% to 40% (He LT et al., Effect of switching from treatment with nucleos(t)ide analogs to pegylated interferonα-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 the reduction of HBsAg during or before PEG-IFNα therapy may significantly increase the rate of HBsAg loss.Thus, a regimen consisting of SIRNA01 and AB01 (both of which were associated with substantial reductions in HBsAg) plus PEG-IFNα increased the functional cure rate beyond that associated with PEG-IFNα monotherapy.

[0644] The combination of SIRNA01 and PEG-IFNα was associated with a significant reduction in HBsAg, exceeding the effects 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, in participants who received 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, few HBsAg loss events were observed. The addition of AB01 may result in greater HBsAg reduction, higher HBsAg loss rates, and higher functional cure rates.

[0645] In addition, PEG-IFNα has an important effect on the immune system, which may increase the potential development of HBV-specific immunity after the administration of AB01. Type I interferon regulates antiviral T cells (Crouse J et al., Regulation of antiviral T cell responses by type I interferons, Nat Rev Immunol. April 2015, 15 (4): 231-42) and infiltrating or liver-resident immune cells (Dill MT et al., Pegylated IFN-αregulates hepatic gene expression through transient Jak / STAT activation, J Clin Invest. April 2014, 124 (4): 1568-81) via the effects on auxiliary cells (such as antigen presenting cells). As recently described, liver-resident immune cells such as Kupffer cells play an important role in eliciting strong HBV-specific T cell responses (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

[0646] Potential for inducing durable T cell responses ("vaccine effect") following combination therapy containing AB01.

[0647] Study Design

[0648] The overall research plan is Fig. 6A and Figure 6B The dosing schedule for all cohorts is shown in Fig. 7A and Figure 7B Shown in.

[0649] Table 6 shows the treatment groups for this study. Cohorts 1a, 2a, and 3a will each enroll approximately 10 participants. Cohorts 4a and 5a will each enroll approximately 15 participants. Additionally, up to 30 floating participants may be added to any cohort. In cohorts 1a, 2a, and 4a, the total study duration for all participants is up to 100 weeks; for cohort 3a it is 92 to 96 weeks; and for cohort 5a it is 104 weeks. This includes a screening period of up to 56 days (8 weeks); the treatment period is 44 weeks for cohorts 1a and 2a, 36 or 40 weeks for cohort 3a, 20 to 44 weeks for cohort 4a, and 20 to 48 weeks for cohort 5a; and a follow-up period of up to 48 weeks.

[0650] Alternatively, for participants in cohort 4a, the treatment period may be 44 weeks, and for participants in cohort 5a it is 48 weeks. Participants in cohorts 4a and 5a may be eligible to stop study intervention (except NRTI) after week 20 and enter the follow-up period at subsequent visits if all of the following criteria are met at 2 consecutive visits before week 20: undetectable HBeAg (based on quantitative HBeAg); undetectable HBsAg (<0.05 IU / mL); and suppressed HBV DNA (<LLOQ). All other participants will continue study intervention until the end of the treatment period and enter the follow-up period 1 week after the end of the treatment period. Alternatively, for participants in cohorts 4a and 5a, the treatment period may be 44 weeks or 48 weeks, with no option to stop study intervention at week 20.

[0651] Once participants complete their respective cohort's treatment period, they will enter the follow-up period. The maximum duration of the follow-up period is 48 weeks after the last dose of study intervention. If participants meet the criteria to stop NRTI based on available data, they will stop NRTI at the F1 or F12 visit during the follow-up period. As indicated in the activity schedule, participants who are required to stop NRTI during the follow-up period will have additional study visits ( FIG. 12A to FIG. 12C). Liver elasticity imaging can be performed using FibroScan or equivalent. In addition, weight (and BMI) will be determined at screening and each study visit. A 12-lead safety electrocardiogram (ECG) will also be recorded at screening and at D8, measured in the supine position after the participant has rested comfortably for approximately 10 minutes. Participants who stop NRTIs at the F1 visit will be asked to return to the site for additional visits at F2, F6, and F10, or will be asked to return to the site for additional visits at F2, F6, F10, F28, F32, F40, and F44. Participants who stop NRTIs at the F12 visit will be asked to return to the site for additional visits at F14, F18, and F22, or will be asked to return to the site for additional visits at F14, F18, F22, F28, F32, F40, and F44. For Cohorts 1a, 2a, and 3a, AB01 PK samples will be collected pre-dose (up to 1 hour pre-dose).

[0652] Table 6. Treatment groups used in clinical studies.

[0653]

[0654] a The dose of AB01 will be determined before participants are enrolled in a cohort.

[0655] b The dosage of tenofovir disoproxil fumarate (TDF) will be 300 mg per label. Supply outside the United States may be tenofovir disoproxil (TD), and the corresponding dosage will be 245 mg per label.

[0656] c Minimum number of doses a participant receives. Participants will continue to receive additional doses of TDF until they are eligible to discontinue NRTIs.

[0657] d The dosing regimen will be finalized before participants are enrolled in the cohort.

[0658] for FIG. 10A to FIG. 10C The queues 4a and FIG. 11A to FIG. 11E For cohort 5a, PK parameters for AB01 and SIRNA01 can be assessed at different time intervals, such as shown in Table 7 below.

[0659] Table 7. Timing of PK assessments for Cohorts 4a and 5a.

[0660]

[0661] AB01 PK sample collection includes samples for free PK assays and total PK assays, as applicable. Pre-dose samples will be collected up to 1 hour prior to dosing.

[0662] Day 2 AB01 PK post-dose samples will be collected between 22 hours and 28 hours after D1 dosing.

[0663] AB01 PK post-dose samples may be collected at any time during the D8 visit.

[0664] SIRNA01PK pre-dose samples will be collected up to 1 hour prior to dosing.

[0665] Day 1, Week 20, and Week 44 SIRNA01 PK post-dose samples will be collected between 2 hours and 6 hours after dosing.

[0666] Day 2 SIRNA01 PK samples will be collected between 22 hours and 28 hours after D1 dosing.

[0667] for FIG. 12A to FIG. 12C The follow-up period shown may include additional visits. For example, activities during the follow-up period may include: FIG. 12D to FIG. 12I shown.

[0668] Participants with baseline HBsAg>3,000IU / mL may participate in the AB01 PK substudy. Participants in this substudy will have up to two additional study visits during the treatment period to collect AB01 PK samples. PK Substudy Visit 1 will be conducted 5 to 7 days after the 3rd, 4th, or 5th dose of AB01. PK Substudy Visit 2 (optional) will be conducted 5 to 7 days after the 7th, 8th, or 9th dose of AB01. In addition to samples for PK, samples for HBsAg, HBV DNA, and liver function tests will be collected at the same visits. Participants enrolled in Cohort 3a are excluded from the optional AB01 PK substudy.

[0669] AB01 is provided as a reconstituted lyophilized powder that is administered subcutaneously (SC) at up to 300 mg every 4 to 12 weeks. SIRNA01 is provided as a liquid that is administered SC at 200 mg every 4 weeks. PEG-IFNα Supplied as a liquid that is administered SC at 180 mcg weekly. Tenofovir disoproxil fumarate (TDF) It is available in tablet form for oral administration at 300 mg daily. Tenofovir is available outside the United States as tenofovir disoproxil (TD), in the United States, tenofovir is available in tablet form for oral administration at 245 mg daily.

[0670] Participants in cohort 1a / 2a will receive up to 300 mg of AB01 every 4 weeks from Day 1 to Week 44, and 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they are eligible to discontinue NRTI or the end of the follow-up period, whichever is later. Participants in cohort 3a will receive 300 mg of AB01 every 8 to 12 weeks from Day 1 to Week 44, and 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they are eligible to discontinue NRTI or the end of the follow-up period, whichever is later.

[0671] Participants in cohort 4a will receive 300 mg of AB01 and 200 mg of SIRNA01 every 4 weeks from Day 1 to Week 20 or Week 44, and 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they are eligible to discontinue NRTI or the end of the follow-up period, whichever is later. Participants in cohort 5a will receive 300 mg of AB01 and 200 mg of SIRNA01 every 4 weeks from Day 1 to Week 20 or Week 48, 180 mcg of PEG-IFNα weekly from Day 1 to Week 48, and 300 mg of TDF (or 245 mg of TD) daily starting from Day 1 until they are eligible to discontinue NRTI or the end of the follow-up period, whichever is later.

[0672] A response-guided approach will be used to evaluate the timing and duration of functional cure achieved with AB01 and TDF with or without SIRNA01. In cohort 4a, AB01 and SIRNA01 will be administered every 4 weeks starting from Day 1, while TDF will be administered daily. Participants who achieve loss of HBsAg and HBeAg and HBV DNA <LLOQ before Week 20 will be eligible to discontinue AB01 and SIRNA01 therapy. Participants who do not achieve loss of HBsAg and HBeAg and HBV DNA <LLOQ before Week 20 will continue to receive AB01 and SIRNA01 therapy until Week 44. TDF will be administered daily starting from Day 1 and will end at most when the NRTI discontinuation criteria are met at Week 12 of the follow-up period.

[0673] A response-guided approach will be used to evaluate the timing and duration of functional cure achieved with AB01 and TDF with or without SIRNA01 and PEG-IFNα. In cohort 5a, AB01 and SIRNA01 will be administered every 4 weeks starting from Day 1, while TDF will be administered daily and PEG-IFNα will be administered weekly.

[0674] Participants who achieve loss of HBsAg and HBeAg and HBV DNA < LLOQ before week 20 may be eligible to discontinue AB01, SIRNA01, and PEG-IFNα therapies, while participants who do not achieve loss of HBsAg and HBeAg and HBV DNA < LLOQ before week 20 will continue to receive AB01, SIRNA01, and PEG-IFNα until week 48. TDF will be administered daily starting from day 1 and may be discontinued at most when the NRTI discontinuation criteria are met during the first or second week of the follow-up period.

[0675] Participants may discontinue NRTI at the F1 or F12 visit during the follow-up period if all of the following criteria are met: a decrease in HBsAg from baseline of HBsAg < 100 IU / mL and ≥ 1 log10 IU / mL; suppressed HBV DNA (defined as < LLOQ); undetectable HBeAg (based on quantitative HBeAg); and ALT ≤ 2 times the upper limit of normal (ULN). Participants who meet the criteria for discontinuation of NRTI therapy will continue according to the follow-up period activity schedule. Based on the investigator's opinion, participants who meet the NRTI discontinuation criteria but are not suitable for NRTI discontinuation for other reasons may continue to receive NRTI therapy.

[0676] For the following participants, the investigator will consider retreatment with NRTI therapy during the follow-up period: an increase in HBV DNA of ≥ 2 log 10 IU / mL within 2 cycles; at any follow-up visit, HBV DNA > 100,000 IU / mL (regardless of other biochemical parameters or ALT values); confirmation of an increase in HBV DNA > 20,000 IU / mL (i.e., in two consecutive collections, regardless of other biochemical parameters or ALT values); HBV DNA > 2,000 IU / mL, in conjunction with any of the following criteria, at the same visit: confirmed total bilirubin > 2x ULN and ALT > ULN, any signs of liver decompensation (including but not limited to confirmation of an increase in prothrombin time [PT] of ≥ 2 or an increase in international normalized ratio [INR] ≥ 0.5, jaundice, ascites, encephalopathy, etc.) from baseline, ALT > 10x ULN, ALT > 2x ULN for ≥ 12 consecutive weeks, and ALT > 5x ULN for ≥ 4 consecutive weeks; confirmed HbeAg seroconversion (i.e., HbeAg positive after being negative at the time of NRTI discontinuation); or any other clinically significant event, to initiate NRTI therapy.

[0677] The HBsAg loss rate will be evaluated at the end of treatment (EOT) and 24 weeks after EOT. The proportion of participants with HBsAg loss (defined as HBsAg < LLOQ or < 0.05 IU / mL) at EOT was determined. The proportion of participants who achieved HBV DNA suppression (< LLOQ) and had HBsAg loss (0.05 < IU / mL) at EOT was also determined. The proportion of participants with HBsAg loss (defined as HBsAg < LLOQ or < 0.05 IU / mL) at 24 weeks after EOT and at the F48 follow-up visit was determined. For HBeAg-positive participants, the proportion of participants with HBeAg loss (undetectable HBeAg), the time to HBeAg loss (undetectable HBeAg), the proportion of participants with anti-HB and anti-HBe seroconversion, and the time to anti-HBe seroconversion were evaluated. At 24 weeks after stopping all treatment and at the F48 follow-up visit, the proportion of participants who achieved sustained suppression of HBV DNA (< LLOQ) and had HBsAg loss (< 0.05 IU / mL); the mean change in serum HBsAg levels relative to baseline across time points in the study; the proportion of participants who achieved HBV DNA < LLOQ across time points in the study; the HBV DNA levels and changes relative to baseline across time points in the study; the nadir and maximum changes in HBV DNA relative to baseline in the study; and the proportion of participants with virological relapse (defined as: (1) an increase of ≥ 1 log10 HBV DNA IU / mL above the nadir at at least 2 consecutive visits, or (2) quantifiable HBV DNA ≥ 1 log10 IU / mL above LLOQ at at least 2 consecutive visits after < LLOQ).

[0678] The proportion of participants who met the NRTI discontinuation or retreatment criteria in the study and the proportion of participants who achieved ALT ≤ ULN across time points in the study were also determined. The pharmacokinetics (PK), immunogenicity, and adverse events of AB01 and / or SIRNA01 were evaluated. The concentrations of AB01 and SIRNA01 PK will be quantified using validated bioanalytical assays. PK parameters may include Cmax, Clast, Tmax, Tlast, AUCinf, AUClast, %AUCexp, t1 / 2, λz, Vz / F, and CL / F. The immunogenicity data for AB01 may include the incidence and titer of anti-drug antibodies and neutralization data. Additional antiviral activities such as anti-HB, HBV RNA, and hepatitis B core-related antigen (HBcrAg) were evaluated.

[0679] 8A to 12C A schedule of activities for the cohort and follow-up period is provided.

[0680] Patient population

[0681] Participants will have chronic HBV infection and will be HBeAg positive or negative, with high HBV DNA (>2000IU / mL) and ALT levels (>ULN and ≤5x ULN). Approximately 40% of participants with baseline HBsAg greater than 10,000IU / mL at screening will be included. In addition, approximately 30% of HBeAg-positive participants will be included in the cohort. For the purposes of the study, chronic HBV infection is defined as 2 serum HBsAg, HBV DNA or HBeAg positive at least 6 months apart (any combination of these tests 6 months apart is acceptable) based on previous or current laboratory documents. The ULN value of ALT can be, for example, 34IU / mL for women and 43IU / mL for men. Participants are aged ≥18 (or legal age of consent, whichever is greater) to <66 years old. Participants also had HBsAg >10 IU / mL and body mass index (BMI) ≥18 kg / m2 to ≤35 kg / m2 at screening 2 .

[0682] Additional inclusion criteria included the following:

[0683] · Must be in good health other than chronic HBV infection, as determined by medical history, with no clinically significant findings on physical examination, vital signs, and laboratory values

[0684] Female participants must have a negative pregnancy test or confirmed postmenopausal status. Postmenopausal status is defined as the absence of menstruation for 12 months without an alternative medical reason. Women of childbearing potential (WOCBP) must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, cannot breastfeed, and must be willing to use a highly effective contraceptive method from 14 days before the study intervention 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 start of the study intervention until 48 weeks after the last dose of AB01, SIRNA01, or PEG-IFNα.

[0685] Male participants with female partners of reproductive potential must agree to meet 1 of the following contraceptive requirements from the start of the study intervention until 48 weeks after the last dose of AB01, SIRNA01, or PEG-IFNα: documented vasectomy or azoospermia, or use of male condoms plus partner use of 1 of the contraceptive options listed for WOCBP contraception. Male participants must also agree not to donate sperm from the start of the first study intervention until 48 weeks after the last dose of AB01, SIRNA01, or PEG-IFNα.

[0686] ·Able to understand and comply with study requirements and able to provide written informed consent.

[0687] Exclusion criteria included the following:

[0688] History of clinically significant liver disease due to non-HBV etiology

[0689] History or current evidence of signs of liver decompensation, including ascites, hepatic encephalopathy, and / or esophageal or gastric varices

[0690] History or current suspicion of malignancy diagnosed or treated within 5 years (topically treated squamous or noninvasive basal cell skin cancer was allowed; in situ cervical cancer was allowed if appropriately treated prior to screening); participants undergoing evaluation for malignancy were not eligible.

[0691] History of bone marrow or solid organ transplantation

[0692] · Within 7 days before Day 1, except for chronic HBV infection or any clinically significant acute

[0693] Known active infection other than symptoms such as fever (>38°C) or acute respiratory or GI illness

[0694] · Coinfected 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 were positive for HCV antibody or HDV antibody but had documented negative HCV RNA or HDV RNA, respectively, were eligible. Participants who were positive for HAV immunoglobulin M (IgM) or HEV IgM but were asymptomatic and positive for HAV immunoglobulin G (IgG) or HEV IgG were eligible.

[0695] · History or clinical evidence of alcohol or drug abuse within 12 months prior to screening, or a positive drug screen at screening, unless explained by prescription medication (diagnosis and prescription must be approved by the investigator). Note: Marijuana use is permitted

[0696] · Received study medication within 90 days or 5 half-lives (if known), whichever is longer, prior to the study intervention, or is active during the follow-up period of another clinical study involving the 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.

[0697] Any clinically significant medical or psychiatric condition that, as determined by the investigator, may interfere with study interventions, assessments, or compliance with the protocol or otherwise render the participant unsuitable for study participation.

[0698] Significant fibrosis or cirrhosis, as defined by liver elastography (FibroScan or equivalent) results >8.5 kPa at screening or Metavir F3 fibrosis or F4 cirrhosis on liver biopsy within 1 year.

[0699] History of immune complex disease

[0700] History of autoimmune disease

[0701] History of HBV-related extrahepatic disease, including but not limited to HBV-related rash, arthritis, or glomerulonephritis

[0702] History of anaphylaxis, hypersensitivity, or intolerance to the monoclonal antibodies, antibody fragments, or any excipients of AB01

[0703] Prior NRTI or PEG-IFNα therapy

[0704] Use of any of the following systemic medications within 14 days prior to study intervention and throughout the study period:

[0705] Acetaminophen (paracetamol) ≥ 3 g / day

[0706] Isoniazid

[0707] Systemic steroids (prednisone equivalents >10 mg / day) or other immunosuppressants (Note: corticosteroid administration is permitted for the treatment of immune-mediated AEs.)

[0708] Cohort 5a only: Theophylline

[0709] Cohort 5a only: Methadone

[0710] Receipt of oligonucleotides with anti-HBV activity (e.g., siRNA, antisense oligonucleotides) within 48 weeks prior to study intervention

[0711] Received AB01 within 24 weeks prior to Day 1

[0712] Participants had the following laboratory parameters at the time of laboratory testing screening:

[0713] Direct bilirubin or INR > 1.5 ULN

[0714] Total bilirubin > 1.5 times ULN

[0715] Platelets <150,000 cells / μL

[0716] Cohort 5a only: serum amylase or lipase ≥ 3 times ULN

[0717] Cohort 5a only: TSH and free T4 above ULN or below LLN

[0718] Cohort 5a only: Absolute neutrophil count (ANC) <1,500 cells / mm 3

[0719] Screening creatinine clearance (CLcr) < 30 mL / min, as calculated by the Cockcroft-Gault formula

[0720] Cohort 5a only: known hypersensitivity or contraindications to interferon products Cohort 5a only: current or past history of psychosis, bipolar disorder, schizophrenia, moderate to severe depression, suicidal ideation, suicide attempt or gesture, or high risk of suicide

[0721] Cohort 5a only: Current or past history of clinically significant retinal disease Cohort 5a only: Current or past history of chronic uncontrolled hypoglycemia or uncontrolled hyperglycemia / diabetes (defined as HbA1c ≥ 8%) at screening Cohort 5a only: Current or past history of colitis

[0722] Clinically significant abnormality on the screening 12-lead ECG (as determined by the investigator) Concomitant therapy

[0723] No concomitant treatment was allowed during the study

[0724] · NRTI use prior to Day 1 is prohibited. During the study, participants will receive TDF per protocol. No other NRTIs are permitted during the study.

[0725] The use of any of the following systemic medications within 14 days prior to the study intervention and throughout the study period is prohibited:

[0726] ○ Systemic steroids (prednisone equivalents >10 mg / day) or other immunosuppressants (Note: corticosteroid administration is permitted for the treatment of immune-mediated AEs.)

[0727] ○ Acetaminophen (paracetamol) ≥ 3 g / day

[0728] ○ Isoniazid

[0729] ○ Cohort 5a only: Theophylline

[0730] ○ Cohort 5a only: Methadone

[0731] In addition, the use of any potentially hepatotoxic medication during the study should only be considered if no therapeutic alternatives can be identified and after careful consideration of the potential risks and benefits to the participants. Medications that are potentially hepatotoxic or have been associated with drug-induced liver injury include, but are not limited to, the following ( 2016): aspirin > 3 g / day or ibuprofen ≥ 1.2 g / day; tricyclic antidepressants; valproic acid; phenytoin; amiodarone; anabolic steroids; allopurinol; amoxicillin-clavulanic acid; minocycline; nitrofurantoin; sulfamethoxazole / trimethoprim; erythromycin; rifampin; azole antifungals; and herbal or natural remedies.

[0732] While specific embodiments have been illustrated and described, it will be readily appreciated that the various embodiments described above may be combined to provide further embodiments, and that various changes may be made therein without departing from the spirit and scope of the invention.

[0733] Unless otherwise indicated, all U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in this specification or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 63 / 343,896 filed on May 19, 2022 and U.S. Provisional Patent Application No. 63 / 353,383 filed on June 17, 2022, are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified to employ concepts of the various patents, applications, and publications to provide yet further embodiments.

[0734] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be interpreted to limit the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full range of equivalents to which the claims are entitled. Therefore, the claims are not limited by the present disclosure.

[0735] sequence

[0736] SEQ ID NO: 1 (Hepatitis B virus genome—NCBI reference sequence NC_003977.2 (GenBank accession number GI: 21326584))

[0737]

[0738] SEQ ID NO: 2 (target sequence, nucleotides 1579-1597 of NC_003977.2 (GenBank Accession No. GI: ​​21326584))

[0739] GTGTGCACTTCGCTTCAC

[0740] SEQ ID NO: 3 (SIRNA01, unmodified positive strand)

[0741] GUGUGCACUUCGCUUCACA

[0742] SEQ ID NO:4 (SIRNA01, unmodified antisense strand)

[0743] UGUGAAGCGAAGUGCACACUU

[0744] SEQ ID NO: 5 (SIRNA01, modified sense strand)

[0745] gsusguGfcAfCfUfucgcuucacaL96

[0746] SEQ ID NO: 6 (SIRNA01, modified antisense strand)

[0747] usGfsuga(Agn)gCfGfaaguGfcAfcacsusu

[0748] SEQ ID NO: 7 (SIRNA02, modified sense strand)

[0749] gsusguGfcAfCfUfucgcuucacaL96

[0750] SEQ ID NO: 8 (SIRNA02, modified antisense strand)

[0751] usGfsugaAfgCfGfaaguGfcAfcacsusu

[0752] SEQ ID NO:9 (Peptide RFGF containing membrane translocation sequence)

[0753] AAVALLPAVLLALLAP

[0754] SEQ ID NO: 10 (peptide RFGF analog containing membrane translocation sequence)

[0755] AALLPVLLAAP

[0756] SEQ ID NO:11 (HIV Tat protein)

[0757] GRKKRRQRRRPPQ

[0758] SEQ ID NO: 12 (Drosophila Antennapedia mutant protein)

[0759] RQIKIWFQNRRMKWK

[0760] SEQ ID NO:13 (HBsAg S domain)

[0761] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS STTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0762] SEQ ID NO: 14 (J02203 (D, ayw3) HBsAg antigen loop sequence)

[0763] QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0764] SEQ ID NO: 15 (FJ899792 (D, adw2) HBsAg antigen loop sequence)

[0765] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0766] SEQ ID NO: 16 (AM282986 (A) HBsAg antigen loop sequence)

[0767] QGMLPVCPLIPGTTTTSTGPCKTCTTPAQGNSMFPSCCCTKPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW

[0768] SEQ ID NO: 17 (D23678 (B1) HBsAg antigen loop sequence)

[0769] QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSW

[0770] SEQ ID NO: 18 (AB117758 (C1) HBsAg antigen loop sequence)

[0771] QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSW

[0772] SEQ ID NO: 19 (AB205192 (E) HBsAg antigen loop sequence)

[0773] QGMLPVCPLIPGSSTTSTGPCRTCTTLAQGTSMFPSCCCSKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLS

[0774] SEQ ID NO:20 (X69798 (F4) HBsAg antigen loop sequence)

[0775] QGMLPVCPLLPGSTTTSTGPCTCTTLAQGTSMFPSCCCSKPSDGNCTCIPIPSSWALGKYLWEWASARFSW

[0776] SEQ ID NO:21 (AF160501 (G) HBsAg antigen loop sequence)

[0777] QGMLPVCPLIPGSSTTSTGPCTCTTPAQGNSMYPSCCCTPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW

[0778] SEQ ID NO:22 (AY090454 (H) HBsAg antigen loop sequence)

[0779] QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFGKYLWEWASARFSW

[0780] SEQ ID NO:23 (AF241409 (I) HBsAg antigen loop sequence)

[0781] QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCTKPSDGNCTCIPIPSSWAFAKYLWEWASARFSW

[0782] SEQ ID NO:24 (AB486012 (J) HBsAg antigen loop sequence)

[0783] QGMLPVCPLLPGSTTTSTGPCRTCTITAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFAKFLWEWASVRFSW

[0784] SEQ ID NO:25 (HBsAg Y100C / P120T HBsAg antigen loop sequence)

[0785] CQGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSS WAFGFLWEWASARFSW

[0786] SEQ ID NO:26 (HBsAg P120T HBsAg antigen loop sequence)

[0787] QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0788] SEQ ID NO:27 (HBsAg P120T / S143L HBsAg antigen loop sequence)

[0789] QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTKPLDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0790] SEQ ID NO:28 (HBsAg C121 S HBsAg antigen loop sequence)

[0791] QGMLPVCPLIPGSSTTGTGPSRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0792] SEQ ID NO:29 (HBsAg R122D HBsAg antigen loop sequence)

[0793] QGMLPVCPLIPGSSTGTGTGPCDTCTTPAQGTSMYPSCCCTKPSDG NCTCIPIPSSWAFGKFLWEWASARFSW

[0794] SEQ ID NO:30 (HBsAg R122I HBsAg antigen loop sequence)

[0795] QGMLPVCPLIPGSSTTGTGPCITCTTPAQGTSMYPSCCCTPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0796] SEQ ID NO:31 (HBsAg T123N HBsAg antigen loop sequence)

[0797] QGMLPVCPLIPGSSTTGTGPCRNCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0798] SEQ ID NO:32 (HBsAg Q129H HBsAg antigen loop sequence)

[0799] QGMLPVCPLIPGSSTTGTGPCRTCTTPAHGTSMYPSCCCTKPSDG NCTCIPIPSSWAFG KFLWEWASARFSW

[0800] SEQ ID NO:33 (HBsAg Q129L HBsAg antigen loop sequence)

[0801] QGMLPVCPLIPGSSTTGTGPCRTCTTPALGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0802] SEQ ID NO:34 (HBsAg M133H HBsAg antigen loop sequence)

[0803] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSHYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0804] SEQ ID NO:35 (HBsAg M133L HBsAg antigen loop sequence)

[0805] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSLYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0806] SEQ ID NO:36 (HBsAg M133T HBsAg antigen loop sequence)

[0807] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSTYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0808] SEQ ID NO:37 (HBsAg K141 E HBsAg antigen loop sequence)

[0809] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTEPSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0810] SEQ ID NO:38 (HBsAg P142S HBsAg antigen loop sequence)

[0811] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKSSDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0812] SEQ ID NO:39 (HBsAg S143K HBsAg antigen loop sequence)

[0813] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPKDGNCTCIPIPSSWAFGKFLWEWASARFSW

[0814] SEQ ID NO:40 (HBsAg D144A HBsAg antigen loop sequence)

[0815] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTPSAGNCTCIPIPSSWAFGKFLWEWASARFSW

[0816] SEQ ID NO:41 (HBsAg G145R HBsAg antigen loop sequence)

[0817] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDRNCTCIPIPSSWAFGKFLWEWASARFSW

[0818] SEQ ID NO:42 (HBsAg N146A HBsAg antigen loop sequence)

[0819] QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGACTCIPIPSSWAFGKFLWEWASARFSW

[0820] SEQ ID NO:43 (HBsAg genotype D S domain (Genbank accession number FJ899792))

[0821] MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSC...

Claims

1. A method for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof, the method comprising: administering to the subject: (a) Anti-HBV antibodies; (b) siRNA targeting HBV mRNA; and (c) nucleoside reverse transcriptase inhibitors (NRTIs); and Wherein: the subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤upper limit of normal (ULN), the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks before treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA.

2. An anti-HBV antibody for use in a method for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: The subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL prior to treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤ the upper limit of normal (ULN), the subject has not been previously administered NRTIs, the subject has not received NRTIs within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA; and The subject was also administered siRNA targeting HBV mRNA and a nucleoside reverse transcriptase inhibitor (NRTI).

3. An anti-HBV antibody; an siRNA targeting HBV mRNA; and a nucleoside (acid) reverse transcriptase inhibitor (NRTI), for use in a method of treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: The subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤upper limit of normal (ULN), the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA.

4. An anti-HBV antibody for use in the manufacture of a medicament for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: The subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤upper limit of normal (ULN), the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA; and The subject was also administered siRNA targeting HBV mRNA and a nucleoside reverse transcriptase inhibitor (NRTI).

5. An anti-HBV antibody for use in the manufacture of a first medicament, an siRNA targeting HBV mRNA for use in the manufacture of a second medicament; and a nucleoside (acid) reverse transcriptase inhibitor (NRTI) for use in the manufacture of a third medicament, wherein the first medicament, the second medicament and the third medicament are used to treat hepatitis B virus (HBV) infection or HBV-related diseases in a subject in need thereof; wherein: The subject is HBeAg negative, the subject has an HBV DNA level of ≤2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level of ≤upper limit of normal (ULN), the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, and / or the subject has not been previously administered siRNA targeting HBV mRNA.

6. The method of claim 1, wherein the subject is HBeAg negative.

7. The method of claim 1, wherein the subject has an HBV DNA level of ≤2000 IU / mL before treatment.

8. The method of claim 1, wherein the subject has an HBV DNA level of <2000 IU / mL prior to treatment.

9. The method of claim 1, wherein the subject is non-cirrhotic and / or has an alanine aminotransferase (ALT) level ≤ the upper limit of normal (ULN).

10. The method of claim 1, wherein the subject is non-cirrhotic and / or has an alanine aminotransferase (ALT) level < the upper limit of normal (ULN).

11. The method of claim 1, wherein the subject has not been previously administered an NRTI.

12. The method of claim 1, wherein the subject has not received an NRTI within 24 weeks prior to treatment.

13. The method of claim 1, wherein the subject has not been previously administered anti-HIV antibodies.

14. The method of claim 1, wherein the subject has not been previously administered siRNA targeting HBV mRNA.

15. A method for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof, the method comprising: administering to the subject: (a) Anti-HBV antibodies; (b) siRNA targeting HBV mRNA; (c) interferon-α; and (d) nucleoside reverse transcriptase inhibitors (NRTIs); and Wherein: the subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks before treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment.

16. An anti-HBV antibody for use in a method of treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: The subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks before treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment; and The subject was also administered siRNA targeting HBV mRNA, interferon-α, and nucleos(t)ide reverse transcriptase inhibitors (NRTIs).

17. An anti-HBV antibody; an siRNA targeting HBV mRNA; an interferon-α; and a nucleoside(t) reverse transcriptase inhibitor (NRTI), for use in a method of treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: The subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment.

18. An anti-HBV antibody for use in the manufacture of a medicament for treating hepatitis B virus (HBV) infection or HBV-related disease in a subject in need thereof; wherein: The subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks before treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment; and The subject was also administered siRNA targeting HBV mRNA, interferon-α, and nucleos(t)ide reverse transcriptase inhibitors (NRTIs).

19. An anti-HBV antibody for use in the manufacture of a first medicament, an siRNA targeting HBV mRNA for use in the manufacture of a second medicament, an interferon-α for use in the manufacture of a third medicament, and a nucleoside (acid) reverse transcriptase inhibitor (NRTI) for use in the manufacture of a fourth medicament, wherein the first medicament, the second medicament, the third medicament and the fourth medicament are used to treat hepatitis B virus (HBV) infection or HBV-related diseases in subjects in need thereof; wherein: The subject is HBeAg negative or HBeAg positive, the subject has an HBV DNA level of >2000 IU / mL before treatment, the subject is non-cirrhotic, the subject has an alanine aminotransferase (ALT) level >the upper limit of normal (ULN) and ≤5 times the ULN, the subject has not been previously administered NRTI, the subject has not received NRTI within 24 weeks prior to treatment, the subject has not been previously administered anti-HIV antibodies, the subject has not been previously administered 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 >10 IU / mL before treatment.

20. The method of claim 15, wherein the subject is HBeAg negative.

21. The method of claim 15, wherein the subject is HBeAg positive.

22. The method of claim 15, wherein the subject has an HBV DNA level of >2000 IU / mL prior to treatment.

23. The method of claim 15, wherein the subject has an alanine aminotransferase (ALT) level > the upper limit of normal (ULN) and ≤ 5 times the ULN.

24. The method of claim 15, wherein the subject has not been previously administered an NRTI.

25. The method of claim 15, wherein the subject has not been previously administered anti-HIV antibodies.

26. The method of claim 15, wherein the subject has not been previously administered siRNA targeting HBV mRNA.

27. The method of claim 15, wherein the subject has not been previously administered interferon-α.

28. The method of claim 15, wherein the subject is HBsAg positive prior to treatment.

29. The method of claim 15, wherein the subject has an HBsAg level of >10 IU / mL prior to treatment.

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

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

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

33. The method of claim 1, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

34. The method of claim 1, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 46 and 47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 50 and 52, respectively.

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

53. H ).

36. The method of claim 1, wherein the anti-HBV antibody comprises: (a) a light chain variable domain (V L ) amino acid sequence; and (b) the heavy chain variable domain (V H ) amino acid sequence.

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

57.

38. The method of claim 1, wherein the anti-HBV antibody comprises: (a) the light chain amino acid sequence according to SEQ ID NO:59, and (b) the heavy chain amino acid sequence according to SEQ ID NO:

57.

39. The method of claim 15, wherein the anti-HBV antibody is a human antibody.

40. The method of claim 15, wherein the antibody is HBC34 or a non-natural variant of HBC34.

41. The method of claim 15, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NO: 44, 45 or 46 and 47, respectively; and (ii) the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NO: 48, 49 or 50 and 52, respectively.

42. The method of claim 15, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively.

43. The method of claim 15, wherein the anti-HBV antibody comprises: (i) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 46 and 47, respectively; and (ii) CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 50 and 52, respectively.

44. The method of claim 15, wherein the anti-HBV antibody comprises: (a) a light chain variable domain (V L) having at least 90%, at least 95% or 100% identity to the amino acid sequence according to SEQ ID NO: 55 L ); and (b) a heavy chain variable domain (V H ).

45. The method of claim 15, wherein the anti-HBV antibody comprises: (a) a light chain variable domain (V L ) amino acid sequence; and (b) the heavy chain variable domain (V H ) amino acid sequence.

46. ​​The method of claim 15, wherein the anti-HBV antibody comprises: (a) a light chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:59, and (b) a heavy chain that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO:

57.

47. The method of claim 15, wherein the anti-HBV antibody comprises: (a) the light chain amino acid sequence according to SEQ ID NO:59, and (b) the heavy chain amino acid sequence according to SEQ ID NO:

57.

48. The method of claim 1, wherein the siRNA comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from nucleotides 1579-1597 of SEQ ID NO: 1 by no more than 3 nucleotides, wherein T is replaced by U.

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

50. The method of claim 49, wherein the sense strand of the siRNA comprises or consists of a nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

51. The method of claim 50, wherein at least one strand of the siRNA comprises a 3' overhang of at least 1 nucleotide.

52. The method of claim 50, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs in length.

53. The method of claim 50, wherein each strand of the RNAi agent has 15 to 30 nucleotides.

54. The method of claim 50, wherein substantially all of the nucleotides of the sense strand of the siRNA and substantially all of the nucleotides of the antisense strand of the siRNA are modified nucleotides, and Wherein the sense strand is conjugated to a ligand attached at the 3' end.

55. The method of claim 54, wherein the ligand is one or more GalNAc derivatives attached via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

56. The method of claim 55, wherein the ligand is 57. The method of claim 56, wherein the siRNA is conjugated to the ligand as shown in the following structure: Wherein X is O or S.

58. The method of claim 57, wherein X is O.

59. The method of claim 50, 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 conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic 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 phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, an adenosine-glycol nucleic acid, or a nucleotide comprising a 5'-phosphate mimetic.

60. The method of claim 50, wherein the siRNA comprises a phosphate backbone modification, a 2' ribose modification, a 5' triphosphate modification, or a GalNAc conjugation modification.

61. The method of claim 50, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand of the siRNA are modified nucleotides.

62. The method of claim 50, wherein the siRNA comprises a sense strand and an antisense strand, the sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), the antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-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; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

63. The method of claim 62, wherein the L96 is conjugated to the sense strand as shown in the following structure: Where X is O.

64. The method of claim 15, wherein the siRNA comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises nucleotides 1579-1580 of SEQ ID NO:

1. 1597 At least 15 consecutive nucleotides differing by no more than 3 nucleotides, wherein T is replaced by U.

65. The method of claim 15, wherein the antisense strand of the siRNA comprises or consists of a nucleotide sequence of 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 4).

66. The method of claim 65, wherein the sense strand of the siRNA comprises or consists of a nucleotide sequence of 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 3).

67. The method of claim 66, wherein at least one strand of the siRNA comprises a 3' overhang of at least 1 nucleotide.

68. The method of claim 66, wherein the double-stranded region of the siRNA is 15 to 30 nucleotide pairs in length.

69. The method of claim 66, wherein each strand of the RNAi agent has 15 to 30 nucleotides.

70. The method of claim 66, wherein substantially all of the nucleotides of the sense strand of the siRNA and substantially all of the nucleotides of the antisense strand of the siRNA are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end.

71. The method of claim 70, wherein the ligand is one or more GalNAc derivatives attached via a monovalent linker, a divalent branched linker, or a trivalent branched linker.

72. The method of claim 71, wherein the ligand is 73. The method of claim 72, wherein the siRNA is conjugated to the ligand as shown in the following structure: Wherein X is O or S.

74. The method of claim 73, wherein X is O.

75. The method of claim 66, 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 conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, an adenosine-glycol nucleic acid, or a nucleotide comprising a 5'-phosphate mimetic.

76. The method of claim 66, wherein the siRNA comprises a phosphate backbone modification, a 2' ribose modification, a 5' triphosphate modification, or a GalNAc conjugation modification.

77. The method of claim 66, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand of the siRNA are modified nucleotides.

78. The method of claim 66, wherein the siRNA comprises a sense strand and an antisense strand, the sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), the antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-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; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

79. The method of claim 78, wherein the L96 is conjugated to the sense strand as shown in the following structure: Where X is O.

80. The method of claim 1, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil (TD), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, fovir dipivoxil, lobcavir, famciclovir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-alpha and ganciclovir, besifovir (ANA-380 / LB-80380) or tenofovir-exaliades (TLX / CMX157).

81. The method of claim 15, wherein the NRTI is tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir disoproxil (TD), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clavudine, ritonavir, fovir dipivoxil, lobcavir, famciclovir, N-acetyl-cysteine ​​(NAC), PC1323, theradigm-HBV, thymosin-alpha and ganciclovir, besifovir (ANA-380 / LB-80380) or tenofovir-exaliades (TLX / CMX157).

82. The method of claim 15, wherein the interferon-α is interferon-α-2a.

83. The method of claim 82, wherein the interferon-α is pegylated.

84. The method of claim 83, wherein the interferon-α is pegylated interferon-α-2a (PEG-IFNα-2a).

85. The method of claim 1, wherein: (i) The anti-HBV antibody comprises: (a) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively, and the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively; and / or (b) a light chain variable domain (V L) that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO: 55 L ); and a heavy chain variable domain (V H ); and / or (c) 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 according to SEQ ID NO:57; and (ii) the siRNA comprises a sense strand and an antisense strand or consists of the sense strand and the antisense strand, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and the antisense strand comprises 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; and (iii) The NRTI is tenofovir or tenofovir disoproxil fumarate (TDF).

86. The method of claim 15, wherein: (i) The anti-HBV antibody comprises: (a) the CDRH1, CDRH2 and CDRH3 amino acid sequences according to SEQ ID NOs: 44, 45 and 47, respectively, and the CDRL1, CDRL2 and CDRL3 amino acid sequences according to SEQ ID NOs: 48, 49 and 52, respectively; and / or (b) a light chain variable domain (V L) that is at least 90%, at least 95% or 100% identical to the amino acid sequence according to SEQ ID NO: 55 L );And a heavy chain variable domain (V H ); and / or (c) a light chain having at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 59, and (b) a light chain having at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID NO: A heavy chain having at least 90%, at least 95%, or 100% identity to the amino acid sequence of 57; and (ii) the siRNA comprises a sense strand and an antisense strand or consists of the sense strand and the antisense strand, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 5), and the antisense strand comprises 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 6), where a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2′-fluoroadenosine-3′-phosphate, 2′-fluorocytidine-3′-phosphate, 2′-fluoroguanosine-3′-phosphate, and 2′-fluorouridine-3′-phosphate, respectively; (Agn) is adenosine-glycol nucleic acid (GNA); s is a phosphorothioate bond; and L96 is N-[tri(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol; (iii) the interferon-α is polyethylene glycol interferon-α-2a (PEG-IFNα-2a); and (iv) The NRTI is tenofovir or tenofovir disoproxil fumarate (TDF).

87. The method of claim 1, wherein the anti-HBV antibody and the siRNA are administered every 4 weeks.

88. The method of claim 15, wherein the anti-HBV antibody and the siRNA are administered every 4 weeks.

89. The method of claim 15, wherein the interferon-α is administered once a week.

90. The method of claim 1 or 15, wherein the NRTI is administered daily.

91. The method of any of the preceding claims, wherein the anti-HBV antibody is administered at a dose of 300 mg.

92. The method of claim 91, wherein the anti-HBV antibody is administered over a treatment period of 44 weeks, 48 ​​weeks or longer.

93. The method of any of the preceding claims, wherein the siRNA is administered at a dose of 200 mg.

94. The method of claim 93, wherein the siRNA is administered over a treatment period of 44 weeks, 48 ​​weeks or longer.

95. The method of any one of claims 15, 20 to 29, 39 to 47, 64 to 79, 81 to 84, 86, and 88 to 94, wherein the interferon-α is administered at a dose of 180 meg.

96. The method of claim 95, wherein the dose of the interferon-α is administered over a treatment period of 44 weeks, 48 ​​weeks, or longer.

97. The method of any of the preceding claims, wherein the NRTI is administered at a dose of 300 mg.

98. The method of any one of claims 1 to 96, wherein the NRTI is administered at a dose of 245 mg.

99. The method of claim 97 or claim 98, wherein the NRTI is administered over a treatment period of 44 weeks, 48 ​​weeks or longer.

100. The method of any of the preceding claims, wherein administration of the siRNA and the anti-HBV antibody to the subject is initiated on the same day.

101. The method of any of the preceding claims, wherein the subject has chronic HBV.

102. The method of any of the preceding claims, wherein the subject has hepatitis delta virus (HDV) infection.

103. The method of any of the preceding claims, wherein the HBV-related disease is chronic HBV.

104. The method of any of the preceding claims, wherein the HBV-related disease is HDV infection.

105. The method of any of the preceding claims, wherein the subject is a human.

106. An anti-HBV antibody; an siRNA targeting HBV mRNA; and an NRTI; for use in the method of any one of claims 1, 6 to 14, 30 to 38, 48 to 63, 80, 85, 87, 89 to 94 and 97 to 105.

107. An anti-HBV antibody; an siRNA targeting HBV mRNA; and an NRTI; for use in the manufacture of a medicament for use in a method according to any one of claims 1, 6 to 14, 30 to 38, 48 to 63, 80, 85, 87, 89 to 94 and 97 to 105.

108. Use of an anti-HBV antibody in the manufacture of a first drug; use of an siRNA targeting HBV mRNA in the manufacture of a second drug; and use of an NRTI in the manufacture of a third drug; wherein The first drug, the second drug and the third drug are to be used in a combination therapy according to any one of claims 1, 6 to 14, 30 to 38, 48 to 63, 80, 85, 87, 89 to 94 and 97 to 105.

109. An anti-HBV antibody; an siRNA targeting HBV mRNA; an interferon-α; and an NRTI; for use in the method of any one of claims 15, 20 to 29, 39 to 47, 64 to 79, 81 to 84, 86 and 88 to 105.

110. An anti-HBV antibody; an siRNA targeting HBV mRNA; an interferon-α; and an NRTI; for use in the manufacture of a medicament for use in a method according to any one of claims 15, 20 to 29, 39 to 47, 64 to 79, 81 to 84, 86 and 88 to 105.

111. Use of an anti-HBV antibody in the manufacture of a first drug; use of an siRNA targeting HBV mRNA in the manufacture of a second drug; use of interferon-α in the manufacture of a third drug; and Use of an NRTI in the manufacture of a fourth medicament; wherein the first medicament, the second medicament, the third medicament and the fourth medicament are to be used in a combination therapy according to any one of claims 15, 20 to 29, 39 to 47, 64 to 79, 81 to 84, 86 and 88 to 105.

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

113. A kit, comprising: A pharmaceutical composition comprising an anti-HBV antibody and a pharmaceutically acceptable excipient; A pharmaceutical composition comprising siRNA targeting HBV mRNA and a pharmaceutically acceptable excipient; A pharmaceutical composition comprising interferon-α and a pharmaceutically acceptable excipient; and A pharmaceutical composition comprising a NRTI and a pharmaceutically acceptable excipient.

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