Pharmaceutical combinations for the treatment of HBV

Abstract

The present invention relates to a pharmaceutical combination for treating Hepatitis B virus (HBV) infection, comprising administering at least two, preferably two or three, different HBV therapeutics. In particular, the present invention relates to a pharmaceutical combination comprising an RNAi oligonucleotide targeting HBV and an anti-PDL1 antisense oligonucleotide.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical combination for treating hepatitis B virus (HBV) infection, comprising administering at least two, preferably two or three, different HBV therapeutic agents. In particular, the present invention relates to a pharmaceutical combination comprising an RNAi oligonucleotide targeting HBV and an anti-PDL1 antisense oligonucleotide. [Background technology]

[0002] HBV infection remains a major health problem worldwide with an estimated 350 million chronic carriers. Approximately 25% of carriers can be expected to die from chronic hepatitis, cirrhosis, or liver cancer. Hepatitis B virus is the second most important carcinogen after tobacco, causing 60% to 80% of all primary liver cancers.

[0003] The outer envelope proteins of HBV are collectively known as hepatitis B surface antigen (HBsAg). HBsAg consists of three related polypeptides, designated S, M, and L, encoded by overlapping open reading frames (ORFs). The smallest envelope protein is the 226-amino acid S, designated the S-ORF. M and L are produced from an upstream translation initiation site and add 55 and 108 amino acids to S, respectively. HBV S, M, and L glycoproteins are found in the viral envelope of intact, infectious HBV virions, called Dane particles, and all three are produced and secreted in vast excess to form noninfectious subviral spherical and filamentous particles (both called decoy particles) found in the blood of patients with chronic HBV infection. The abundance of HBsAg on the surface of decoy particles is thought to inhibit humoral immunity and spontaneous clearance in patients with chronic HBV infection (CHB).

[0004] The current standard of care for chronic HBV infection is treatment with oral nucleoside(t) analogs such as entecavir or tenofovir, which inhibit HBV DNA synthesis to suppress HBV replication but do not directly act on viral antigens such as HBsAg. Nucleoside(t) analogs only show low levels of HBsAg clearance, even with long-term treatment. In this regard, patients with chronic hepatitis B exhibit very weak HBV T cell responses and lack anti-HBs antibodies, which is thought to be one of the reasons why these patients are unable to clear the virus.

[0005] A clinically important goal is to achieve functional cure of chronic HBV infection, defined as HBsAg seroconversion and elimination of serum HBV DNA. This is expected to result in a durable response, thereby preventing the development of cirrhosis and liver cancer and prolonging survival. Currently, chronic HBV infection cannot be completely eradicated due to the long-term or persistence of the viral genome as covalently closed circular DNA (cccDNA) within the nuclei of infected hepatocytes. Complete cure from chronic HBV infection would require the removal of this cccDNA from infected hepatocytes.

[0006] The review article Soriano et al. 2017 Expert Opinion on Investigational Drugs Vol. 26, pp. 843, describes the current state of drug development aimed at achieving either a functional or complete cure for HBV. The article highlights some of the more than 30 drugs currently being tested in HBV therapy and notes that any effective treatment that results in a cure will likely require a combination of virus-targeted therapy and immunotherapy.

[0007] Antisense oligonucleotides are essentially single-stranded oligonucleotides that can modulate the expression of target genes by hybridizing to target nucleic acids. Target modulation can be down-regulation via RNase H-mediated degradation or transcription blockade. Antisense oligonucleotides can also up-regulate targets, for example, through splice switching or microRNA suppression. For targets in the liver, GalNAc conjugation has proven highly effective for delivering antisense oligonucleotides. WO 2014 / 179627 and WO 2015 / 173208 describe HBV treatment by degrading HBV mRNA in hepatocytes using single-stranded antisense oligonucleotides in combination with GalNAc conjugation. Various combination therapies, including the TLR7 agonist GS-9620, are briefly mentioned in WO 2015 / 173208.

[0008] WO 2016 / 077321 describes HBV treatment by degrading HBV mRNA in hepatocytes using double-stranded siRNA in combination with GalNAc conjugation on the sense strand. Various combination therapies, including TLR7 agonists, are briefly mentioned.

[0009] WO 2017 / 157899 describes single-stranded LNA oligonucleotide conjugates for reducing PD-L1 expression. WO 2019 / 079781 describes RNAi therapeutics targeting HBsAg.

[0010] To the best of the inventors' knowledge, no specific combination of therapeutic oligonucleotides against HBV has been tested in vitro or in vivo in the art.

[0011] Object of the invention The present invention identifies novel pharmaceutical combinations of HBV therapeutic agents that offer advantages over monotherapy treatment. In particular, the present invention identifies novel pharmaceutical combinations of HBV-targeting RNAi oligonucleotides and anti-PDL1 antisense oligonucleotides, and advantageous dosing regimens thereof. The specific combination of HBV-targeting RNAi oligonucleotides and anti-PDL1 antisense oligonucleotides achieves surprising synergistic effects on HBV serum markers beyond those expected from these individual therapeutic agents alone. Summary of the Invention

[0012] The invention is defined by the claims.The present description provides further illustrations of embodiments and alternatives according to the present invention.

[0013] In one aspect, the present invention provides a pharmaceutical combination comprising at least two HBV therapeutic agents. As used herein, an HBV therapeutic agent is any drug or treatment useful for treating HBV infection. The HBV therapeutic agent may be an active ingredient, a prodrug, a composition, a conjugate, or any other form that results in the realization of the therapeutic effect of the drug when that form is administered to a patient.

[0014] In a preferred embodiment, the pharmaceutical combination comprises an RNAi oligonucleotide targeting HBV and an anti-PDL1 oligonucleotide.

[0015] In one embodiment, the pharmaceutical combination comprises an RNAi oligonucleotide targeting HBV, defined herein as Therapeutic Agent T1, and an anti-PDL1 antisense oligonucleotide, defined herein as Therapeutic Agent T2.

[0016] In a further aspect, the present invention provides a composition comprising the pharmaceutical combination of the present invention.Preferably, in the pharmaceutical combination, the RNAi oligonucleotide targeting HBV is contained in a first composition, the anti-PDL1 antisense oligonucleotide is contained in a second composition, and any additional HBV therapeutic agent is optionally contained in a third composition.

[0017] In a further aspect, the present invention provides a kit of parts comprising a first HBV therapeutic agent comprised in a pharmaceutical combination as defined herein for treating hepatitis B virus infection, and instructions for co-administration with a second HBV therapeutic agent comprised in a pharmaceutical combination as defined herein, in some embodiments, the kit comprises both or all of the HBV therapeutic agents comprised in the combination.

[0018] In a further aspect, the present invention provides the use of a pharmaceutical combination, composition or kit of the present invention for treating hepatitis B virus infection.

[0019] In a further aspect, the present invention provides a pharmaceutical combination, composition or kit of the invention for use in medicine.

[0020] In a further aspect, the present invention provides a pharmaceutical combination, composition or kit of the present invention for use in the treatment of hepatitis B virus infection.

[0021] In a further aspect, the present invention provides the use of a pharmaceutical combination, composition or kit of the present invention in the manufacture of a medicament.

[0022] In a further aspect, the present invention provides the use of a pharmaceutical combination, composition or kit of the invention in the manufacture of a medicament for treating hepatitis B virus infection.

[0023] In a further aspect, the present invention provides a method for treating hepatitis B virus infection, comprising administering a therapeutically effective amount of the pharmaceutical combination, composition, or kit of the present invention to a subject infected with hepatitis B virus infection.

[0024] In a further aspect, the present invention provides a method of reducing the expression of hepatitis B virus surface antigen in a cell, comprising delivering to the cell a pharmaceutical combination or composition of the present invention.

[0025] The present invention further provides advantageous dosing regimens for administering the pharmaceutical combinations of the present invention. [Brief explanation of the drawings]

[0026] [Figure 1] Serum levels of HBsAg (Panel A) and changes in HBsAg serum levels (Panel B) over the course of the study herein are shown. [Figure 2] Serum levels of HBeAg (Panel A) and changes in HBeAg serum levels (Panel B) over the course of the study herein are shown. [Figure 3] Serum levels of HBV-DNA (Panel A) and changes in HBV-DNA serum levels (Panel B) over the course of the study herein are shown. [Figure 4] Figure 1 shows the changes in HBsAg, HBeAg and HBV-DNA serum levels over the course of the study herein compared to day 0 of the study. HBV siRNA surrogate = sT1, PDL1 LNA = sT2. Results for the study group administered the pharmaceutical combination of the present invention (G10) are shown in contrast to the study groups administered a vehicle control (G1), an equivalent dose of an RNAi oligonucleotide targeting HBV as monotherapy (G03), and an equivalent dose of an anti-PDL1 antisense oligonucleotide as monotherapy (G06). [Figure 5-1] A specific definition of the therapeutic agent T1, an RNAi oligonucleotide targeting HBV, used in the preferred pharmaceutical combination of the present invention is provided. [Figure 5-2] A specific definition of the therapeutic agent T1, an RNAi oligonucleotide targeting HBV, used in the preferred pharmaceutical combination of the present invention is provided. [Figure 5-3] A specific definition of the therapeutic agent T1, an RNAi oligonucleotide targeting HBV, used in the preferred pharmaceutical combination of the present invention is provided. [Figure 5-4] A specific definition of the therapeutic agent T1, an RNAi oligonucleotide targeting HBV, used in the preferred pharmaceutical combination of the present invention is provided. [Figure 6-1]

[0023] Figure 2 shows the results of Example 2, including the change in serum HBsAg and HBV-DNA levels when comparable alternative therapeutic agents T1 and T3 are administered alone and in combination. The inventive combination of T1 and T3 shows a significant reduction in HBsAg and HBV-DNA. [Figure 6-2]

[0023] Figure 2 shows the results of Example 2, including the change in serum HBsAg and HBV-DNA levels when comparable alternative therapeutic agents T1 and T3 are administered alone and in combination. The inventive combination of T1 and T3 shows a significant reduction in HBsAg and HBV-DNA. [Figure 7-1]

[0023] Figure 1 shows the results of Example 3, including changes in serum HBsAg, HBV-DNA, and HBeAg levels upon administration of comparable alternative treatments T1 and T5 as defined in Example 3. The inventive combination of T1 and T5 resulted in a significant reduction in serum markers, and in particular, this combination significantly reduced serum levels of HBsAg and HBV-DNA during the T5 treatment period from days 21 to 33. [Figure 7-2]

[0023] Figure 1 shows the results of Example 3, including changes in serum HBsAg, HBV-DNA, and HBeAg levels upon administration of comparable alternative treatments T1 and T5 as defined in Example 3. The inventive combination of T1 and T5 resulted in a significant reduction in serum markers, and in particular, this combination significantly reduced serum levels of HBsAg and HBV-DNA during the T5 treatment period from days 21 to 33. [Figure 7-3]

[0023] Figure 1 shows the results of Example 3, including changes in serum HBsAg, HBV-DNA, and HBeAg levels upon administration of comparable alternative treatments T1 and T5 as defined in Example 3. The inventive combination of T1 and T5 resulted in a significant reduction in serum markers, and in particular, this combination significantly reduced serum levels of HBsAg and HBV-DNA during the T5 treatment period from days 21 to 33. [Figure 8-1]

[0023] Figure 1 shows the results of Example 3, including changes in serum HBsAg, HBV-DNA, and HBeAg levels upon administration of comparable alternative treatments T1 and T5 as defined in Example 3. The inventive combination of T1 and T5 resulted in a significant reduction in serum markers, and in particular, this combination significantly reduced serum levels of HBsAg and HBV-DNA during the T5 treatment period from days 21 to 33. [Figure 8-2]

[0023] Figure 1 shows the results of Example 3, including changes in serum HBsAg, HBV-DNA, and HBeAg levels upon administration of comparable alternative treatments T1 and T5 as defined in Example 3. The inventive combination of T1 and T5 resulted in a significant reduction in serum markers, and in particular, this combination significantly reduced serum levels of HBsAg and HBV-DNA during the T5 treatment period from days 21 to 33. [Figure 8-3]

[0023] Figure 1 shows the results of Example 3, including changes in serum HBsAg, HBV-DNA, and HBeAg levels upon administration of comparable alternative treatments T1 and T5 as defined in Example 3. The inventive combination of T1 and T5 resulted in a significant reduction in serum markers, and in particular, this combination significantly reduced serum levels of HBsAg and HBV-DNA during the T5 treatment period from days 21 to 33.

[0027] definition Oligonucleotides The term "oligonucleotide," as used herein, is defined as a molecule containing two or more covalently linked nucleosides, as commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are typically produced in a laboratory by solid-phase chemical synthesis, followed by purification and isolation. Reference to the sequence of an oligonucleotide refers to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or modifications thereof. The oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides or nucleotides, such as 2'-sugar-modified nucleosides.

[0028] Furthermore, an oligonucleotide is a short nucleic acid, for example, less than 100 nucleotides in length. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide may or may not have a double-stranded region. As a non-limiting example, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0029] synthesis As used herein, the term "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or is not otherwise derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0030] double-stranded oligonucleotides As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide in a substantially duplex form. In some embodiments, the complementary base pairing in the duplex region of the double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides in covalently separated nucleic acid strands. In some embodiments, the complementary base pairing in the duplex region of the double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides in covalently linked nucleic acid strands. In some embodiments, the complementary base pairing in the duplex region of the double-stranded oligonucleotide is formed from a single-stranded nucleic acid that folds back (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides that base pair together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are fully duplexed with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are partially duplexed, e.g., have overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of partially complementary nucleotides and therefore may have one or more mismatches, which may include internal or terminal mismatches.

[0031] chain As used herein, the term "strand" refers to a single, continuous sequence of nucleotides linked together via internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, e.g., a 5' end and a 3' end.

[0032] double stranded As used herein, the term "duplex" with respect to nucleic acids (eg, oligonucleotides) refers to the structure formed by complementary base pairing of two antiparallel sequences of nucleotides.

[0033] overhang As used herein, the term "overhang" refers to terminal non-base-paired nucleotide(s) originating from one strand or region that extends beyond the end of the complementary strand with which it forms a duplex. In some embodiments, the overhang comprises one or more unpaired nucleotides extending from the duplex region at the 5'-end or 3'-end of the double-stranded oligonucleotide. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand of the double-stranded oligonucleotide.

[0034] loop As used herein, the term "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) that is adjacent to two antiparallel regions of nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell), the two antiparallel regions flanking the unpaired region hybridize to form a duplex (called a "stem").

[0035] RNAi oligonucleotides As used herein, the term "RNAi oligonucleotide" refers to either (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand, or a portion thereof, is used by Argonaute 2 (Ago2) endonuclease in cleaving a target mRNA, or (b) a single-stranded oligonucleotide having a single-stranded antisense strand, where the antisense strand (or a portion thereof) is used by Ago2 endonuclease in cleaving a target mRNA.

[0036] RNAi agents The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," used interchangeably herein, refer to agents, e.g., RNAi oligonucleotides, that contain RNA nucleosides and mediate targeted cleavage of RNA transcripts by the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, target nucleic acid expression in cells, e.g., cells within a subject, such as a mammalian subject. RNAi agents include single-stranded RNAi agents and double-stranded siRNAs, as well as short hairpin RNAs (shRNAs). The oligonucleotides of the present invention, or their consecutive nucleotide sequences, can be in the form of an RNAi agent or can form part of an RNAi agent, such as an siRNA or shRNA. In some embodiments of the present invention, the oligonucleotides of the present invention, or their consecutive nucleotide sequences, are RNAi agents, such as siRNAs.

[0037] siRNA The term "siRNA" refers to a small interfering ribonucleic acid (RNAi) agent, a type of double-stranded RNA molecule also known in the art as short interfering RNA or silencing RNA. siRNA typically comprises a sense strand (also called a passenger strand) and an antisense strand (also called a guide strand), each strand being 17-30 nucleotides long, typically 19-25 nucleosides long. The antisense strand is complementary, e.g., perfectly complementary, to a target nucleic acid (preferably a mature mRNA sequence), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a duplex or duplex region. The siRNA strands can form a blunt-ended duplex, or advantageously, the 3' ends of the sense and antisense strands can form a 3' overhang of, for example, 1, 2, or 3 nucleosides. In some embodiments, both the sense and antisense strands have a 2-nt 3' overhang. Thus, the duplex region can be, for example, 17 to 25 nucleotides in length, such as 21 to 23 nucleotides in length.

[0038] Once inside the cell, the antisense strand is incorporated into the RISC complex, which mediates targeted degradation or target inhibition of the target nucleic acid. siRNA typically contains modified nucleosides in addition to RNA nucleosides, or in some embodiments, all of the nucleotides in the siRNA strand can be modified (sense 2' sugar-modified nucleosides, such as LNA (see, e.g., WO2004083430, WO2007085485), 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl, can be incorporated into the siRNA). In some embodiments, the passenger strand of the siRNA can be discontinuous (see, e.g., WO2007107162). It has been reported that the incorporation of thermolabile nucleotides in the seed region of the antisense strand of siRNA is useful for reducing the off-target activity of siRNA (see, e.g., WO18098328).

[0039] In some embodiments, the dsRNA agent, such as the siRNA of the present invention, comprises at least one modified nucleotide.In some embodiments, substantially all of the nucleotides of sense strand comprise modification; substantially all of the nucleotides of antisense strand comprise modification, or substantially all of the nucleotides of sense strand and substantially all of the nucleotides of antisense strand comprise modification.In yet another embodiment, all of the nucleotides of sense strand comprise modification; all of the nucleotides of antisense strand comprise modification; or all of the nucleotides of sense strand and all of the nucleotides of antisense strand comprise modification.

[0040] In some embodiments, the modified nucleotides may be independently selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, unlinked nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, glycol modified nucleotides, and 2-O-(N-methylacetamido) modified nucleotides, and combinations thereof. Preferably, the siRNA comprises a 5' phosphate group or a 5' phosphate mimetic at the 5' end of the antisense strand. In some embodiments, the 5' end of the antisense strand is an RNA nucleoside.

[0041] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage can be at the 3'-end of one or both strands (e.g., the antisense strand or the sense strand), or the phosphorothioate or methylphosphonate internucleoside linkage can be at the 5'-end of one or both strands (e.g., the antisense strand or the sense strand), or the phosphorothioate or methylphosphonate internucleoside linkage can be at both the 5'-end and the 3'-end of one or both strands (e.g., the antisense strand or the sense strand). In some embodiments, the remaining internucleoside linkages are phosphodiester linkages.

[0042] The dsRNA agent can further comprise a ligand. In some embodiments, the ligand is attached to the 3' end of the sense strand. For biological distribution, the siRNA can be bound to a targeting ligand and / or formulated into, for example, lipid nanoparticles.

[0043] Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA, such as siRNA molecules, suitable for therapeutic use, and methods of inhibiting target gene expression by administering dsRNA, such as siRNA, molecules of the invention, for the treatment of various disease conditions, e.g., as disclosed herein.

[0044] Tetraloop As used herein, the term "tetraloop" refers to a loop that increases the stability of a contiguous duplex formed by hybridization of an adjacent sequence of nucleotides. The increase in stability is the T of the contiguous stem duplex that would be expected on average from a set of loops of equivalent length consisting of a randomly selected sequence of nucleotides. m The melting temperature (T m) is detectable. For example, a tetraloop can confer a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 58°C, at least 60°C, at least 65°C, or at least 75°C in 10 mM NaHPO4 to a hairpin comprising a duplex at least two base pairs in length. In some embodiments, the tetraloop can stabilize the base pairs of the adjacent stem duplex through stacking interactions. Furthermore, interactions between nucleotides in the tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16;346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12;253(5016):191-4). In some embodiments, the tetraloop comprises 4 to 5 nucleotides. In certain embodiments, the tetraloop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In one embodiment, the tetraloop consists of 4 nucleotides. Any nucleotide may be used in the tetraloop, and the standard IUPAC-IUB symbols for such nucleotides may be used, as described in Cornish-Bowden (1985) Nucl. Acids Res. 13:3021-3030. For example, the letter "N" may be used to indicate that any base can be at that position, the letter "R" may be used to indicate that A (adenine) or G (guanine) can be at that position, and "B" may be used to indicate that C (cytosine), G (guanine), or T (thymine) can be at that position.Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., Proc Natl Acad Sci USA. 1990 November;87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 November;19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA)), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example, Nakano et al. Biochemistry, 41(48), 14281-14292, 2002. Shinji et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000), which are incorporated herein by reference for their relevant disclosures. In some embodiments, the tetraloop is comprised within a nicked tetraloop structure.

[0045] Nicked tetraloop structure A "nicked tetraloop structure" is a structure of an RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, where the sense strand has a region of complementarity with the antisense strand, and at least one of the strands, generally the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed within at least one strand.

[0046] antisense oligonucleotides As used herein, the term "antisense oligonucleotide" is defined as an oligonucleotide that can regulate the expression of target gene by hybridizing to target nucleic acid, particularly to the continuous sequence on target nucleic acid.Antisense oligonucleotide is not essentially double-stranded, and therefore is not siRNA or shRNA.Preferably, the antisense oligonucleotide of the present invention is single-stranded.It is understood that the single-stranded oligonucleotide of the present invention can form hairpin or intermolecular duplex structure (duplex between two molecules of the same oligonucleotide), as long as the degree of complementarity between itself or itself is less than 50% over the entire length of the oligonucleotide.

[0047] Advantageously, the single-stranded antisense oligonucleotides of the present invention do not contain RNA nucleosides to reduce nuclease resistance.

[0048] Advantageously, the oligonucleotides of the invention comprise one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides. Furthermore, it is advantageous for the unmodified nucleosides to be DNA nucleosides.

[0049] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "contiguous nucleobase sequence" and "oligonucleotide motif sequence." In some embodiments, all nucleotides of an oligonucleotide constitute a contiguous nucleotide sequence. In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence, e.g., an FG-F' gapmer region, and may optionally include a nucleotide linker region that can be used to attach additional nucleotide(s), e.g., a functional group, to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. It is understood that the contiguous nucleotide sequence of an oligonucleotide cannot be longer than the oligonucleotide itself, and that the oligonucleotide cannot be shorter than the contiguous nucleotide sequence.

[0050] nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides. Nucleotides, such as DNA and RNA nucleotides, naturally contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides). Nucleosides and nucleotides can also be referred to interchangeably as "units" or "monomers."

[0051] Deoxyribonucleotides As used herein, the term "deoxyribonucleotide" refers to a nucleotide that, compared to a ribonucleotide, has a hydrogen instead of a hydroxyl at the 2' position of its pentose sugar. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of the sugar, phosphate group, or base.

[0052] Ribonucleotides As used herein, the term "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar and containing a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide with one or more modifications or substitutions of an atom other than the 2' position, including modifications or substitutions of the ribose, phosphate group, or base.

[0053] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that has been modified relative to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications to the sugar or (nucleic acid) base moiety. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably with the terms "nucleoside analog" or modified "unit" or modified "monomer." Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA nucleosides if they are capable of Watson-Crick base pairing.

[0054] Modified Nucleotides As used herein, the term "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. In some embodiments, the modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0055] Modified internucleoside linkages The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) bond that covalently links two nucleosides together, as commonly understood by those skilled in the art. Thus, oligonucleotides of the present invention may contain modified internucleoside linkages. In some embodiments, modified internucleoside linkages increase the nuclease resistance of the oligonucleotide compared to phosphodiester linkages. In naturally occurring oligonucleotides, modified internucleoside linkages containing a phosphate group that creates a phosphodiester bond between adjacent nucleosides are particularly useful for stabilizing oligonucleotides for in vivo use and may serve to protect against nuclease cleavage in regions of DNA or RNA nucleosides of the oligonucleotides of the present invention, such as the gap region G of a gapmer oligonucleotide, and regions F and F' of modified nucleosides.

[0056] In one embodiment, the oligonucleotide comprises one or more modified internucleoside linkages modified from natural phosphodiester, e.g., to be more resistant to nuclease attack. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)), both of which are well known in the art. An internucleoside linkage that can enhance the nuclease resistance of an oligonucleotide is referred to as a nuclease-resistant internucleoside linkage. In some embodiments, at least 50% of the internucleoside linkages of the oligonucleotide or its contiguous nucleotide sequence are modified, e.g., at least 60%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, or e.g., at least 90% of the internucleoside linkages of the oligonucleotide or its contiguous nucleotide sequence are modified. In some embodiments, all of the internucleoside linkages of the oligonucleotide or its contiguous nucleotide sequence are modified. It will be appreciated that in some embodiments, the nucleosides linking the oligonucleotides of the invention to non-nucleotide functional groups, e.g., conjugates, can be phosphodiesters. In some embodiments, all of the internucleoside linkages of the oligonucleotide or its contiguous nucleotide sequence are nuclease-resistant internucleoside linkages.

[0057] The oligonucleotides of the invention advantageously use phosphorothioate internucleoside linkages.

[0058] Phosphorothioate internucleoside linkages are particularly useful due to their nuclease resistance, favorable pharmacokinetics, and ease of manufacture. In some embodiments, at least 50% of the internucleoside linkages of an oligonucleotide or its consecutive nucleotide sequence are phosphorothioate, and at least 60%, for example at least 70%, for example at least 75%, for example at least 80%, or for example at least 90% of the internucleoside linkages of an oligonucleotide or its consecutive nucleotide sequence are phosphorothioate. In some embodiments, all of the internucleoside linkages of an oligonucleotide or its consecutive nucleotide sequence are phosphorothioate.

[0059] In some embodiments, oligonucleotides of the invention contain, in addition to phosphorodithioate linkage(s), both phosphorothioate internucleoside linkages and at least one phosphodiester linkage, such as 2, 3, or 4 phosphodiester linkages. In gapmer oligonucleotides, phosphodiester linkages, if present, are not properly positioned between consecutive DNA nucleosides within the gap region G.

[0060] Nuclease-resistant linkages such as phosphorothioate linkages are particularly useful in regions of an oligonucleotide that can recruit nucleases when duplexed with a target nucleic acid, e.g., region G of a gapmer. However, phosphorothioate linkages may also be useful in non-nuclease recruiting and / or affinity-enhancing regions, e.g., regions F and F' of a gapmer. A gapmer oligonucleotide may, in some embodiments, contain one or more phosphodiester linkages in region F or F', or in both regions F and F', and all of the internucleoside linkages in region G may be phosphorothioate.

[0061] Advantageously, all internucleoside linkages of the consecutive nucleotide sequence of the oligonucleotide are phosphorothioate, or all internucleoside linkages of the oligonucleotide are phosphorothioate, in particular, all internucleoside linkages of the consecutive nucleotide sequence of the antisense oligonucleotide are phosphorothioate, or all internucleoside linkages of the antisense oligonucleotide are phosphorothioate.

[0062] As disclosed in EP 2742135, it is recognized that therapeutic oligonucleotides may contain other internucleoside linkages (other than phosphodiester and phosphorothioate), such as alkylphosphonate / methylphosphonate internucleoside linkages, which according to EP 2742135 may be tolerated, for example, within the gap region of another DNA phosphorothioate.

[0063] Nucleic acid bases The term "nucleobase" includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term "nucleobase" also encompasses modified nucleobases that may differ from naturally occurring nucleobases but function during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, vol. 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Suppl. 37, 1.4.1.

[0064] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0065] Nucleobase moieties may be represented by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, where each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplary oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides may be used.

[0066] Modified Oligonucleotides The term modified oligonucleotide refers to an oligonucleotide containing one or more sugar-modified nucleosides and / or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term used in the literature to describe oligonucleotides having modified nucleosides.

[0067] Complementarity As used herein, "complementary" refers to a structural relationship between two nucleotides or two sequences of nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides or two sequences of nucleotides to base pair with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid can base pair by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). Oligonucleotides may contain nucleosides having modified nucleobases, e.g., 5-methylcytosine is often substituted for cytosine, and it will be understood that the term complementarity therefore encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0068] The term "% complementary," as used herein, refers to the percentage of nucleotides in a contiguous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) across the contiguous nucleotide sequence. Thus, the percentage of complementarity is calculated by counting the number of aligned nucleobases that are complementary (e.g., by Watson-Crick base pairing) between two sequences (aligning the target sequence 5'-3' with the oligonucleotide sequence from 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (e.g., form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in calculating the percent complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of a nucleobase will be disregarded so long as the nucleobase retains its functional ability to form, for example, Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

[0069] The term "fully complementary" refers to 100% complementarity.

[0070] In some embodiments, two nucleic acids can have a region of multiple nucleotides that are complementary to one another to form a region of complementarity, as described herein.

[0071] Areas of complementarity As used herein, the term "region of complementarity" refers to a sequence of nucleotides in a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides to allow hybridization between the two sequences of nucleotides under appropriate hybridization conditions, e.g., in a phosphate buffer, in a cell, etc.

[0072] identity The term "identity" as used herein refers to the percentage (expressed as a percentage) of nucleotides in a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is identical to a reference sequence (e.g., a sequence motif) across the contiguous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleobases between two sequences (in the contiguous nucleotide sequence of the compound of the present invention and the reference sequence), dividing this number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (number of matches x 100) / length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percentage identity of a contiguous nucleotide sequence. It should be understood that in determining identity, chemical modifications of nucleobases are ignored as long as the nucleobase retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating percent identity).

[0073] Hybridization As used herein, the term "hybridize" or "hybridizing" should be understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is determined by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) is often explained by the following: Under physiological conditions, T m is not strictly proportional to affinity (Mergny and Lacroix (2003) Oligonucleotides 13:515-537). The standard state Gibbs free energy, ΔG°, more accurately represents binding affinity, ΔG°=-RTln(K d ) to calculate the dissociation constant (K d), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction at an aqueous concentration of 1M, pH 7, and temperature of 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be experimentally measured, for example, by isothermal titration calorimetry (ITC) method, as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discovery Today. Those skilled in the art will know that commercially available devices are available for measuring ΔG°. ΔG° can also be numerically estimated using the nearest neighbor model described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, or by using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. To ensure the potential for hybridization modulation of their intended nucleic acid targets, oligonucleotides of the present invention hybridize to target nucleic acids with an estimated ΔG° value of less than -10 kcal for oligonucleotides 10-30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard-state Gibbs free energy ΔG°. The oligonucleotides may hybridize to the target nucleic acid with an estimated ΔG° value in the range of less than −10 kcal, for example less than −15 kcal, for example less than −20 kcal, and for example less than −25 kcal for oligonucleotides 8 to 30 nucleotides in length, hi some embodiments, the oligonucleotides hybridize to the target nucleic acid with an estimated ΔG° value of −10 to −60 kcal, for example −12 to −40, for example −15 to −30 kcal, or −16 to −27 kcal, for example −18 to −25 kcal.

[0074] target nucleic acid According to the present invention, the target nucleic acid can be, for example, a gene, RNA, mRNA, viral mRNA, or cDNA sequence.

[0075] For in vivo or in vitro applications, the oligonucleotides of the present invention are typically capable of inhibiting expression of an HBV target nucleic acid in cells that express the HBV target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotides of the present invention is typically complementary to the HBV target nucleic acid, measured over the length of the oligonucleotide, optionally except for one or two mismatches, and optionally except for a nucleotide-based linker region or other non-complementary terminal nucleotide (e.g., D' or D'') that may link the oligonucleotide to any functional group, such as a conjugate.

[0076] Target sequence The term "target sequence" as used herein refers to a sequence of nucleotides present in a target nucleic acid, which comprises a nucleobase sequence complementary to an oligonucleotide of the present invention. In some embodiments, the target sequence consists of a region on the target nucleic acid that has a nucleobase sequence complementary to the continuous nucleotide sequence of an oligonucleotide of the present invention. This region of the target nucleic acid can be interchangeably referred to as a target nucleotide sequence, a target sequence, or a target region. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide, and can represent a preferred region of the target nucleic acid that can be targeted, for example, by several oligonucleotides of the present invention.

[0077] target cell As used herein, the term "target cell" refers to a cell expressing a target nucleic acid. In some embodiments, the target cell can be in vivo or in vitro. In some embodiments, the target cell is a rodent cell, such as a mouse cell or a human cell, particularly an HBV-infected mammalian cell, such as an HBV-infected hepatocyte.

[0078] In a preferred embodiment, the target cells express HBV mRNA and secrete HBsAg and HBeAg.

[0079] liver cells As used herein, the term "hepatocyte" or "hepatocytes" refers to cells of the liver parenchyma. These cells comprise approximately 70-85% of the liver's mass and produce serum albumin, fibrinogen, and the prothrombin group of clotting factors (excluding factors 3 and 4). Markers of hepatocyte lineage cells may include, but are not limited to, transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers of mature hepatocytes may include, but are not limited to, cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, for example, Huch et al., (2013), Nature, 494(7436):247-250, the contents of which are incorporated herein by reference.

[0080] Decreased expression As used herein, the term "reduced expression" of a gene refers to a decrease in the amount of RNA transcript or protein encoded by the gene and / or a decrease in the amount of gene activity in a cell or subject compared to a suitable reference cell or subject. For example, treating cells with a pharmaceutical combination or a double-stranded oligonucleotide (e.g., one having an antisense strand complementary to an HBsAg mRNA sequence) can result in a decrease in the amount of RNA transcript, protein, and / or enzyme activity (e.g., encoded by the S gene of the HBV genome) compared to cells not treated with the pharmaceutical combination or double-stranded oligonucleotide, respectively. Similarly, as used herein, "reducing expression" refers to an action that results in a decrease in the expression of a gene (e.g., the S gene of the HBV genome).

[0081] Naturally occurring variants The term "naturally occurring variant thereof" refers to a variant of a target nucleic acid that occurs naturally within a defined taxonomic group, such as HBV genotypes A-H. Typically, when referring to a "naturally occurring variant" of a polynucleotide, the term can encompass any allelic variant of the target sequence encoding genomic DNA found by chromosomal translocation or duplication, and RNA derived therefrom, such as mRNA. A "naturally occurring variant" can also include variants resulting from alternative splicing of the target sequence mRNA. For example, when referring to a specific polypeptide sequence, the term also includes naturally occurring forms of the protein that can be processed by co- or post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc.

[0082] High-affinity modified nucleosides High affinity modified nucleosides are modified nucleotides that, when incorporated into an oligonucleotide, e.g., increase the melting temperature (T m The high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature of +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0083] sugar modification Oligomers of the invention may contain one or more nucleosides which have modified sugar moieties, ie, sugar moieties which are modified compared to the ribose sugar moiety found in DNA and RNA.

[0084] Numerous nucleosides with modifications in the ribose sugar moiety have been created primarily with the goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0085] Such modifications include those in which the ribose ring structure has been modified, for example, by replacing it with a hexose ring (HNA) or bicyclic ring (typically having a biradical bridge between the C2 and C4 carbons of the ribose ring (LNA)), or an unlinked ribose ring (e.g., UNA), which typically lacks a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0086] Sugar modifications also include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally occurring in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0087] 2' sugar-modified nucleosides A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (2' substituted nucleoside), or a nucleoside containing a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon of the ribose ring, such as an LNA (2'-4' biradical bridge) nucleoside.

[0088] Indeed, much attention has been focused on the development of 2'-sugar-substituted nucleosides, and many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can confer enhanced binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted modified nucleosides include 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides. For further examples, see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavy and Damha, Chemistry and Biology 2012, 19, 937. Below are examples of some 2'-substituted modified nucleosides. [ka]

[0089] In the context of the present invention, 2'-substituted sugar modified nucleosides do not include 2'-bridged nucleosides such as LNA.

[0090] Locked nucleic acid nucleosides (LNA nucleosides) "LNA nucleosides" are 2'-modified nucleosides containing a biradical (also referred to as a "2'-4' bridge") linking the C2' and C4' ends of the ribose sugar ring of the nucleoside, which restricts or fixes the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Fixation of the ribose conformation is associated with improved hybridization affinity (duplex stabilization) when LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0091] Non-limiting exemplary LNA nucleosides include those described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al. al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0092] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1. [ka]

[0093] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA. A particularly preferred LNA is beta-D-oxy-LNA.

[0094] Phosphate analogues As used herein, the term "phosphate mimetic" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is placed on the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5'-phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include 5'-phosphonates, such as 5'-methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar (referred to as a "4'-phosphate analog") on the 5'-terminal nucleotide. An example of a 4'-phosphate analog is an oxymethyl phosphonate or an analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., its 4'-carbon). See, for example, U.S. Provisional Patent Application No. 62 / 383,207, filed September 2, 2016, and U.S. Provisional Patent Application No. 62 / 393,401, filed September 12, 2016, the contents of each of which regarding phosphate analogs are incorporated herein by reference. Other modifications to the 5' end of oligonucleotides have been developed (see, for example, WO 2011 / 133871; U.S. Pat. No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011, the contents of each of which regarding phosphate analogs are incorporated herein by reference).

[0095] Nuclease-mediated degradation Nuclease-mediated degradation refers to an oligonucleotide that, when duplexed with a complementary nucleotide sequence, is capable of mediating the degradation of such sequence.

[0096] In some embodiments, antisense oligonucleotides can function through nuclease-mediated degradation of target nucleic acids, and the oligonucleotides of the invention can recruit nucleases, particularly endonucleases, preferably endoribonucleases (RNases), such as RNase H. Examples of oligonucleotide designs that act via a nuclease-mediated mechanism are oligonucleotides that typically contain a region of at least five or six consecutive DNA nucleosides and are flanked on one or both sides by affinity-enhancing nucleosides, such as gapmers, headmers, and tailmers.

[0097] RNase H activity and recruitment In one embodiment, the therapeutic oligonucleotide is an antisense oligonucleotide capable of recruiting RNase H. RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when duplexed with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity that can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate measured in pmol / l / min that is at least 5%, e.g., at least 10% or more than 20% of the initial rate determined when using an oligonucleotide that has the same base sequence as the modified oligonucleotide being tested but contains only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, using the methodology provided in Examples 91-95 of WO 01 / 23613 (incorporated herein by reference). For use in determining RNase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland.

[0098] Gapmar In some embodiments in which the therapeutic oligonucleotide of the present invention is an antisense oligonucleotide, the nucleic acid molecule of the present invention or a contiguous nucleotide sequence thereof is a gapmer antisense oligonucleotide. Antisense gapmers are typically used to inhibit target nucleic acids via RNase H-mediated degradation. In one embodiment of the present invention, the antisense oligonucleotide of the present invention is capable of recruiting RNase H.

[0099] Gapmer antisense oligonucleotides contain at least three distinct structural regions: a 5'-flank, a gap, and a 3'-flank, FG-F', in a 5'->3' orientation. The "gap" region (G) contains a stretch of consecutive DNA nucleotides that allows the oligonucleotide to recruit RNase H. The gap region is flanked by a 5'-flanking region (F) containing one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides, and a 3'-flanking region (F') containing one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides. The one or more sugar-modified nucleosides in regions F and F' improve the affinity of the oligonucleotide for the target nucleic acid (i.e., are affinity-enhancing sugar-modified nucleosides). In some embodiments, the one or more sugar-modified nucleosides in regions F and F' are 2'-sugar-modified nucleosides, such as high-affinity 2'-sugar modifications independently selected from, for example, LNA and 2'-MOE.

[0100] In a gapmer design, the 5'- and 3'-most nucleosides of the gap region are DNA nucleosides, positioned adjacent to sugar-modified nucleosides in the 5' (F) or 3' (F') regions, respectively. Flanks may be further defined by having at least one sugar-modified nucleoside at the end furthest from the gap region, i.e., at the 5'-end of the 5' flank and at the 3'-end of the 3' flank.

[0101] The region FG-F' forms a contiguous nucleotide sequence. The antisense oligonucleotide of the present invention or the contiguous nucleotide sequence thereof may comprise a gapmer region of the formula FG-F'.

[0102] The total length of the gapmer design FG-F' can be, for example, 12 to 30 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, such as 13 to 17, such as 14 to 16 nucleosides.

[0103] By way of example, a gapmer oligonucleotide of the invention can be represented by the following formula: F1-6 -G 6-16 -F' 1-6 ,for example F 1-4 -G 7-10 -F' 2-4 However, the total length of the gapmer region FG-F' is at least 12, for example at least 13, nucleotides in length.

[0104] In one embodiment of the present invention, the antisense oligonucleotide or its contiguous nucleotide sequence consists of or comprises a gapmer of the formula 5'-FG-F'-3', in which regions F and F' independently comprise or consist of 1 to 8 nucleosides, 1 to 4 of which are 2' sugar modified, defining the 5' and 3' ends of the F and F' regions, and G is a region of 6 to 16 nucleosides capable of recruiting RNase H.

[0105] In one embodiment of the invention, the contiguous nucleotide sequence is a gapmer of the formula 5'-FG-F'-3', where regions F and F' independently consist of 2-4 2' sugar-modified nucleotides and define the 5' and 3' ends of the F and F' regions, and G is a 6-10 DNA nucleoside region capable of recruiting RNase H.

[0106] In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive phosphorothioate-linked DNA nucleosides. In some embodiments, the gap region G consists of 7 to 10 DNA nucleosides. In some embodiments, all internucleoside linkages in the gap are phosphorothioate linkages.

[0107] In some embodiments, regions F and F' independently consist of or comprise a contiguous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F may be independently selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units.

[0108] In some embodiments, regions F or F', or all nucleosides of F and F', are LNA nucleosides, e.g., independently selected from beta-D-oxyLNA, ENA, or ScET nucleosides. In some embodiments, region F consists of 1 to 5, e.g., 2 to 4, e.g., 3 to 4, e.g., 1, 2, 3, 4, or 5 contiguous LNA nucleosides. In some embodiments, all nucleosides of regions F and F' are beta-D-oxyLNA nucleosides.

[0109] In some embodiments, all nucleosides in regions F or F', or F and F', are 2'-substituted nucleosides, e.g., OMe or MOE nucleosides. In some embodiments, region F consists of 1, 2, 3, 4, 5, 6, 7, or 8 consecutive OMe or MOE nucleosides. In some embodiments, only one of the flanking regions can consist of 2'-substituted nucleosides, e.g., OMe or MOE nucleosides. In some embodiments, it is the 5' (F) flanking region that consists of 2'-substituted nucleosides, e.g., OMe or MOE nucleosides, while the 3' (F') flanking region comprises at least one LNA nucleoside, e.g., a beta-D-oxyLNA nucleoside or a cET nucleoside. In some embodiments, it is the 3' (F') flanking region that consists of 2' substituted nucleosides, such as OMe or MOE nucleosides, while the 5' (F) flanking region comprises at least one LNA nucleoside, such as a beta-D-oxyLNA nucleoside or a cET nucleoside.

[0110] Further gapmer designs are disclosed in WO 2004 / 046160, WO 2007 / 146511 and WO 2008 / 113832, which are incorporated herein by reference.

[0111] LNA gapmers An LNA gapmer is a gapmer which comprises or consists of LNA nucleosides in one or both of regions F and F'. A beta-D-oxy gapmer is a gapmer which comprises or consists of beta-D-oxy LNA nucleosides in one or both of regions F and F'.

[0112] In some embodiments, the LNA gapmer has the formula: [LNA] 1-5 -[Area G] 6-10 -[LNA] 1-5 and region G is as defined in the definition of gapmer region G.

[0113] MOE Gapmar An MOE gapmer is a gapmer in which regions F and F' consist of MOE nucleosides. In some embodiments, an MOE gapmer has the design [MOE] 1-8 -[Area G] 5-16 -[MOE] 1-8 , e.g. [MOE] 2-7 -[Area G] 6-14 -[MOE] 2-7 , e.g. [MOE] 3-6 -[Area G] 8-12 -[MOE] 3-6 where region G is as defined in the gapmer definition. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

[0114] Mixed Wing Gappa A mixed-wing gapmer is an LNA gapmer in which one or both of regions F and F' comprise 2'-substituted nucleosides, e.g., MOE nucleosides, independently selected from the group consisting of 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units. In some embodiments in which at least one of regions F and F', or both of regions F and F', comprise at least one LNA nucleoside, the remaining nucleosides in regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments in which at least one of regions F and F', or both of regions F and F', comprise at least two LNA nucleosides, the remaining nucleosides in regions F and F' are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of regions F and F' may further comprise one or more DNA nucleosides.

[0115] Mixed wing gapmer designs are disclosed in WO 2008 / 049085 and WO 2012 / 109395, both of which are incorporated herein by reference.

[0116] Region D' or D'' within the oligonucleotide

[0117] Oligonucleotides of the invention, in some embodiments, can comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, e.g., a gapmer FG-F', as well as additional 5' and / or 3' nucleosides. The additional 5' and / or 3' nucleosides may or may not be fully complementary to the target nucleic acid. Such additional 5' and / or 3' nucleosides may be referred to herein as regions D' and D''.

[0118] The addition of region D' or D" can be used for the purpose of linking a contiguous nucleotide sequence, such as a gapmer, to a conjugate moiety or another functional group. When used to link a conjugate moiety to a conjugate moiety, it can serve as a biocleavable linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0119] Regions D' and D" can be attached to the 5' end of region F or the 3' end of region F', respectively, to generate designs of the following formula: D'-FG-F', FG-F'-D" or D'-FG-F'-D" where FG-F' is the gapmer portion of the oligonucleotide and regions D' or D" constitute separate portions of the oligonucleotide. The transitions between regions D' and F and between regions F' and D" are characterized by nucleosides having a phosphodiester bond toward the D' or D" region and a phosphorothioate bond toward the F or F' region, which nucleosides are considered to be part of a gapmer (a contiguous nucleotide sequence complementary to a target nucleic acid).

[0120] Region D' or D" independently comprises or consists of 1, 2, 3, 4, or 5 additional nucleotides and may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides, such as DNA or RNA, or base-modified versions thereof. The D' or D" region can serve as a nuclease-sensitive biocleavable linker (see definition of linker). In some embodiments, the additional 5' and / or 3' terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biocleavable linkers suitable for use as region D' or D" are disclosed in WO 2014 / 076195, including, by way of example, phosphodiester-linked DNA dinucleotides. In some embodiments, region D' or D" is not complementary to the target nucleic acid or comprises at least a 50% mismatch.

[0121] In some embodiments, region D' or D'' comprises or consists of a dinucleotide of the sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, where C may be 5-methylcytosine and / or T may be replaced by U. The internucleoside linkages in the dinucleotide are phosphodiester bonds. In some embodiments, region D' or D" consists of or consists of the trinucleotides of the sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, and GGG, wherein C may be 5-methylcytosine and / or T may be replaced with U. The internucleoside linkages are phosphodiester bonds. When referring to the (naturally occurring) nucleobases A (adenine, T (thymine), U (uracil), G (guanine), C (cytosine), it will be understood that these can be substituted with nucleobase analogs that function as equivalent natural nucleobases (e.g., base pair with complementary nucleosides).

[0122] In one embodiment, the antisense oligonucleotide of the present invention comprises regions D' and / or D'' in addition to the contiguous nucleotide sequence that constitutes the gapmer.

[0123] In some embodiments, the antisense oligonucleotides of the invention can be represented by the following formula: D'-FG-F', especially D' 1-3 -F 1-4 -G 6-10 -F' 2-4 FG-F'-D'', especially F 1-4 -G 6-10 -F' 2-4 -D'' 1-3 D'-FG-F'-D'', especially D' 1-3 -F 1-4 -G 6-10 -F' 2-4 -D'' 1-3 .

[0124] In some embodiments, the internucleoside linkage located between region D' and region F is a phosphodiester bond. In some embodiments, the internucleoside linkage located between region F' and region D'' is a phosphodiester bond.

[0125] Conjugates The term conjugate as used herein refers to a non-nucleotide moiety (conjugate), such as a GalNAc cluster, that can be covalently linked to a therapeutic oligonucleotide. The terms conjugate and cluster or conjugate moiety can be used interchangeably. In some instances, a conjugated therapeutic oligonucleotide can also be referred to as an oligonucleotide conjugate. In one embodiment, the conjugate is a targeting ligand.

[0126] Targeting Ligands As used herein, the term "targeting ligand" refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) in a tissue or cell of interest and can be conjugated to other substances for the purpose of targeting the other substances to the tissue or cell of interest. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, the targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, the targeting ligand, when conjugated to the oligonucleotide, facilitates delivery of the oligonucleotide to a specific cell by selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, targeting ligand, and receptor. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after or during cellular internalization, such that the oligonucleotide is released from the targeting ligand within the cell.

[0127] Oligonucleotide Linkers A bond or linker is a connection between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds.The conjugate group can be directly or via a linking moiety (e.g., a linker or tether) attached to the oligonucleotide.The linker serves to covalently attach the conjugate group to the oligonucleotide or consecutive nucleotide sequence that is complementary to the target nucleic acid.

[0128] In some embodiments of the invention, the therapeutic oligonucleotide optionally comprises a linker region located between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid and the conjugate.

[0129] Such a linker may be a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions normally encountered or similar to those encountered in a mammalian body, hi one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage.

[0130] For a biocleavable linker disposed between the conjugate and the therapeutic oligonucleotide, it is preferred that the cleavage rate observed in the target tissue (e.g., muscle, liver, kidney, or tumor) is greater than that observed in serum. In some embodiments, the biocleavable linker is cleaved by at least about 20% when compared to a standard, such as at least about 30% cleaved, for example, at least about 40% cleaved, for example, at least about 50% cleaved, for example, at least about 60% cleaved, for example, at least about 70% cleaved, for example, at least about 75% cleaved.

[0131] In a preferred embodiment, the nuclease-sensitive linker comprises between 1 and 10 nucleosides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, more preferably between 2 and 6 nucleosides, and most preferably between 2 and 4 linked nucleosides, comprising at least two consecutive phosphodiester bonds, e.g., at least three, four, or five consecutive phosphodiester bonds. Preferably, the nucleosides are DNA or RNA. Phosphodiester-containing biocleavable linkers (PO linkers) are described in more detail in WO 2014 / 076195, which is incorporated herein by reference.

[0132] Additional or alternative linkers, which are not necessarily biocleavable but primarily serve to covalently link the conjugate to the oligonucleotide, may also be used alone or in combination with the PO linker. Non-cleavable linkers may include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. In some embodiments, the non-cleavable linker is an aminoalkyl, such as a C2-C36 aminoalkyl group, including a C6-C12 aminoalkyl group. In a preferred embodiment, the linker is a C6 aminoalkyl group.

[0133] Hepatitis B virus As used herein, "hepatitis B virus" or "HBV" refers to a member of the Hepadnaviridae family, which has a small, double-stranded DNA genome of approximately 3,200 base pairs and a tropism for hepatocytes. "HBV" includes hepatitis B viruses that infect any of a variety of mammalian (e.g., humans, non-human primates, etc.) and avian (e.g., duck) hosts. "HBV" includes any known HBV genotype, such as serotypes A, B, C, D, E, F, and G; any HBV serotype or HBV subtype; any HBV isolate; HBV variants, such as HBeAg-negative variants, drug-resistant HBV variants (e.g., lamivudine-resistant variants; adefovir-resistant mutants; tenofovirus-resistant mutants; entecavir-resistant mutants, etc.); and the like.

[0134] HBV is a small DNA virus belonging to the family Hepadnaviridae, classified as the type species of the genus Orthohepadnavirus. The HBV virus particle (virion) contains an outer lipid envelope and an icosahedral nucleocapsid core composed of proteins. The nucleocapsid generally encloses viral DNA and a DNA polymerase with reverse transcriptase activity similar to that of retroviruses. The HBV outer envelope contains embedded proteins involved in viral binding and entry into susceptible cells. HBV, which attacks the liver, is classified according to at least 10 genotypes (A-J) based on its sequence. Generally, there are four genes encoded by the genome, designated C, P, S, and X. The core protein is encoded by gene C (HBcAg), whose start codon is preceded by an upstream in-frame AUG initiation codon, from which the precore protein is produced. HBeAg is produced by proteolytic processing of the precore protein. The DNA polymerase is encoded by gene P. Gene S encodes the surface antigen (HBsAg). The HBsAg gene is one long open reading frame, but contains three in-frame "start" (ATG) codons that divide the gene into three sections (pre-S1, pre-S2, and S). Due to the multiple start codons, three different sized polypeptides (pre-S1 + pre-S2 + S, pre-S2 + S, or S) are produced, termed large, medium, and small. These may have a ratio of 1:1:4 (Heermann et al., 1984).

[0135] Hepatitis B virus (HBV) proteins can be organized into several categories and functions. The polymerase functions as a reverse transcriptase (RT) to generate viral DNA from pregenomic RNA (pgRNA) and as a DNA-dependent polymerase to generate covalently closed circular DNA (cccDNA) from viral DNA. They are covalently attached to the 5' end of the minus strand. The core protein generates the viral capsid and secreted E antigen. The surface antigen is a hepatocyte-internalized ligand and also the main component of viral spherical and filamentous particles. Aviral particles are produced in greater than 1,000 times greater quantities than Dane particles (infectious virions) and can act as immune decoys.

[0136] Hepatitis B virus surface antigen As used herein, the term "hepatitis B virus surface antigen" or "HBsAg" refers to the S domain protein encoded by gene S (e.g., ORF S) of the HBV genome. Hepatitis B virus particles harbor viral nucleic acid within a core particle enveloped by three proteins encoded by gene S: the large surface protein, the intermediate surface protein, and the major surface protein. Of these proteins, the major surface protein is generally about 226 amino acids and contains only the S domain.

[0137] Hepatitis B e antigen (HBeAg): As used herein, the term "hepatitis B e antigen" or "HBeAg" is an indicator of viral replication, although some variants of the virus do not express HBeAg. Active infection can be described as HBeAg positive or HBeAg negative, depending on whether HBeAg is secreted.

[0138] infection As used herein, the term "infection" refers to the pathogenic invasion and / or proliferation of a microorganism, such as a virus, in a subject. Infection may be lysogenic, e.g., viral DNA remains dormant within the cell. Alternatively, infection may be lytic, e.g., the virus actively replicates, causing destruction of infected cells. Infection may or may not cause clinically apparent symptoms. Infection may remain localized or may be transmitted, e.g., via the subject's blood or lymphatic system. For example, individuals with HBV infection can be identified by detecting one or more of viral load, surface antigen (HBsAg), e antigen (HBeAg), and various other assays for detecting HBV infection known in the art. Assays for detecting HBV infection may include testing serum or blood samples for the presence of HBsAg and / or HBeAg, and optionally further screening for the presence of one or more viral antibodies (e.g., IgM and / or IgG) to account for any periods during which HBV antigens may be at undetectable levels.

[0139] HBV infection The terms "hepatitis B virus infection" or "HBV infection" are commonly known in the art and refer to an infectious disease caused by the hepatitis B virus (HBV) and affecting the liver. HBV infection can be acute or chronic. Some infected individuals have no symptoms during the initial infection and rapidly develop illness with vomiting, yellowish skin, fatigue, dark urine, and abdominal pain ("Hepatitis B Fact Sheet #204," who.int. July 2014, retrieved November 4, 2014). In many cases, these symptoms last for several weeks and can be fatal. Symptoms can take 30 to 180 days to begin. Ninety percent of people infected around birth develop chronic hepatitis B infection, while fewer than 10% of those infected after age 5 develop the disease ("Hepatitis B FAQs on Public Transmission," Centers for Disease Control and Prevention (CDC), retrieved November 29, 2011). Although most people with chronic disease do not have symptoms, cirrhosis and liver cancer can eventually develop (Chang, 2007, Semin Fetal Neonatal Med, 12:160-167). These complications result in death in 15-25% of people with chronic disease ("Hepatitis B Fact Sheet #204," who.int. July 2014, retrieved November 4, 2014). As used herein, the term "HBV infection" includes acute and chronic hepatitis B infection. The term "HBV infection" also includes the asymptomatic stage of early infection, the symptomatic stage, and the asymptomatic chronic stage of HBV infection.

[0140] Liver inflammation The term "liver inflammation" or "hepatitis" used herein refers to the physical condition that the liver becomes swollen, dysfunctional and / or painful as a result of damage or infection, which may be caused by exposure to hepatotoxic drugs in particular.Symptoms may include jaundice (yellowing of skin or eyes), fatigue, weakness, nausea, vomiting, loss of appetite and weight loss.If left untreated, liver inflammation may progress to fibrosis, cirrhosis, liver failure or liver cancer.

[0141] Liver fibrosis As used herein, the term "liver fibrosis" or "fibrosis of the liver" refers to the excessive accumulation of extracellular matrix proteins in the liver, which may include collagen (I, III, and IV), fibronectin, undulin, elastin, laminin, hyaluronan, and proteoglycans, resulting from inflammation and liver cell death. If left untreated, liver fibrosis can progress to cirrhosis, liver failure, or liver cancer.

[0142] TLR7 As used herein, "TLR7" refers to Toll-like receptor 7 of any species of origin (e.g., human, mouse, woodchuck, etc.).

[0143] TLR7 agonists As used herein, the term "TLR7 agonist" refers to a compound that acts as an agonist of TLR7. Unless otherwise specified, a TLR7 agonist can include any pharmaceutically acceptable form of the compound, including any isomers (e.g., diastereomers or enantiomers), salts, solvates, polymorphs, etc. TLR agonism for a particular compound can be determined in any suitable manner. For example, assays for detecting TLR agonism of test compounds are described, for example, in U.S. Provisional Patent Application No. 60 / 432,650, filed December 11, 2002, and recombinant cell lines suitable for use in such assays are described, for example, in U.S. Provisional Patent Application No. 60 / 432,651, filed December 11, 2002. A further assay for evaluating TLR7 agonists is the HEK293-Blue-hTLR-7 cell assay described in Example 43 of WO 2016 / 091698 (this assay is incorporated herein by reference).

[0144] Diastereomer Diastereomer As used herein, the term "diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, activity, and reactivity.

[0145] The compounds of general formulas (I) to (V) containing one or several chiral centers can exist either as racemates, diastereomeric mixtures, or optically active single isomers. The racemates can be separated into enantiomers according to known methods. In particular, diastereomeric salts, which can be separated by crystallization, are formed from the racemic mixtures by reaction with optically active acids such as D- or L-tartaric acid, mandelic acid, malic acid, lactic acid, or camphorsulfonic acid.

[0146] Pharmaceutically acceptable salts The compounds according to the invention may exist in the form of their pharmaceutically acceptable salts.

[0147] The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the free base or free acid, which are not biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, especially hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, and the like.

[0148] Alternatively, these salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resins. The compound of formula (I) can also exist in the form of a zwitterion. Particularly preferred pharmaceutically acceptable salts of the compound of formula (I) are salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and methanesulfonic acid.

[0149] Chemical modification of pharmaceutical compounds into salts is a technique well known to medicinal chemists to improve the physical and chemical stability, hygroscopicity, flowability and solubility of the compounds. For example, it is described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or Ansel, In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compounds provided herein can be a sodium salt.

[0150] Drug combinations As used herein, a pharmaceutical combination is understood to mean a combination of at least two different HBV therapeutic agents, such as active compounds or prodrugs (medicinal compounds or drugs), for treating disease. A pharmaceutical combination may include compounds that are physically, chemically, or otherwise combined (e.g., in the same vial); compounds that are packaged together (e.g., as two separate entities in the same package (kit of parts) for either simultaneous or separate administration); or compounds that are provided separately but intended to be used together (e.g., the combination is explicitly stated on the compound label, instructions, or package insert). In one embodiment, the pharmaceutical combination consists of a medicinal compound formulated for oral administration and a medicinal compound formulated for subcutaneous injection.

[0151] approximately As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise specified or otherwise clear from the context, refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value (except where such number would exceed 100% of possible values).

[0152] Administration As used herein, the term "administer" or "administration" means providing a substance (e.g., a pharmaceutical combination or an oligonucleotide) to a subject in a pharmacologically useful manner (e.g., to treat a condition in the subject).

[0153] Asialoglycoprotein receptor (ASGPR) As used herein, the term "asialoglycoprotein receptor" or "ASGPR" refers to a bicomponent C-type lectin formed by a major 48 kDa subunit (ASGPR-1) and a minor 40 kDa subunit (ASGPR-2). ASGPR is expressed primarily on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins containing terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins).

[0154] Prodrug The term "prodrug" as used herein refers to a compound form or derivative that is metabolized by a subject after administration, for example, by biological fluids or enzymes, into a pharmacologically active form of the compound in vivo to produce a desired pharmacological effect. Prodrugs are described, for example, in "Organic Chemistry of Drug Design and Drug Action" by Richard B. Silverman, Academic Press, San Diego, 2004, Chapter 8 Prodrugs and Drug Delivery Systems, pp. 497-558.

[0155] subject As used herein, the term "subject" refers to any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or non-human primate. The terms "individual" or "patient" may be used interchangeably with "subject."

[0156] treatment As used herein, the terms "treatment," "treating," "treating," and the like generally refer to obtaining a desired pharmacological and / or physiological effect. This effect is therapeutic in that it partially or completely cures a disease and / or adverse effects resulting from the disease. The effect is provided by administering a therapeutic agent (e.g., a pharmaceutical combination or oligonucleotide) to a subject with the intent of improving the subject's health and / or well-being with respect to an existing condition (e.g., an existing HBV infection) or with the intent of preventing or reducing the likelihood of the condition occurring (e.g., preventing liver fibrosis, hepatitis, liver cancer, or other conditions associated with HBV infection). As used herein, the term "treatment" encompasses any treatment of HBV infection in a subject, including: (a) inhibiting the disease, i.e., halting its development, such as inhibiting the increase of HBsAg and / or HBeAg; or (b) ameliorating (i.e., palliating) the disease, i.e., causing regression of the disease, such as suppressing HBsAg and / or HBeAg production. Thus, a compound or a combination of compounds that ameliorates and / or inhibits HBV infection is a compound or a combination of compounds that treats HBV infection. Preferably, the term "treatment" as used herein relates to medical intervention of an already manifested disorder, such as the treatment of an already defined and manifested HBV infection, in particular chronic HBV infection.

[0157] In some embodiments, treatment includes reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., an HBV infection or an associated condition) experienced by a subject. During HBV infection, a subject may exhibit symptoms such as yellowing of the skin and eyes (jaundice), dark urine, extreme fatigue, nausea, vomiting, and abdominal pain. Thus, in some embodiments, a treatment, e.g., a pharmaceutical combination, provided herein may result in a reduction in the frequency or severity of one or more of such symptoms. However, HBV infection may progress to one or more liver conditions, such as cirrhosis, liver fibrosis, liver inflammation, or liver cancer. Thus, in some embodiments, a treatment, e.g., a pharmaceutical combination, provided herein may result in a reduction in the frequency or severity of, or prevention or alleviation of, one or more of such symptoms.

[0158] Therapeutically effective dose The term "therapeutically effective amount" refers to an amount of a compound or pharmaceutical combination of the present invention that, when administered to a subject, (i) treats or prevents a particular disease, condition, or disorder, (ii) reduces, ameliorate, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. A therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

[0159] excipients As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in one or more of the compositions containing pharmaceuticals that are part of a pharmaceutical combination, for example, to provide or contribute a desired consistency or stabilizing effect. DETAILED DESCRIPTION OF THE INVENTION

[0160] Detailed Description of the Invention The present invention relates to a pharmaceutical combination comprising at least two HBV therapeutic agents. More particularly, the present invention relates to a pharmaceutical combination comprising an RNAi oligonucleotide targeting HBV and an anti-PDL1 antisense oligonucleotide as defined herein.

[0161] The HBV therapeutic agents and dosing regimens used in the pharmaceutical combinations of the present invention will now be described in detail.

[0162] RNAi oligonucleotides targeting HBV In one embodiment, the therapeutic agent used in the pharmaceutical combination of the present invention is an RNAi oligonucleotide targeting HBV, which can be used to achieve a therapeutic benefit by reducing the expression of HBsAg mRNA.

[0163] In one embodiment, the RNAi oligonucleotide in the pharmaceutical combination of the present invention is an oligonucleotide that targets HBsAg mRNA.

[0164] In one embodiment, the RNAi oligonucleotide in the pharmaceutical combination of the present invention is an oligonucleotide that targets HBsAg mRNA, thereby reducing the expression of HBsAg mRNA.

[0165] Through examination of HBV surface antigen mRNA and testing of different oligonucleotides, potent oligonucleotides have been developed to reduce expression of HBV surface antigen (HBsAg) to treat HBV infection. The RNAi oligonucleotides provided herein, in some embodiments, are designed to target HBsAg mRNA sequences that cover >95% of the known HBV genome across all known genotypes. In some embodiments, such oligonucleotides, when used as part of a pharmaceutical combination of the present invention, result in a greater than 90% reduction in HBV pregenomic RNA (pgRNA) and HBsAg mRNA in the liver. In some embodiments, the reduction in HBsAg expression persists long-term following the pharmaceutical combination treatment regimen.

[0166] Thus, in some embodiments, the RNAi oligonucleotides provided herein are designed to have a region of complementarity to HBsAg mRNA for the purpose of targeting the transcript in a cell and inhibiting its expression. The region of complementarity is generally of a length and base content suitable to allow the oligonucleotide (or a strand thereof) to anneal to HBsAg mRNA for the purpose of inhibiting its expression. In some embodiments, the region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides in length. In some embodiments, the oligonucleotides provided herein have a region of complementarity to HBsAg mRNA that is in the range of 12 to 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, the RNAi oligonucleotides provided herein have a region of complementarity to HBsAg mRNA that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0167] In some embodiments, the RNAi oligonucleotides provided herein are designed to target an mRNA sequence encoding HBsAg. For example, in some embodiments, RNAi oligonucleotides are provided having an antisense strand with a region of complementarity to the following sequence: ACAANAAUCCUCACAAUA (SEQ ID NO: 1), where N refers to any nucleotide (A, G, T / U, or C). In some embodiments, the oligonucleotide further comprises a sense strand that forms a duplex region with the antisense strand. In some embodiments, the sense strand has a region of complementarity to the following sequence: UUNUUGUGAGGAUUN (SEQ ID NO: 2). In some embodiments, the sense strand includes a region of complementarity to the following sequence (shown 5' to 3'): UUAUUGUGAGGAUUNUUGUC (SEQ ID NO: 3).

[0168] In some embodiments, the antisense strand comprises or consists of the sequence shown below: UUAUUGUGAGGAUUNUUGUCGG (SEQ ID NO: 4). In some embodiments, the antisense strand comprises or consists of the sequence shown below: UUAUUGUGAGGAUUCUUGUCGG (SEQ ID NO: 5). In some embodiments, the antisense strand comprises or consists of the sequence shown below: UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 6). In some embodiments, the sense strand comprises or consists of the sequence shown below: ACAANAAUCCUCACAAUAA (SEQ ID NO: 7). In some embodiments, the sense strand comprises or consists of the sequence shown below: GACAANAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 8). In some embodiments, the sense strand comprises or consists of the sequence shown below: GACAAAAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 9). In some embodiments, the sense strand comprises or consists of the sequence shown below: GACAAGAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 10).

[0169] In some embodiments, an RNAi oligonucleotide for reducing HBsAg mRNA expression comprises a sense strand that forms a duplex region with an antisense strand, wherein the sense strand comprises a sequence set forth in any one of SEQ ID NOs: 7-10, and the antisense strand comprises a sequence set forth in any one of SEQ ID NOs: 4-6. In some embodiments, the sense strand comprises 2'-fluoro and 2'-O-methyl modified nucleotides and at least one phosphorothioate internucleotide linkage. In some embodiments, the sense strand is conjugated to an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the antisense strand comprises 2'-fluoro and 2'-O-methyl modified nucleotides and at least one phosphorothioate internucleotide linkage. In some embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the antisense strand and the sense strand each comprise 2'-fluoro and 2'-O-methyl modified nucleotides and at least one phosphorothioate internucleotide linkage, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog, and the sense strand is conjugated to an N-acetylgalactosamine (GalNAc) moiety.

[0170] In some embodiments, the sense strand comprising a sequence set forth in any one of SEQ ID NOs: 8-10 comprises 2'-fluoro-modified nucleotides at positions 3, 8-10, 12, 13, and 17. In some embodiments, the sense strand comprises 2'-O-methyl-modified nucleotides at positions 1, 2, 4-7, 11, 14-16, 18-26, and 31-36. In some embodiments, the sense strand comprises one phosphorothioate internucleotide linkage. In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between the nucleotides at positions 1 and 2. In some embodiments, the sense strand is conjugated to an N-acetylgalactosamine (GalNAc) moiety.

[0171] In some embodiments, the antisense strand comprising any one of SEQ ID NOs: 4-6 comprises 2'-fluoro-modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16, and 19. In some embodiments, the antisense strand comprises 2'-O-methyl-modified nucleotides at positions 1, 4, 6, 9, 11, 13, 15, 17, 18, and 20-22. In some embodiments, the antisense strand comprises three phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 3 and 4, between the nucleotides at positions 20 and 21, and between the nucleotides at positions 21 and 22. In some embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog.

[0172] In one embodiment of the pharmaceutical combination of the present invention, the RNAi oligonucleotide is an oligonucleotide comprising an antisense strand 19 to 30 nucleotides in length, wherein the antisense strand comprises a region of complementarity to the sequence of HBsAg mRNA represented as ACAANAAUCCUCACAAUA (SEQ ID NO: 1) (N can represent any nucleotide A, G, C, or T / U). In some embodiments, the oligonucleotide further comprises a sense strand that forms a duplex region with the antisense strand. In some embodiments, the sense strand has a region of complementarity to the sequence represented as UUNUUGUGAGGAUUN (SEQ ID NO: 2); in some embodiments, the sense strand comprises a region of complementarity to the sequence represented (shown 5' to 3') as UUAUUGUGAGGAUUNUUGUC (SEQ ID NO: 3).

[0173] In one embodiment, the RNAi oligonucleotide in the pharmaceutical combination of the present invention is an oligonucleotide for reducing expression of hepatitis B virus surface antigen (HBsAg) mRNA, the oligonucleotide comprising a sense strand that forms a duplex region with an antisense strand, the sense strand consists of the sequence GACAAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 9), containing 2'-fluoro modified nucleotides at positions 3, 8-10, 12, 13, and 17, 2'-O-methyl modified nucleotides at positions 1, 2, 4-7, 11, 14-16, 18-26, and 31-36, and a phosphorothioate linkage between the nucleotide at position 1 and 2, wherein each nucleotide of the -GAAA- sequence on the sense strand is conjugated to a monovalent GalNac moiety; the antisense strand is comprised of the sequence UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 6), which contains 2'-fluoro modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16, and 19, 2'-O-methyl modified nucleotides at positions 1, 4, 6, 9, 11, 13, 15, 17, 18, and 20-22, and phosphorothioate linkages between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 3 and 4, between the nucleotides at positions 20 and 21, and between the nucleotides at positions 21 and 22; The 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a methoxyphosphonate (MOP).

[0174] In a preferred embodiment, the RNAi oligonucleotide in the pharmaceutical combination of the present invention is an oligonucleotide comprising a sense strand that forms a duplex region with an antisense strand, the sense strand comprises the sequence GACAAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 9), containing 2'-fluoro modified nucleotides at positions 3, 8-10, 12, 13, and 17, 2'-O-methyl modified nucleotides at positions 1, 2, 4-7, 11, 14-16, 18-26, and 31-36, and one phosphorothioate internucleotide linkage between the nucleotide at position 1 and 2, wherein each nucleotide of the -GAAA- sequence on the sense strand is conjugated to a monovalent GalNac moiety, and the -GAAA- sequence has the following structure: [ka] Including, The antisense strand comprises the sequence set forth in UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 6), which contains 2'-fluoro modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16, and 19, 2'-O-methyl modified nucleotides at positions 1, 4, 6, 9, 11, 13, 15, 17, 18, and 20-22, and five phosphorothioate internucleotide linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, and the 5'-nucleotide of the antisense strand has the following structure: [ka] or a pharmaceutically acceptable salt thereof. This definition of the RNAi oligonucleotide targeting HBV used in the pharmaceutical combination of the present invention is referred to herein as "T1" or "Therapeutic Agent T1."

[0175] In certain specific embodiments, T1 may be further defined as the molecule of FIG.

[0176] In one embodiment, the RNAi oligonucleotide is administered subcutaneously.

[0177] In one embodiment, the RNAi oligonucleotide is administered at an initial dose of about 0.1 mg / kg to about 12 mg / kg, preferably about 0.1 mg / kg to about 9 mg / kg, more preferably about 0.5 mg / kg to about 7 mg / kg, more preferably about 0.5 mg / kg to about 6.5 mg / kg, more preferably about 1 mg / kg to about 6 mg / kg, more preferably about 1.5 mg / kg to about 6 mg / kg, more preferably about 2 mg / kg to about 6 mg / kg, and most preferably about 3 mg / kg or about 6 mg / kg.

[0178] In one embodiment, the RNAi oligonucleotide is administered at an initial dose of about 6 to about 800 mg, preferably about 100 mg, about 200 mg, or about 400 mg.

[0179] In one embodiment, the initial dose is a single dose or the only dose administered.

[0180] In one embodiment, one or more subsequent doses of RNAi oligonucleotide are administered in an amount of about 0.1 mg / kg to about 12 mg / kg, hi one embodiment, the subsequent dose(s) is about 1.5 mg / kg, about 3 mg / kg, or about 6 mg / kg.

[0181] In one embodiment, one or more subsequent doses of oligonucleotide are administered in an amount between about 6 mg and about 800 mg, hi one embodiment, the subsequent dose(s) is about 100 mg, about 200 mg, or about 400 mg.

[0182] In one embodiment, each dose is administered at least about once every 2 weeks, at least about once every 3 weeks, at least about once every 4 weeks, at least about once every 5 weeks, at least about once every 6 weeks, at least about once every 7 weeks, or at least about once every 8 weeks. In one embodiment, the doses are separated in time from each other by at least about 4 weeks. In one embodiment, doses of about 1 mg / kg to 6 mg / kg are administered, each separated by at least about 4 weeks.

[0183] In one embodiment, the doses are separated in time from each other by about 4 weeks and are administered over a period of about 48 weeks, about 24 weeks, about 3 months, or about 12 weeks.

[0184] In one embodiment, the period between each of the doses is independently selected from the group consisting of about 4 weeks, about 1 month, about 2 months, about 3 months, or about 6 months.

[0185] Further useful definitions and substitutions for RNAi oligonucleotides targeting HBV in the pharmaceutical combinations of the present invention are provided below.

[0186] I. Double-stranded oligonucleotides for targeting HBsAg mRNA There are various structures of oligonucleotides useful for targeting HBsAg mRNA expression in the pharmaceutical combinations of the present disclosure, including RNAi, miRNA, etc. Any of the structures described herein or elsewhere can be used as a framework for incorporating or targeting the sequences described herein. Double-stranded oligonucleotides for targeting HBV antigen expression (e.g., via the RNAi pathway) generally have a sense strand and an antisense strand that form a duplex with each other. In some embodiments, the sense strand and the antisense strand are not covalently linked. However, in some embodiments, the sense strand and the antisense strand are covalently linked.

[0187] In some embodiments of the present invention, double-stranded oligonucleotides for reducing HBsAg mRNA expression involve RNA interference (RNAi). For example, RNAi oligonucleotides have been developed with each strand having a size of 19 to 25 nucleotides with at least one 3' overhang of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides that are processed by Dicer to generate active RNAi products have also been developed (see, e.g., U.S. Pat. No. 8,883,996). Further research has produced extended double-stranded oligonucleotides in which at least one end of at least one strand extends beyond the duplex targeting region, with one of the strands containing a thermodynamically stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, and International Publication No. WO2010033225, the disclosures of which are incorporated herein by reference). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

[0188] In some embodiments, the oligonucleotides provided herein are cleavable by Dicer enzyme. Such oligonucleotides may have an overhang (e.g., 1, 2, or 3 nucleotides in length) at the 3' end of the sense strand. Such oligonucleotides (e.g., siRNA) may comprise a 21-nucleotide guide strand that is antisense to the target RNA and a complementary passenger strand, with the two strands annealing to form a 19-bp duplex and a two-nucleotide overhang at either or both of the 3' ends. Longer oligonucleotide designs are also available, including oligonucleotides with a 23-nucleotide guide strand and a 21-nucleotide passenger strand, with a blunt end on the right side of the molecule (3' end of the passenger strand / 5' end of the guide strand) and a two-nucleotide 3' guide strand overhang on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). Such molecules have a 21-base pair duplex region. See, for example, U.S. Patent Nos. 9,012,138, 9,012,621, and 9,193,753. each of which is incorporated herein for their relevant disclosure.

[0189] In some embodiments, oligonucleotides disclosed herein can comprise a sense strand and an antisense strand, both ranging from 17 to 26 (e.g., 17 to 26, 20 to 25, 19 to 21, or 21 to 23) nucleotides in length. In some embodiments, the sense and antisense strands are equal in length. In some embodiments, for oligonucleotides having a sense strand and an antisense strand, both ranging from 21 to 23 nucleotides in length, the 3' overhangs of the sense strand, the antisense strand, or both the sense and antisense strands are 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has a 23-nucleotide guide strand and a 21-nucleotide passenger strand, with a blunt end on the right side of the molecule (3' end of the passenger strand / 5' end of the guide strand) and a 2-nucleotide 3' guide strand overhang on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). Such molecules have a 21-base pair duplex region. In some embodiments, the oligonucleotide comprises a 25 nucleotide sense strand and a 27 nucleotide antisense strand that, when acted upon by the dicer enzyme, results in the antisense strand being incorporated into mature RISC.

[0190] Other oligonucleotide designs for use in the compositions and methods disclosed herein include 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., with stems of 19 bp or shorter; see, e.g., Moore et al. Methods Mol. Biol. 2010;629:141-158)), blunt siRNAs (e.g., 19 bp in length; see, e.g., Kraynack and Baker, RNA Vol. 12, pp. 163-176 (2006)), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric short duplex siRNAs (see, e.g., Chang et al., Mol. Ther. 2009). Apr;17(4):725-32), forked siRNA (see, e.g., Hohjoh, FEBS Letters, Vol 557, issues 1-3; Jan 2004, p 193-198), single-stranded siRNA (Elsner; Nature Biotechnology 30, 1063(2012)), dumbbell-shaped circular siRNA (see, e.g., Abe et al. J Am Chem Soc 129:15108-15109(2007)), and small internal segmented interfering RNA (sisiRNA; see, e.g., Bramsen et al., Nucleic Acids Res. 2007 Sep;35(17):5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the relevant disclosures therein. Further non-limiting examples of oligonucleotide structures that may be used in pharmaceutical combinations in some embodiments to reduce or inhibit expression of HBsAg are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, e.g., Hamilton et al., Embo J., 2002, 21(17):4671-4679; see also U.S. Patent Application No. 20090099115).

[0191] a. antisense strand In some embodiments, the antisense strand of oligonucleotide can be called " guide strand ".For example, if antisense strand can be associated with RNA-induced silencing complex (RISC) and bind to Argonaute protein, or can be associated with one or more similar factors and directly silence target gene, it can be called guide strand.In some embodiments, the sense strand that is complementary to guide strand can be called " passenger strand ".

[0192] In some embodiments, the oligonucleotides provided herein comprise an antisense strand that is up to 50 nucleotides in length (e.g., up to 30, up to 27, up to 25, up to 21, or up to 19 nucleotides in length). In some embodiments, the oligonucleotides provided herein comprise an antisense strand that is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, or at least 27 nucleotides in length). In some embodiments, the antisense strand of the oligonucleotides disclosed herein is within the range of 12 to 50 or 12 to 30 (e.g., 12 to 30, 11 to 27, 11 to 25, 15 to 21, 15 to 27, 17 to 21, 17 to 25, 19 to 27, or 19 to 30) nucleotides in length. In some embodiments, the antisense strand of any one of the oligonucleotides disclosed herein is 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, or 50 nucleotides in length.

[0193] B sense strand In some embodiments, a double-stranded oligonucleotide can have a sense strand up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide can have a sense strand at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide can have a sense strand in the range of 12 to 50 nucleotides in length (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, the oligonucleotide may have a sense strand that is 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, or 40 nucleotides in length. In some embodiments, the sense strand of the oligonucleotide is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In some embodiments, the sense strand of the oligonucleotide is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides).

[0194] In some embodiments, the sense strand comprises a stem-loop at its 3'-end. In some embodiments, the sense strand comprises a stem-loop at its 5'-end. In some embodiments, the strand comprising the stem-loop is 2 to 66 nucleotides in length (e.g., 2 to 66, 10 to 52, 14 to 40, 2 to 30, 4 to 26, 8 to 22, 12 to 18, 10 to 22, 14 to 26, or 14 to 30 nucleotides in length). In some embodiments, the strand comprising the stem-loop is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the stem comprises a duplex that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the stem loop provides better protection of the molecule against degradation (e.g., enzymatic degradation) and facilitates targeting properties for delivery to target cells. For example, in some embodiments, the loop provides an additional nucleotide that can be modified without substantially affecting the gene expression inhibitory activity of the oligonucleotide. In certain embodiments, provided herein are oligonucleotides whose sense strand comprises (e.g., at its 3' end) a stem loop as described below: S1-L-S2, where S1 is complementary to S2, and L forms a loop between S1 and S2 of up to 10 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length).

[0195] In some embodiments, the loop (L) of the stem-loop is a tetraloop (e.g., in a nicked tetraloop structure). The tetraloop can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, the tetraloop has 4-5 nucleotides.

[0196] c. length of the duplex In some embodiments, the duplex formed between the sense strand and the antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is in the range of 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In some embodiments, the duplex formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand does not span the entire length of the sense strand and / or the antisense strand. In some embodiments, the duplex formed between the sense strand and the antisense strand spans the entire length of either the sense strand or the antisense strand. In certain embodiments, the duplex formed between the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand.

[0197] d. oligonucleotide terminus In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, whereby a 3'-overhang is present on either the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, the oligonucleotides provided herein have one 5'-end that is thermodynamically less stable than the other 5'-end. In some embodiments, asymmetric oligonucleotides are provided that comprise a blunt end on the 3'-end of the sense strand and an overhang on the 3'-end of the antisense strand. In some embodiments, the 3'-overhang on the antisense strand is 1 to 8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length).

[0198] Typically, RNAi oligonucleotides have a two-nucleotide overhang at the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang comprising 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length. However, in some embodiments, the overhang is a 5' overhang comprising 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0199] In some embodiments, one or more (e.g., 2, 3, 4) terminal nucleotides at the 3'-end or 5'-end of the sense strand and / or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides at the 3'-end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3'-end of the antisense strand is modified, for example, comprises a 2'-modification, such as 2'-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3'-end of the antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3'-end of the antisense strand are not complementary to the target.

[0200] In some embodiments, a double-stranded oligonucleotide is provided, which has a nicked tetraloop structure at the 3'-end of the sense strand, and has two terminal overhang nucleotides at the 3'-end of its antisense strand.In some embodiments, the two terminal overhang nucleotides are GG.Typically, one or both of the two terminal GG nucleotides of the antisense strand are complementary or not complementary to the target.

[0201] In some embodiments, the 5' and / or 3' ends of the sense or antisense strand have inverted cap nucleotides.

[0202] In some embodiments, one or more (e.g., 2, 3, 4, 5, 6) modified internucleotide linkages are provided between the terminal nucleotides at the 3' or 5' ends of the sense and / or antisense strands, hi some embodiments, modified internucleotide linkages are provided between overhanging nucleotides at the 3' or 5' ends of the sense and / or antisense strands.

[0203] e. Mismatch In some embodiments, the oligonucleotide may have one or more (e.g., 1, 2, 3, 4, 5) mismatches between the sense strand and the antisense strand. When there are two or more mismatches between the sense strand and the antisense strand, they may be arranged consecutively (e.g., 2, 3, or more in a row) or may be scattered throughout the region of complementarity. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated into the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of a segment at the 3' end of the sense strand of the oligonucleotide improved the efficacy of synthetic duplexes in RNAi, possibly by facilitating processing by Dicer.

[0204] In some embodiments, the antisense strand may have a region of complementarity to an HBsAg transcript that contains one or more mismatches compared to the corresponding transcript sequence. The region of complementarity on the oligonucleotide may have up to 1, up to 2, up to 3, up to 4, up to 5, etc. mismatches, so long as it maintains the ability to form complementary base pairs with the transcript under appropriate hybridization conditions. Alternatively, the region of complementarity of the oligonucleotide may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches, so long as it maintains the ability to form complementary base pairs with HBsAg mRNA under appropriate hybridization conditions. In some embodiments, when there are two or more mismatches in the region of complementarity, they may be arranged consecutively (e.g., 2, 3, 4, or more consecutively) or interspersed throughout the region of complementarity, so long as the oligonucleotide maintains the ability to form complementary base pairs with HBsAg mRNA under appropriate hybridization conditions.

[0205] II. Single-stranded oligonucleotides In some embodiments, the RNAi oligonucleotide for reducing HBsAg expression described herein is a single-stranded oligonucleotide that has complementarity with HBsAg mRNA.Such structure can include, but is not limited to, single-stranded RNAi oligonucleotide.Recent attempts have demonstrated the activity of single-stranded RNAi oligonucleotide (see, for example, Matsui et al. (May 2016), Molecular Therapy, Vol.24(5), 946-955).

[0206] Such single-stranded RNAi oligonucleotides may technically be considered antisense oligonucleotides, but may still function through the mechanism of RNA interference and have the characteristics as described herein for RNAi oligonucleotides.

[0207] III. Oligonucleotide Modification The modifications discussed in this section are particularly preferred for implementation in the RNAi oligonucleotides of the invention.

[0208] Oligonucleotides can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base-pairing properties, RNA distribution and cellular uptake, and other characteristics relevant to therapeutic or research use. See, for example, Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen and Kjems (Frontiers in Genetics, 3(2012):1-22). Thus, in some embodiments, therapeutic oligonucleotides of the present disclosure may contain one or more appropriate modifications. In some embodiments, modified nucleotides have modifications in their base (or nucleobase), sugar (e.g., ribose, deoxyribose), or phosphate group.

[0209] The number of modifications on an oligonucleotide and the location of those nucleotide modifications can affect the properties of the oligonucleotide. For example, oligonucleotides can be delivered in vivo by conjugating or incorporating them into lipid nanoparticles (LNPs) or similar carriers. However, if the oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of its nucleotides to be modified. Thus, in certain embodiments of any of the therapeutic oligonucleotides provided herein, all or substantially all of the nucleotides of the oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In certain embodiments, less than half of the nucleotides are modified. Typically, for naked delivery, all sugars are modified at the 2' position. These modifications can be reversible or irreversible. In some embodiments, the oligonucleotides disclosed herein have a sufficient number and type of modified nucleotides to induce desired characteristics (e.g., protection from enzymatic degradation, ability to target desired cells after in vivo administration, and / or thermodynamic stability).

[0210] IV. Sugar modification In some embodiments, modified sugars (also referred to herein as sugar analogs) comprise modified deoxyribose or ribose moieties, e.g., one or more modifications occur at the 2', 3', 4', and / or 5' carbon positions of the sugar. In some embodiments, modified sugars can also comprise unnatural alternative carbon structures, such as those found in locked nucleic acids ("LNAs") (see, e.g., Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids ("UNAs") (see, e.g., Snead et al. (2013), Molecular Therapy-Nucleic Acids, 2, e103), and bridged nucleic acids ("BNAs") (see, e.g., Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated herein by reference for their disclosures regarding sugar modifications.

[0211] In some embodiments, the nucleotide modification in the sugar comprises a 2'-modification. The 2'-modification can be 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, or 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. Typically, the modification is 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl. In some embodiments, the sugar modification comprises a modification of the sugar ring, which can include modification of one or more carbons of the sugar ring. For example, the sugar modification of the nucleotide can include the 2'-oxygen of the sugar being linked to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen being linked to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking a 2'-carbon-3'-carbon bond. In some embodiments, the modified nucleotide has a thiol group, for example, at the 4'-position of the sugar.

[0212] In some embodiments, the terminal 3'-end group (e.g., 3'-hydroxyl) is a phosphate group or other group that can be used, for example, to attach a linker, adapter, or label, or to directly ligate the oligonucleotide to another nucleic acid.

[0213] V. 5'-Terminal Phosphate In some embodiments, the 5'-terminal phosphate group of an oligonucleotide enhances its interaction with Argonaute 2. However, oligonucleotides containing a 5'-phosphate group are susceptible to degradation via phosphatases or other enzymes, potentially limiting their bioavailability in vivo. In some embodiments, the oligonucleotide comprises a 5'-phosphate analog that is resistant to such degradation. In some embodiments, the phosphate analog can be an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5'-end of the oligonucleotide chain is linked to a chemical moiety that mimics the electrostatic and steric properties of a natural 5'-phosphate group (a "phosphate mimetic") (see, e.g., Prakash et al. (2015), Nucleic Acids Res., Nucleic Acids Res. 2015 Mar 31;43(6):2993-3011, the contents of which regarding phosphate analogs are incorporated herein by reference). Many phosphate mimetics that can be attached to the 5' end have been developed (see, for example, U.S. Patent No. 8,927,513, the contents of which regarding phosphate analogs are incorporated herein by reference). Other modifications to the 5' end of oligonucleotides have been developed (see, for example, WO 2011 / 133871, the contents of which regarding phosphate analogs are incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5' end of the oligonucleotide.

[0214] In some embodiments, oligonucleotides have a phosphate analog at the 4'-carbon position of the sugar (referred to as a "4'-phosphate analog"). See, for example, U.S. Provisional Patent Application No. 62 / 383,207, filed September 2, 2016, entitled "4'-Phosphate Analogs and Oligonucleotides Comprising the Same," and U.S. Provisional Patent Application No. 62 / 393,401, filed September 12, 2016, entitled "4'-Phosphate Analogs and Oligonucleotides Comprising the Same." The contents of each of these applications regarding phosphate analogs are incorporated herein by reference. In some embodiments, the oligonucleotides provided herein include a 4'-phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethyl phosphonate or an analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., the 4'-carbon). In other embodiments, the 4' phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate (wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4'-carbon of the sugar moiety), or an analog thereof. In certain embodiments, the 4' phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, where R is independently selected from H, a CH3 alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CHOCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3.

[0215] In certain embodiments, the phosphate analog attached to the oligonucleotide is methoxyphosphonate (MOP). In certain embodiments, the phosphate analog attached to the oligonucleotide is 5' monomethyl-protected MOP. In some embodiments, the following uridine nucleotides containing phosphate analogs can be used, for example, at position 1 of the guide (antisense) strand: [ka] This modified nucleotide is called [Mephosphonate-4O-mU] or 5'-methoxy, phosphonate-4'oxy-2'-O-methyluridine.

[0216] VI. Modified Internucleoside Linkages In some embodiments, the phosphate modification or substitution can result in an oligonucleotide comprising at least one (e.g., at least one, at least two, at least three, or at least five) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

[0217] The modified internucleotide bond can be a phosphorothioate bond, a phosphorothioate bond, a phosphotriester bond, a thionoalkylphosphonate bond, a thionoalkylphosphotriester bond, a phosphoramidite bond, a phosphonate bond, or a boranophosphate bond. In some embodiments, at least one modified internucleotide bond of any one of the oligonucleotides disclosed herein is a phosphorothioate bond.

[0218] VII. Base Modifications In some embodiments, the oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as a base analog) is linked to the 1' position of the nucleotide sugar moiety. In certain embodiments, the modified nucleobase is a nitrogenous base. In certain embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 20080274462. In some embodiments, the modified nucleotide contains a universal base. However, in certain embodiments, the modified nucleotide does not contain a nucleobase (abasic).

[0219] In some embodiments, a universal base is a heterocyclic moiety located at the 1'-position of a nucleotide sugar moiety in a modified nucleotide, or an equivalent position of a nucleotide sugar moiety substitute that, when present in a duplex, can be located opposite two or more bases without substantially altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is perfectly complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base exhibits a lower T than a duplex formed with a complementary nucleic acid. m However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base is replaced with a base resulting in a single mismatch, the single-stranded nucleic acid containing the universal base has a higher T than the duplex formed with the nucleic acid containing the mismatched base. m It forms a duplex with a target nucleic acid having the formula:

[0220] Non-limiting examples of universal binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (Quay et al., U.S. Patent Application Publication No. 20070254362; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside, Nucleic Acids Res. 1995 Nov 11;23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR, Nucleic Acids Res. 1995 Jul 11;23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue, Nucleic Acids Res. 1994 Oct 11). 11;22(20):4039-43. Each of the above is incorporated herein by reference for their disclosure regarding base modifications.

[0221] VIII. Reversible modification Certain modifications can be made to protect the oligonucleotide from the in vivo environment before it reaches the target cell, but this can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made so that the molecule retains desirable properties outside the cell, and are then removed once it enters the cytosolic environment of the cell. Reversible modifications can be removed, for example, by the action of intracellular enzymes or intracellular chemical conditions (e.g., via reduction by intracellular glutathione).

[0222] In some embodiments, the reversibly modified nucleotide comprises a glutathione-sensitive moiety. Typically, nucleic acid molecules are chemically modified with a cyclic disulfide moiety to mask the negative charge generated by the internucleotide diphosphate bond and improve cellular uptake and nuclease resistance. See U.S. Patent Application Publication No. 2011 / 0294869 ("Traversa"), originally assigned to Traversa Therapeutics, Inc.; PCT Publication No. WO2015 / 188197 ("Solstice"), to Solstice Biologics, Ltd.; Meade et al., Nature Biotechnology, 2014, 32:1256-1263 ("Meade"); and PCT Publication No. WO2014 / 088920, to Merck Sharp & Dohme Corp., each of which is incorporated by reference for its disclosure of such modifications. This reversible modification of the internucleotide diphosphate bridge is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). Previous examples include neutralizing phosphotriester modifications that have been reported to be cleavable intracellularly (Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

[0223] In some embodiments, such reversible modifications allow for protection during in vivo administration (e.g., passage through the blood and / or lysosomal / endosomal compartments of cells), where the oligonucleotide is exposed to nucleases and other harsh environmental conditions (e.g., pH). Upon release into the cytosol of cells, where glutathione levels are higher compared to the extracellular space, the modification is reversed, yielding a cleaved oligonucleotide. The use of reversible glutathione-sensitive moieties allows for the introduction of sterically larger chemical groups into the oligonucleotide of interest compared to options available using irreversible chemical modifications. This is because these larger chemical groups are removed in the cytosol and therefore do not interfere with the biological activity of the oligonucleotide within the cytosol of cells. As a result, these larger chemical groups can be engineered to confer various advantages on the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to alter its release kinetics.

[0224] In some embodiments, the glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety is attached to the 2'-carbon of the sugar of the modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, particularly when the modified nucleotide is the 5'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3'-carbon of the sugar, particularly when the modified nucleotide is the 3'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., U.S. Provisional Patent Application No. 62 / 378,635, filed August 23, 2016, entitled "Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof," the contents of which are incorporated herein by reference for their relevant disclosures.

[0225] IX. Targeting Ligands In some embodiments, it may be desirable to target the oligonucleotides of the present disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs or may avoid excessive loss of oligonucleotides to cells, tissues, or organs that are not beneficial to the oligonucleotide. Thus, in some embodiments, the oligonucleotides disclosed herein may be modified to facilitate targeting of specific tissues, cells, or organs, for example, to facilitate delivery of oligonucleotides to the liver. In certain embodiments, the oligonucleotides disclosed herein may be modified to facilitate delivery of oligonucleotides to hepatocytes in the liver. In some embodiments, the oligonucleotides comprise nucleotides conjugated to one or more targeting ligands.

[0226] The targeting ligand may comprise a carbohydrate, an amino sugar, cholesterol, a peptide, a polypeptide, a protein, or a portion of a protein (e.g., an antibody or antibody fragment) or a lipid. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand may be an RGD peptide used to target tumor vasculature or glioma cells, a CREKA peptide for targeting tumor vasculature or stoma, an aptamer for targeting transferrin, lactoferrin, or the transferrin receptor expressed on the CNS vasculature, or an anti-EGFR antibody for targeting EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

[0227] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to 2 to 4 nucleotides at either the end of the sense strand or the antisense strand, such that the targeting ligand resembles the bristles of a toothbrush and the oligonucleotide resembles the toothbrush (e.g., the ligand is conjugated to an overhang or extension of 2 to 4 nucleotides on the 5' or 3' end of the sense or antisense strand). For example, the oligonucleotide may comprise a stem loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem loop may be individually conjugated to a targeting ligand.

[0228] In some embodiments, it is desirable to target the oligonucleotide that reduces expression of an HBV antigen to hepatocytes in the liver of a subject. Any suitable hepatocyte targeting moiety may be used for this purpose.

[0229] GalNAc is a high-affinity ligand for the asialoglycoprotein receptor (ASGPR), which is expressed primarily on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins containing terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (indirectly or directly) of a GalNAc moiety to the oligonucleotides of the present disclosure can be used to target these oligonucleotides to the ASGPR expressed on these hepatocytes.

[0230] In some embodiments, the oligonucleotide of the present disclosure is directly or indirectly conjugated to a monovalent GalNAc. In some embodiments, the oligonucleotide is directly or indirectly conjugated to two or more monovalent GalNAc moieties (i.e., conjugated to two, three or four monovalent GalNAc moieties, typically conjugated to three or four monovalent GalNAc moieties). In some embodiments, the oligonucleotide of the present disclosure is conjugated to one or more divalent, trivalent or tetravalent GalNAc moieties.

[0231] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to a separate GalNAc. In some embodiments, a targeting ligand is conjugated to 2 to 4 nucleotides at either the sense or antisense strand end, such that the GalNAc moieties resemble the bristles of a toothbrush and the oligonucleotide resembles a toothbrush (e.g., the ligand is conjugated to an overhang or extension of 2 to 4 nucleotides on the 5' or 3' end of the sense or antisense strand). For example, the oligonucleotide can comprise a stem-loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem loop can be individually conjugated to a GalNAc moiety. In some embodiments, the GalNAc moiety is conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand, with each GalNAc moiety conjugated to one nucleotide.

[0232] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc linked to a guanidine nucleotide, designated [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanidine-GalNAc, as shown below. [ka]

[0233] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc linked to an adenine nucleotide, designated [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as shown below. [ka]

[0234] An example of such a conjugation is shown below for a loop containing the nucleotide sequence GAAA from 5' to 3' (L = linker, X = heteroatom). The stem attachment points are indicated. [ka] is the point of attachment to the oligonucleotide chain. [ka]

[0235] The targeting ligand can be linked to the nucleotide using an appropriate method or chemistry (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate the targeting ligand to any one of the nucleotides of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO2016100401 A1, published June 23, 2016, the contents of which regarding such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is fairly stable.

[0236] An example of a loop containing the nucleotides GAAA 5' to 3', in which the GalNAc moiety is attached to the nucleotide of the loop using an acetal linker, is shown below: [ka] is the point of attachment to the oligonucleotide chain. [ka]

[0237] Anti-PDL1 antisense oligonucleotides In one embodiment, the therapeutic agent used in the pharmaceutical combination of the present invention is an anti-PDL1 antisense oligonucleotide.

[0238] In one embodiment, the anti-PDL1 antisense oligonucleotide is an N-acetylgalactosamine (GalNAc) conjugated locked nucleic acid (LNA) single-stranded oligonucleotide (SSO) that induces RNAse H-mediated degradation of PDL1 mRNA.

[0239] In one embodiment, the anti-PDL1 antisense oligonucleotide in the pharmaceutical combination of the present invention is disclosed in WO 2017 / 157899, which is incorporated herein by reference in its entirety.

[0240] In a preferred embodiment, the anti-PDL1 antisense oligonucleotide in the pharmaceutical combination of the present invention is CMP No. 768_2 disclosed in WO 2017 / 157899 or a pharmaceutically acceptable salt thereof.

[0241] In one embodiment, the anti-PDL1 antisense oligonucleotide in the pharmaceutical combination of the present invention comprises the sequence CCTATTTAACATCAGAC (SEQ ID NO: 11).

[0242] In a preferred embodiment, the anti-PDL1 antisense oligonucleotide in the pharmaceutical combination of the present invention has the formula GN2-C6 o c o a o CCtatttaacatcAGAC (where C6 represents an aminoalkyl group having 6 carbons, uppercase letters represent β-D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA C's are 5-methylcytosines, and subscripts o represents a phosphodiester internucleoside linkage, unless otherwise indicated, all internucleoside linkages are phosphorothioate internucleoside linkages, and GN2 represents the following trivalent GalNAc cluster: [ka]

[0243] Additionally, the wavy line in the trivalent GalNAc cluster indicates the conjugation site of the trivalent GalNAc cluster to a C6 aminoalkyl group) or a pharmaceutically acceptable salt thereof. This definition of the anti-PDL1 antisense oligonucleotide used in the pharmaceutical combination of the present invention is referred to herein as "T2" or "therapeutic agent T2."

[0244] In one embodiment, the anti-PDL1 antisense oligonucleotide is administered subcutaneously. In one embodiment, the anti-PDL1 antisense oligonucleotide is administered at a dose of about 0.1 mg / kg to about 35 mg / kg, or about 0.1 mg / kg to about 15 mg / kg, or about 0.1 mg / kg to about 10 mg / kg, or about 0.2 mg / kg to about 10 mg / kg, or about 0.25 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 5 mg / kg, or about 0.2 mg / kg to about 5 mg / kg, or about 0.25 mg / kg to about 5 mg / kg.

[0245] In one embodiment, the anti-PDL1 antisense oligonucleotide is administered at a dose of about 7 mg / kg to about 35 mg / kg.

[0246] In one embodiment, the dose of anti-PDL1 antisense oligonucleotide is administered once a week, once every two weeks, once every three weeks, or once a month.

[0247] In a more preferred embodiment of the pharmaceutical combination of the present invention, particularly when it further comprises the RNAi oligonucleotide targeting HBV, anti-PDL1 antisense oligonucleotide is administered up to 5 times.Preferably, each dose is about 3mg / kg.Preferably, the dose is administered Q2W (every 2 weeks).

[0248] I. Antisense Oligonucleotide Modifications The modifications discussed in this section are particularly preferred for implementation in the antisense oligonucleotides of the invention.

[0249] It is understood that the consecutive nucleobase sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid.

[0250] In one embodiment, the continuous nucleobase sequence of the oligonucleotide comprises at least one modified internucleoside linkage. Suitable internucleoside modifications are described in the "Definitions" section under "Modified Internucleoside Linkages." It is advantageous if at least 75%, for example, all, of the internucleoside linkages in the continuous nucleotide sequence are internucleoside linkages. In some embodiments, all internucleoside linkages in the continuous sequence of the oligonucleotide are phosphorothioate linkages.

[0251] The oligonucleotides of the invention are designed using modified nucleosides and DNA nucleosides. It is advantageous to use high affinity modified nucleosides.

[0252] In one embodiment, the oligonucleotide comprises at least three modified nucleosides, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In one embodiment, the oligonucleotide comprises 3 to 8 modified nucleosides, e.g., 4 to 6 modified nucleosides, e.g., 4, 5, or 6 nucleosides, e.g., 5 or 6 modified nucleosides. Suitable modifications are described in the "Definitions" sections of "Modified Nucleosides," "High-Affinity Modified Nucleosides," "Sugar Modifications," "2' Sugar Modifications," and Locked Nucleic Acid (LNA).

[0253] In one embodiment, the oligonucleotide comprises one or more sugar-modified nucleosides, for example, 2'-sugar-modified nucleosides. Preferably, the oligonucleotide of the present invention comprises one or more 2'-sugar-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides. It is advantageous if one or more or all of the modified nucleosides are locked nucleic acids (LNA).

[0254] In some embodiments, the oligonucleotides of the invention, e.g., the contiguous nucleotide sequence, comprise at least one LNA nucleoside, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides, e.g., 2-6 LNA nucleosides, e.g., 3-6 LNA nucleosides, 4-6 LNA nucleosides, or 4, 5, or 6 LNA nucleosides.

[0255] In some embodiments, at least 75% of the modified nucleosides of the oligonucleotide are LNA nucleosides, for example, at least 80%, for example, at least 85%, for example, at least 90% of the modified nucleosides. In yet further embodiments, all of the modified nucleosides of the oligonucleotide are LNA nucleosides. In further embodiments, the LNA nucleosides are selected from beta-D-oxyLNA, thioLNA, aminoLNA, oxyLNA, ScET, and / or ENA, in the beta-D or alpha-L configuration, or a combination thereof. In further embodiments, all LNA nucleosides are beta-D-oxyLNA. In further embodiments, the cytosine units are 5-methyl-cytosine. For nuclease stability of an oligonucleotide or a consecutive nucleotide sequence, it is advantageous to have at least one LNA nucleoside at the 5'-end and at least two LNA nucleosides at the 3'-end of the nucleotide sequence.

[0256] TLR7 agonists In one embodiment, the therapeutic agent used in the pharmaceutical combination of the present invention is a TLR7 agonist.

[0257] In one embodiment, the TLR7 agonist in the pharmaceutical combination of the present invention is a 3-substituted 5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione compound or a prodrug thereof having Toll-like receptor agonist activity. WO 2006 / 066080, WO 2016 / 055553 and WO 2016 / 091698 describe such TLR7 agonists and their prodrugs, as well as their preparation, which are incorporated herein by reference.

[0258] In one embodiment, the TLR7 agonist in the pharmaceutical combination of the present invention has formula (I): [ka] wherein X is CH or S; R1 is —OH or —H; R2 is 1-hydroxypropyl or hydroxymethyl. or formula (II): [ka] wherein X is CH or S; R1 is -OH or -H or acetoxy; R2 is 1-acetoxypropyl or 1-hydroxypropyl or 1-hydroxymethyl or acetoxy(cyclopropyl)methyl or acetoxy(propyn-1-yl)methyl or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. The compounds of formula (I) are active TLR7 agonists.

[0259] In one embodiment, a subset of the active TLR7 agonists of formula (I) in the pharmaceutical combination of the present invention are those of formula (V): [ka] (wherein R1 is —OH and R2 is 1-hydroxypropyl or hydroxymethyl) or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

[0260] In one embodiment, the substituent at R2 in formula (I) or (V) is [ka] is selected from.

[0261] The compound of formula (II) is a TLR7 agonist prodrug. In one embodiment, the prodrug has at R2: [ka] A single prodrug having a substituent selected from:

[0262] In one embodiment, the prodrug has at R2: [ka] and a double prodrug having a substituent selected from:

[0263] In one embodiment, a subset of the TLR7 agonist prodrugs of formula (II) in the pharmaceutical combination of the present invention is represented by formula (III): [ka] wherein R1 is —OH or acetoxy, and R2 is 1-acetoxypropyl, 1-hydroxypropyl, 1-hydroxymethyl, or [ka] is) or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof; or formula (IV): [ka] wherein R1 is acetoxy(cyclopropyl)methyl or acetoxy(propyn-1-yl)methyl, or [ka] is) or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

[0264] The compound of formula (IV) is a double prodrug, similar to the compound of formula (III) where R1 is OH and R2 is 1-acetoxypropyl. The compound of formula (III) where R1 is acetoxy and R2 is a triple prodrug.

[0265] After administration, compounds of formula (II), (III) or (IV) are metabolized to their active forms which are useful TLR7 agonists.

[0266] In one embodiment, the TLR7 agonist in the pharmaceutical combination of the present invention is [(1S)-1-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]acetate (CMP No. VI); 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1S)-1-hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione (CMP No. VII); 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1S)-1-hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one (CMP No. VIII); 5-amino-3-(3'-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one (CMP No. IX); 5-amino-3-(2'-O-acetyl-3'-deoxy-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one (CMP No. X); 5-amino-3-(3'-deoxy-β-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione (CMP No. XI); [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-1,3-oxathiolan-2-yl]-cyclopropyl-methyl]acetate (CMP No. XII); and (1S)-1-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-1,3-oxathiolan-2-yl]but-2-ynyl]acetate (CMP No. XIII) and pharmaceutically acceptable salts, enantiomers or diastereomers thereof.

[0267] Table 1 lists TLR7 agonists in pharmaceutical combination embodiments of the present invention, including literature references describing their preparation. [Table 1-1] [Table 1-2]

[0268] In a particularly preferred embodiment of the pharmaceutical combination of the present invention, the TLR7 agonist is CMP No. VI. This definition of the TLR7 agonist used in the pharmaceutical combination of the present invention is referred to herein as "T3" or "therapeutic T3."

[0269] In one embodiment, the TLR7 agonist is administered orally.

[0270] In one embodiment, T3 is administered orally as a unit dose in the range of 150-170 mg every other day (QOD) for 8 to 26 weeks, such as 10 to 24 weeks, for example 12 or 13 weeks, followed by weekly (QW) administration for 24 to 48 weeks, such as 30 to 40 weeks, for example 35 weeks. The number of doses of T3 administered is 60 to 100 doses, for example 75 to 90 doses, for example 81, 82, 83 or 84 doses over the treatment period.

[0271] Preferably, the TLR7 agonist is administered for a period of 12 weeks or less.

[0272] In one embodiment, the pharmaceutical combination of the present invention comprising T1 or T2 and T3, T1 and T3, or T2 and T3, is administered less than one month apart, for example less than one week apart, for example 2 days apart, for example on the same day.

[0273] In one embodiment, the TLR7 agonist in the pharmaceutical combination of the present invention is administered enterally (e.g., orally or through the digestive tract). The TLR7 agonist compound of the present invention can be administered in any convenient dosage form, such as a unit dose of tablet, powder, capsule, solution, dispersion, suspension, syrup, spray, suppository, gel, or emulsion. In particular, oral unit dosage forms such as tablets and capsules can be used. In one example, a pharmaceutically effective amount of the TLR7 agonist compound of the present invention ranges from about 75 to 250 mg, e.g., 100 to 200 mg, e.g., 150 to 170 mg pr. dose. Administration can be daily, every other day (QOD), or weekly (QW).

[0274] In a preferred embodiment of the pharmaceutical combination of the present invention comprising a TLR7 agonist, the TLR7 agonist is administered at a dose of at least about 100 mg, or about 100 mg, or preferably about 150 mg. In one embodiment, the TLR7 agonist is administered at least QW (weekly), or QW, or more preferably QOD (every other day).

[0275] Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C., Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005.

[0276] Interferon-alpha In one embodiment, the therapeutic agent used in the pharmaceutical combination of the present invention is interferon-alpha (IFNα).

[0277] In various embodiments, the interferon-alpha in the pharmaceutical combination of the present invention can be interferon alpha-2b, interferon alpha-2a, and interferon alpha-1 (pegylated and non-pegylated).

[0278] In further various embodiments, the IFN-α in the pharmaceutical combination of the present invention is Pegasys® (Roche), PEG-Intron® (Merck & Co., Inc.), or Y-pegylated recombinant interferon alpha-2a (YPEG-IFNα-2a, Xiamen Amoytop Biotech Co., Ltd.).

[0279] In one embodiment, the IFNα in the pharmaceutical combination of the present invention is pegylated IFNα. This definition of IFNα used in the pharmaceutical combination of the present invention is referred to herein as "T4" or "therapeutic T4".

[0280] In one embodiment, the interferon-alpha is administered subcutaneously.

[0281] Anti-HBV antibody In one embodiment, the therapeutic agent used in the pharmaceutical combination of the present invention is an anti-HBV antibody.

[0282] In one embodiment, the anti-HBV antibody in the pharmaceutical combination of the present invention is an antibody that binds to hepatitis B surface antigen (anti-HBsAg).

[0283] In one embodiment, a combination comprising an oligonucleotide therapeutic and an anti-HBV antibody may result in seroclearance of HBsAg in a patient.

[0284] In one embodiment, the anti-HBV antibody in the pharmaceutical combination of the present invention is monoclonal.

[0285] In one embodiment, the anti-HBV antibodies in the pharmaceutical combination of the present invention are monoclonal and human.

[0286] In one embodiment, the anti-HBV antibody in the pharmaceutical combination of the present invention is an anti-HBsAg monoclonal antibody. This definition of the anti-HBV antibody used in the pharmaceutical combination of the present invention is referred to herein as "T5" or "therapeutic T5."

[0287] In preferred embodiments of the pharmaceutical combination of the present invention comprising T5, specifically in any of the embodiments of combinations C4, C11, C17, C22, C27, C28, C29, C30, C39, C45, C50, C66, C71, C86, C55, C56, C57, C58, C76, C77, C78, ​​C79, C91, C92, C93, C94, C101, C102, C103, C104, C111, C112, C113, C114, C115 or C116 identified in Tables 2 and 3, T5 comprises (a) a CDR-H comprising the amino acid sequence of NYGMQ (SEQ ID NO: 12), 1. An anti-HBsAg antibody comprising: (b) a heavy chain variable domain (VH) comprising a CDR-H2 comprising the amino acid sequence of IIWADGTKQYYGDSVKG (SEQ ID NO: 13) and (c) a CDR-H3 comprising the amino acid sequence of DGLYASAPNDV (SEQ ID NO: 14); and (d) a light chain variable domain (VL) comprising a CDR-L1 comprising the amino acid sequence of RASQRISTYLN (SEQ ID NO: 15), (e) a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 16), and (f) a CDR-L3 comprising the amino acid sequence of QQTYTLPPN (SEQ ID NO: 17).

[0288] In one embodiment of the pharmaceutical combination of the present invention comprising T5, specifically any of the combinations C4, C11, C17, C22, C27, C28, C29, C30, C39, C45, C50, C66, C71, C86, C55, C56, C57, C58, C76, C77, C78, ​​C79, C91, C92, C93, C94, C101, C102, C103, C104, C111, C112, C113, C114, C115 or C116 identified in Tables 2 and 3, T5 comprises (a) a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 18); An anti-HBsAg antibody comprising: a heavy chain variable domain (VH) comprising (c) a CDR-H2 comprising the amino acid sequence of FSGTGGSTYYADSVKG (SEQ ID NO: 19), and a CDR-H3 comprising the amino acid sequence of DPGHTSNWRDNYQYYQMDV (SEQ ID NO: 20); and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 21), (e) a CDR-L2 comprising the amino acid sequence of AASSLQS (SEQ ID NO: 22), and (f) a CDR-L3 comprising the amino acid sequence of LQHNSYPRT (SEQ ID NO: 23).

[0289] In one embodiment of the pharmaceutical combination of the present invention comprising T5, specifically in any of the embodiments of combinations C4, C11, C17, C22, C27, C28, C29, C30, C39, C45, C50, C66, C71, C86, C55, C56, C57, C58, C76, C77, C78, ​​C79, C91, C92, C93, C94, C101, C102, C103, C104, C111, C112, C113, C114, C115 or C116 identified in Tables 2 and 3, T5 comprises (a) a CDR-H1 comprising the amino acid sequence of NYHIH (SEQ ID NO: 24), (b) a heavy chain variable domain (VH) comprising (b) a CDR-H2 comprising the amino acid sequence of IINPRRLSTAYAPKFQG (SEQ ID NO: 25), and (c) a CDR-H3 comprising the amino acid sequence of DAGDDTSGPFDS (SEQ ID NO: 26); and (d) a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of RASQSINTWLA (SEQ ID NO: 27), (e) a CDR-L2 comprising the amino acid sequence of KASSLES (SEQ ID NO: 28), and (f) a CDR-L3 comprising the amino acid sequence of QQYNTFS (SEQ ID NO: 29).

[0290] In one embodiment of the pharmaceutical combination of the present invention comprising T5, specifically in any of the embodiments of combinations C4, C11, C17, C22, C27, C28, C29, C30, C39, C45, C50, C66, C71, C86, C55, C56, C57, C58, C76, C77, C78, ​​C79, C91, C92, C93, C94, C101, C102, C103, C104, C111, C112, C113, C114, C115 or C116 identified in Tables 2 and 3, T5 comprises (a) a CDR-H1 comprising the amino acid sequence of TNNWWS (SEQ ID NO: 30); An anti-HBsAg antibody comprising a heavy chain variable domain (VH) comprising (b) a CDR-H2 comprising the amino acid sequence of EIHHIGSTNYNPSLKS (sequence number 31) and (c) a CDR-H3 comprising the amino acid sequence of GRLGITRDRYYFDS (sequence number 32), and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of QASQDISNYLN (sequence number 33), (e) a CDR-L2 comprising the amino acid sequence of DTSSLER (sequence number 34), and (f) a CDR-L3 comprising the amino acid sequence of QQYYNLPHT (sequence number 35).

[0291] In preferred embodiments of the pharmaceutical combinations of the present invention comprising T5, in particular in any of the embodiments of combinations C4, C11, C17, C22, C27, C28, C29, C30, C39, C45, C50, C66, C71, C86, C55, C56, C57, C58, C76, C77, C78, ​​C79, C91, C92, C93, C94, C101, C102, C103, C104, C111, C112, C113, C114, C115 or C116 identified in Tables 2 and 3, T5 is selected from the group consisting of: QVQLVESGGGVVQPGRSLRLSCEASGFTFSNYGMQWVRQAPGKGLE This is an anti-HBsAg antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence WVAIIWADGTKQYYGDSVKGRFTISRDNFKNTLYLQMNSLRGEDTAMYFCARDGLYASAPNDVWGQGTLVTVSS (SEQ ID NO: 39) and a light chain variable domain (VL) comprising the amino acid sequence DIQMTQSPSSLSAYVGDRVTITCRASQRISTYLNWYHQRPGKSPSLLIYGASSLQSGVPSRFSASASGTDFTLTISSLRPEDLGTYYCQQTYTLPPNSGGGTKVEIK (SEQ ID NO: 37).

[0292] In one embodiment of the pharmaceutical combination of the present invention comprising T5, specifically in any of the embodiments of combinations C4, C11, C17, C22, C27, C28, C29, C30, C39, C45, C50, C66, C71, C86, C55, C56, C57, C58, C76, C77, C78, ​​C79, C91, C92, C93, C94, C101, C102, C103, C104, C111, C112, C113, C114, C115 or C116 identified in Tables 2 and 3, T5 is selected from the group consisting of QVQLVESGGGVVQPGRSLRLS CEASGFTFSNYGMQWVRQAPGKGLEWVAIIWADGTKQYYGDSVKGRFTISRDNFKNTLYLQMNSLRGEDTAMYFCARDGLYASAPNDVWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLY A heavy chain variable domain (VH) of ITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38), and and a light chain variable domain (VL) of RVTITCRASQRISTYLNWYHQRPGKSPSLLIYGASSLQSGVPSRFSASASGTDFTLTISSLRPEDLGTYYCQQTYTLPPNSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 36).

[0293] In one embodiment, the anti-HBV antibodies are administered subcutaneously.

[0294] Antibodies that antagonize PD1 signaling In one embodiment, the therapeutic agent used in the pharmaceutical combination of the present invention is an antibody that antagonizes PD1 signaling. In one embodiment, the antibody is an anti-PDL1 antibody. In one embodiment, the antibody is an anti-PD1 antibody.

[0295] In a preferred embodiment, the anti-PD1 antibody in the pharmaceutical combination of the present invention is nivolumab (trade name OPDIVO®, available from Bristol Myers Squibb). This definition of the anti-PD1 antibody used in the pharmaceutical combination of the present invention is referred to herein as "T6" or "therapeutic T6."

[0296] In one embodiment, the anti-PDL1 antibody in the pharmaceutical combination of the present invention is atezolizumab (trade name Tecentriq®, available from Genentech / Roche). This definition of the anti-PDL1 antibody used in the pharmaceutical combination of the present invention is referred to herein as "T7" or "therapeutic T7."

[0297] The most preferred pharmaceutical combination of the present invention comprising an antibody that antagonizes PD1 signaling comprises only one antibody that antagonizes PD1 signaling. In one embodiment, the antibody that antagonizes PD1 signaling is administered subcutaneously. In one embodiment, the antibody is T6 and is administered subcutaneously.

[0298] Nucleotide Analogues In one embodiment, the therapeutic agent used in the pharmaceutical combination of the present invention is a nucleotide analogue.

[0299] In one embodiment, the nucleotide analogue in the pharmaceutical combination of the present invention is selected from among the following: lamivudine, telbivudine, entecavir, adefovir, tenofovir, clevudine, tenofovir alafenamide, CMX157 and AGX-1009.

[0300] In one embodiment, the nucleotide analog in the pharmaceutical combination of the present invention is entecavir. This definition of the nucleotide analog used in the pharmaceutical combination of the present invention is referred to herein as "T8" or "therapeutic T8."

[0301] In one embodiment, the nucleotide analog in the pharmaceutical combination of the present invention is tenofovir. This definition of the nucleotide analog used in the pharmaceutical combination of the present invention is referred to herein as "T9" or "therapeutic T9."

[0302] In one embodiment, the nucleotide analogue is administered subcutaneously.

[0303] Drug combinations The present invention provides various pharmaceutical combinations comprising at least two HBV therapeutic agents, preferably two or three HBV therapeutic agents.

[0304] Pharmaceutical combinations of the present invention that include two specific HBV therapeutic agents are set forth in Table 2 below. [Table 2]

[0305] Pharmaceutical combinations of the present invention comprising three specific HBV therapeutic agents are set forth in Table 3 below. [Table 3-1] [Table 3-2]

[0306] Having described the pharmaceutical combinations of the present invention, certain preferred embodiments of the pharmaceutical combinations of the present invention are described herein. In a preferred embodiment, the pharmaceutical combinations of the present invention are those that do not include both therapeutic agent T6 and therapeutic agent T7 in the same combination. In a preferred embodiment, the pharmaceutical combinations of the present invention include therapeutic agent T1 in combination with one or more additional HBV therapeutic agents. In a preferred embodiment, the pharmaceutical combinations of the present invention include therapeutic agents T1 and T2, optionally in combination with an additional third HBV therapeutic agent, preferably any one of T3, T4, T5, T6, T7, T8, or T9. In a preferred embodiment, the pharmaceutical combinations of the present invention include T1 and T2, optionally in combination with T3.

[0307] Typically, the above combinations include the recited elements, i.e., include the recited HBV therapeutics, but do not exclude the inclusion of additional, unrecited HBV therapeutics. However, in another embodiment, the combinations defined above are limited to the recited elements, i.e., the pharmaceutical combination consists essentially of the recited elements to the exclusion of any other HBV therapeutics. This does not exclude the presence of any carrier, excipient, adjuvant, diluent, or salt in the combination. Thus, in another embodiment, the pharmaceutical combination of the present invention consists essentially of the relevant elements listed for that combination in Table 2 or 3.

[0308] In a preferred embodiment, each of the HBV therapeutic agents in the pharmaceutical combination of the present invention is formulated in a pharmaceutically acceptable carrier. More preferably, each HBV therapeutic agent is formulated in a pharmaceutically acceptable carrier suitable for administration of the HBV therapeutic agent in question.

[0309] The pharmaceutical combination of the present invention can be used to treat HBV infection more effectively than the individual HBV therapeutic agents contained therein alone. In one embodiment, the pharmaceutical combination of the present invention can be used to inhibit HBV more rapidly, inhibit HBV for a longer duration, and / or inhibit HBV more effectively than the individual HBV therapeutic agents contained therein alone. These effects can be measured by a decrease in HBsAg, HBeAg, or HBV-DNA titer. In one embodiment, the pharmaceutical combination of the present invention causes a more rapid decrease in HBsAg, HBeAg, or HBV-DNA titer than the individual HBV therapeutic agents contained therein alone. In one embodiment, the pharmaceutical combination of the present invention causes a longer-lasting decrease in HBsAg, HBeAg, or HBV-DNA titer than the individual HBV therapeutic agents contained therein alone. In one embodiment, the pharmaceutical combination of the present invention causes a greater decrease in HBsAg, HBeAg, or HBV-DNA titer than the individual HBV therapeutic agents contained therein alone. HBsAg is primarily measured for this purpose.

[0310] The pharmaceutical combinations of the present invention may also be present in a kit or kit of parts. The term "kit" or "kit of parts" refers to an assembly of materials used in carrying out treatment of an HBV-infected individual, including instructions on how to carry out the treatment.

[0311] One aspect of the present invention is a kit of parts containing two or more therapeutically active ingredients (eg, medicinal components or medicaments) selected from the HBV treatments described herein.

[0312] One embodiment of the present invention is a kit of parts comprising a first HBV therapeutic agent described herein and a second HBV therapeutic agent described herein, and optionally further comprising a third HBV therapeutic agent described herein as a medical component.

[0313] In one embodiment, the kit of the present invention includes a first pharmaceutical agent, an RNAi oligonucleotide targeting HBV formulated for subcutaneous injection, and a second pharmaceutical agent, an anti-PDL1 antisense oligonucleotide, also formulated for subcutaneous administration. The RNAi oligonucleotide targeting HBV and the anti-PDL1 antisense oligonucleotide are formulated separately. Each of the RNAi oligonucleotide targeting HBV and the anti-PDL1 antisense oligonucleotide can be formulated as a liquid in a vial containing one or more doses, or in a pre-filled syringe containing a single pharmaceutically effective dose. Alternatively, each of the RNAi oligonucleotide targeting HBV and the anti-PDL1 antisense oligonucleotide can be in the form of a lyophilized powder, and the kit includes a solvating agent for preparation for injection. It is understood that all pharmaceutical agents for injection are sterile. If a TLR7 agonist is included in the kit, it can be in tablet form (or an alternative unit dose form for oral administration, such as a capsule or gel) containing a single pharmaceutically effective dose, and the kit can include multiple tablets.

[0314] In a further embodiment, the kit of parts of the invention further comprises instructions for administering an RNAi oligonucleotide targeting HBV in combination with an anti-PDL1 antisense oligonucleotide to treat hepatitis B virus infection. In particular, the instructions describe treatment of chronic hepatitis B virus infection.

[0315] The kit may include only one of the medical components and instructions for use in combination with the other medical components. In one embodiment, the kit of parts of the present invention includes or contains a first medical agent that is an RNAi oligonucleotide targeting HBV and instructions for use in combination with an anti-PDL1 antisense oligonucleotide, purchased separately, as a second medical agent. In another embodiment, the kit of parts of the present invention includes or contains a first medical agent that is an anti-PDL1 antisense oligonucleotide and instructions for use in combination with an RNAi oligonucleotide targeting HBV, purchased separately, as a second medical agent.

[0316] In some embodiments, the pharmaceutical combination of the present invention can be used in combination with a third or additional therapeutic agent(s), which can be included in a kit of parts or supplied separately. In one embodiment, the additional therapeutic agent is any of T3, T4, T5, T6, T7, T8, or T9. Preferably, the additional therapeutic agent is T3.

[0317] Administration sequence of drug combinations The specific sequence of administration of the HBV therapeutic agents within the pharmaceutical combinations of the present invention (combinations C1 to C120) defined above is described in this section.

[0318] It should be noted that the "element" designations used above (element A, element B, and element C) are purely for reference purposes only and do not imply anything regarding the order in which the therapeutic agents in a particular pharmaceutical combination should be administered. Rather, the order of administration of the therapeutic agents in the pharmaceutical combinations of the present invention is expressly set forth herein in terms of which elements are administered first and second (and third, if relevant).

[0319] For example, in an embodiment directed to pharmaceutical combination "C1" of the invention, component A is administered first and component B is administered second. In this embodiment of combination C1, the first or initial dose of component A of combination C1 (the HBV therapeutic agent defined herein as T1) is administered before the first or initial dose of component B of combination C1 (the HBV therapeutic agent defined herein as T2).

[0320] Herein, any designated order for administration of elements of a particular pharmaceutical combination of the present invention relates only to those elements that are explicitly part of that pharmaceutical combination. For example, an element designated as "administered first" does not necessarily exclude that a patient may not have previously received a different HBV therapeutic agent that was not administered as part of the administration of a pharmaceutical combination of the present invention.

[0321] It should also be understood herein that the elements of the pharmaceutical combination of the present invention can be administered at a single time point, for example, as a single dose, or as multiple doses given over a period of time. Thus, a reference herein to an element being "administered" can refer either to the specific time at which the element is administered (one dose given at a single time point) or to the time at which administration of the element begins (multiple doses given over a period of time). Thus, it is contemplated that the pharmaceutical combination of the present invention may include overlapping dosing regimens. For example, in a pharmaceutical combination of the present invention in which element A is administered first and element B is administered second, if element A is administered as several doses over a period of time, the administration of a further dose of element A may overlap with the administration of element B if the first dose of element A is administered as a single dose or before the first dose of element B.

[0322] Combination C1 In one embodiment of the present invention, the pharmaceutical combination is combination C1, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0323] Combination C2 In one embodiment of the present invention, the pharmaceutical combination is combination C2, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0324] Combination C3 In one embodiment of the present invention, the pharmaceutical combination is combination C3, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0325] Combination C4 In one embodiment of the present invention, the pharmaceutical combination is combination C4, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0326] Combination C5 In one embodiment of the present invention, the pharmaceutical combination is combination C5, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0327] Combination C6 In one embodiment of the present invention, the pharmaceutical combination is combination C6, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0328] Combination C7 In one embodiment of the present invention, the pharmaceutical combination is combination C7, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0329] Combination C8 In one embodiment of the present invention, the pharmaceutical combination is combination C8, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0330] Combination C9 In one embodiment of the present invention, the pharmaceutical combination is combination C9, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0331] Combination C10 In one embodiment of the present invention, the pharmaceutical combination is combination C10, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0332] Combination C11 In one embodiment of the present invention, the pharmaceutical combination is combination C11, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0333] Combination C12 In one embodiment of the present invention, the pharmaceutical combination is combination C12, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0334] Combination C13 In one embodiment of the present invention, the pharmaceutical combination is combination C13, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0335] Combination C14 In one embodiment of the present invention, the pharmaceutical combination is combination C14, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0336] Combination C15 In one embodiment of the present invention, the pharmaceutical combination is combination C15, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0337] Combination C16 In one embodiment of the present invention, the pharmaceutical combination is combination C16, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0338] Combination C17 In one embodiment of the present invention, the pharmaceutical combination is combination C17, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0339] Combination C18 In one embodiment of the present invention, the pharmaceutical combination is combination C18, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0340] Combination C19 In one embodiment of the present invention, the pharmaceutical combination is combination C19, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0341] Combination C20 In one embodiment of the present invention, the pharmaceutical combination is combination C20, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0342] Combination C21 In one embodiment of the present invention, the pharmaceutical combination is combination C21, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0343] Combination C22 In one embodiment of the present invention, the pharmaceutical combination is combination C22, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0344] Combination C23 In one embodiment of the present invention, the pharmaceutical combination is combination C23, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0345] Combination C24 In one embodiment of the present invention, the pharmaceutical combination is combination C24, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0346] Combination C25 In one embodiment of the present invention, the pharmaceutical combination is combination C25, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0347] Combination C26 In one embodiment of the present invention, the pharmaceutical combination is combination C26, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0348] Combination C27 In one embodiment of the present invention, the pharmaceutical combination is combination C27, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0349] Combination C28 In one embodiment of the present invention, the pharmaceutical combination is combination C28, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0350] Combination C29 In one embodiment of the present invention, the pharmaceutical combination is combination C29, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0351] Combination C30 In one embodiment of the present invention, the pharmaceutical combination is combination C30, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0352] Combination C31 In one embodiment of the present invention, the pharmaceutical combination is combination C31, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0353] Combination C32 In one embodiment of the present invention, the pharmaceutical combination is combination C32, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0354] Combination C33 In one embodiment of the present invention, the pharmaceutical combination is combination C33, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0355] Combination C34 In one embodiment of the present invention, the pharmaceutical combination is combination C34, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0356] Combination C35 In one embodiment of the present invention, the pharmaceutical combination is combination C35, comprising component A and component B as defined in Table 2 above. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0357] Combination C36 In one embodiment of the present invention, the pharmaceutical combination is combination C36, comprising component A and component B as defined above in Table 2. In one embodiment of this combination, component A is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A.

[0358] Combination C37 In one embodiment of the present invention, the pharmaceutical combination is combination C37, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0359] Combination C38 In one embodiment of the present invention, the pharmaceutical combination is combination C38, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0360] Combination C39 In one embodiment of the present invention, the pharmaceutical combination is combination C39, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0361] Combination C40 In one embodiment of the present invention, the pharmaceutical combination is combination C40, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0362] Combination C41 In one embodiment of the present invention, the pharmaceutical combination is combination C41 comprising component A, component B, and component C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0363] Combination C42 In one embodiment of the present invention, the pharmaceutical combination is combination C42, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0364] Combination C43 In one embodiment of the present invention, the pharmaceutical combination is combination C43, comprising components A, B, and C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0365] Combination C44 In one embodiment of the present invention, the pharmaceutical combination is combination C44, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0366] Combination C45 In one embodiment of the present invention, the pharmaceutical combination is combination C45, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0367] Combination C46 In one embodiment of the present invention, the pharmaceutical combination is combination C46, ​​comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0368] Combination C47 In one embodiment of the present invention, the pharmaceutical combination is combination C47, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0369] Combination C48 In one embodiment of the present invention, the pharmaceutical combination is combination C48, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0370] Combination C49 In one embodiment of the present invention, the pharmaceutical combination is combination C49, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0371] Combination C50 In one embodiment of the present invention, the pharmaceutical combination is combination C50, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0372] Combination C51 In one embodiment of the present invention, the pharmaceutical combination is combination C51 comprising component A, component B, and component C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0373] Combination C52 In one embodiment of the present invention, the pharmaceutical combination is combination C52, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0374] Combination C53 In one embodiment of the invention, the pharmaceutical combination is combination C53, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0375] Combination C54 In one embodiment of the present invention, the pharmaceutical combination is combination C54, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0376] Combination C55 In one embodiment of the invention, the pharmaceutical combination is combination C55, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0377] Combination C56 In one embodiment of the present invention, the pharmaceutical combination is combination C56, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0378] Combination C57 In one embodiment of the present invention, the pharmaceutical combination is combination C57, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0379] Combination C58 In one embodiment of the present invention, the pharmaceutical combination is combination C58, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0380] Combination C59 In one embodiment of the invention, the pharmaceutical combination is combination C59, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0381] Combination C60 In one embodiment of the present invention, the pharmaceutical combination is combination C60, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0382] Combination C61 In one embodiment of the present invention, the pharmaceutical combination is combination C61 comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0383] Combination C62 In one embodiment of the invention, the pharmaceutical combination is combination C62, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0384] Combination C63 In one embodiment of the invention, the pharmaceutical combination is combination C63, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0385] Combination C64 In one embodiment of the present invention, the pharmaceutical combination is combination C64, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0386] Combination C65 In one embodiment of the present invention, the pharmaceutical combination is combination C65, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0387] Combination C66 In one embodiment of the present invention, the pharmaceutical combination is combination C66, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0388] Combination C67 In one embodiment of the invention, the pharmaceutical combination is combination C67, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0389] Combination C68 In one embodiment of the present invention, the pharmaceutical combination is combination C68, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0390] Combination C69 In one embodiment of the invention, the pharmaceutical combination is combination C69, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0391] Combination C70 In one embodiment of the present invention, the pharmaceutical combination is combination C70, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0392] Combination C71 In one embodiment of the present invention, the pharmaceutical combination is combination C71, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0393] Combination C72 In one embodiment of the invention, the pharmaceutical combination is combination C72, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0394] Combination C73 In one embodiment of the present invention, the pharmaceutical combination is combination C73, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0395] Combination C74 In one embodiment of the present invention, the pharmaceutical combination is combination C74, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0396] Combination C75 In one embodiment of the invention, the pharmaceutical combination is combination C75, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0397] Combination C76 In one embodiment of the present invention, the pharmaceutical combination is combination C76, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0398] Combination C77 In one embodiment of the present invention, the pharmaceutical combination is combination C77, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0399] Combination C78 In one embodiment of the present invention, the pharmaceutical combination is combination C78, ​​comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0400] Combination C79 In one embodiment of the invention, the pharmaceutical combination is combination C79, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0401] Combination C80 In one embodiment of the present invention, the pharmaceutical combination is combination C80, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0402] Combination C81 In one embodiment of the invention, the pharmaceutical combination is combination C81, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0403] Combination C82 In one embodiment of the present invention, the pharmaceutical combination is combination C82, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0404] Combination C83 In one embodiment of the invention, the pharmaceutical combination is combination C83, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0405] Combination C84 In one embodiment of the invention, the pharmaceutical combination is combination C84, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0406] Combination C85 In one embodiment of the present invention, the pharmaceutical combination is combination C85, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0407] Combination C86 In one embodiment of the present invention, the pharmaceutical combination is combination C86, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0408] Combination C87 In one embodiment of the present invention, the pharmaceutical combination is combination C87, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0409] Combination C88 In one embodiment of the invention, the pharmaceutical combination is combination C88, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0410] Combination C89 In one embodiment of the invention, the pharmaceutical combination is combination C89, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0411] Combination C90 In one embodiment of the present invention, the pharmaceutical combination is combination C90, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0412] Combination C91 In one embodiment of the invention, the pharmaceutical combination is combination C91, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0413] Combination C92 In one embodiment of the invention, the pharmaceutical combination is combination C92, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0414] Combination C93 In one embodiment of the invention, the pharmaceutical combination is combination C93, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0415] Combination C94 In one embodiment of the invention, the pharmaceutical combination is combination C94, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0416] Combination C95 In one embodiment of the present invention, the pharmaceutical combination is combination C95, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0417] Combination C96 In one embodiment of the present invention, the pharmaceutical combination is combination C96, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0418] Combination C97 In one embodiment of the present invention, the pharmaceutical combination is combination C97, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0419] Combination C98 In one embodiment of the invention, the pharmaceutical combination is combination C98, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0420] Combination C99 In one embodiment of the invention, the pharmaceutical combination is combination C99, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0421] Combination C100 In one embodiment of the present invention, the pharmaceutical combination is combination C100, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0422] Combination C101 In one embodiment of the present invention, the pharmaceutical combination is combination C101, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0423] Combination C102 In one embodiment of the present invention, the pharmaceutical combination is combination C102, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0424] Combination C103 In one embodiment of the present invention, the pharmaceutical combination is combination C103, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0425] Combination C104 In one embodiment of the present invention, the pharmaceutical combination is combination C104, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0426] Combination C105 In one embodiment of the present invention, the pharmaceutical combination is combination C105, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0427] Combination C106 In one embodiment of the present invention, the pharmaceutical combination is combination C106, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0428] Combination C107 In one embodiment of the present invention, the pharmaceutical combination is combination C107, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0429] Combination C108 In one embodiment of the invention, the pharmaceutical combination is combination C108, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0430] Combination C109 In one embodiment of the invention, the pharmaceutical combination is combination C109, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0431] Combination C110 In one embodiment of the present invention, the pharmaceutical combination is combination C110, comprising component A, component B, and component C, as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, which is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, which is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, which is administered prior to the administration of component A.

[0432] Combination C111 In one embodiment of the present invention, the pharmaceutical combination is combination C111, comprising component A, component B, and component C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, and component B is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, and component C is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, and component A is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component C, and component C is administered prior to the administration of component A. In a further embodiment of this combination, component C is administered prior to the administration of component A, and component A is administered prior to the administration of component B. In a further embodiment of this combination, component C is administered prior to the administration of component B, and component B is administered prior to the administration of component A.

[0433] Combination C112 In one embodiment of the present invention, the pharmaceutical combination is combination C112, comprising components A, B, and C as defined in Table 3 above. In one embodiment of this combination, component A is administered prior to the administration of component B, which is administered prior to the administration of component C. In a further embodiment of this combination, component A is administered prior to the administration of component C, which is administered prior to the administration of component B. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is administered prior to the administration of component A, which is administered prior to the administration of component C. In a further embodiment of this combination, component B is admini...

Claims

1. A pharmaceutical combination for treating HBV, comprising an RNAi oligonucleotide targeting HBV and an anti-PDL1 antisense oligonucleotide, wherein the RNAi oligonucleotide is an siRNA oligonucleotide that targets HBsAg mRNA and reduces expression of HBsAg mRNA.

2. 2. The pharmaceutical combination of claim 1, wherein the RNAi oligonucleotide is an oligonucleotide comprising an antisense strand 19 to 30 nucleotides in length, and the antisense strand comprises a region complementary to the sequence of HBsAg mRNA shown as ACAANAAUCCUCACAAUA (SEQ ID NO: 1).

3. 2. The pharmaceutical combination of claim 1, wherein the RNAi oligonucleotide comprises a sense strand having a region of complementarity to the sequence set forth as UUNUUGUGAGGAUUN (SEQ ID NO: 2).

4. 2. The pharmaceutical combination of claim 1, wherein the RNAi oligonucleotide comprises a sense strand comprising the sequence GACAANAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 8), wherein one or more of the nucleotides of the -GAAA- sequence on the sense strand are conjugated to a GalNac moiety; and preferably, the RNAi oligonucleotide further comprises an antisense strand comprising the sequence UUAUUGUGAGGAUUNUUGUCGG (SEQ ID NO: 4).

5. the RNAi oligonucleotide is an oligonucleotide comprising a sense strand that forms a duplex region with an antisense strand, the sense strand consists of the sequence GACAAAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 9) containing 2'-fluoro modified nucleotides at positions 3, 8-10, 12, 13, and 17, 2'-O-methyl modified nucleotides at positions 1, 2, 4-7, 11, 14-16, 18-26, and 31-36, and a phosphorothioate linkage between the nucleotide at position 1 and 2, wherein each nucleotide of the -GAAA- sequence on the sense strand is conjugated to a monovalent GalNac moiety; the antisense strand consists of the sequence UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 6) containing 2'-fluoro modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16, and 19, 2'-O-methyl modified nucleotides at positions 1, 4, 6, 9, 11, 13, 15, 17, 18, and 20-22, and phosphorothioate linkages between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 3 and 4, between the nucleotides at positions 20 and 21, and between the nucleotides at positions 21 and 22; the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a methoxyphosphonate (MOP); Preferably, the -GAAA- sequence has the following structure: 【Chemistry 1】 Including, Preferably, the 5'-nucleotide of the antisense strand has the following structure: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, The pharmaceutical combination of claim 1.

6. the anti-PDL1 antisense oligonucleotide comprises the sequence CCTATTTAACATCAGAC (SEQ ID NO: 11); Preferably, the anti-PDL1 antisense oligonucleotide has the formula GN2-C6 o c o a o CCtatttaacatcAGAC wherein C6 represents an aminoalkyl group having 6 carbons, uppercase letters represent β-D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, the subscript o represents a phosphodiester nucleoside linkage, and unless otherwise indicated, all internucleoside linkages are phosphorothioate internucleoside linkages, and GN2 represents a trivalent GalNAc cluster as follows: 【Transformation 3】 represents Additionally, the wavy line in the trivalent GalNAc cluster indicates the conjugation site of the trivalent GalNAc cluster to the C6 aminoalkyl group. or a pharmaceutically acceptable salt thereof, The pharmaceutical combination of claim 1.

7. 2. The pharmaceutical combination of claim 1, wherein the combination is capable of reducing serum HBsAg, HBeAg and / or HBV-DNA in a patient, and the reduction is greater than the sum of a) the reduction provided by the same dose of an RNAi oligonucleotide targeting HBV when administered without the anti-PDL1 antisense oligonucleotide and b) the reduction provided by the same dose of an anti-PDL1 antisense oligonucleotide when administered without the RNAi oligonucleotide targeting HBV.

8. the RNAi oligonucleotide targeting HBV is present in an amount that results in a dose of at least about 0.1 mg / kg to about 12 mg / kg, or a dose of at least about 0.5 mg / kg, or a dose of at least about 1 mg / kg, or a dose of at least about 1.5 mg / kg, or a dose of at least about 2 mg / kg, or a dose of at least about 3 mg / kg, or a dose of at least about 6 mg / kg, or a dose of at least about 9 mg / kg; and / or the anti-PDL1 antisense oligonucleotide is present in an amount resulting in a dose of at least about 0.1 mg / kg to about 35 mg / kg, or at least about 0.5 mg / kg, or at least about 1 mg / kg, or at least about 1.5 mg / kg, or at least about 2 mg / kg, or at least about 3 mg / kg, or at least about 6 mg / kg, or at least about 9 mg / kg; The pharmaceutical combination of claim 1.

9. 2. The pharmaceutical combination of claim 1, further comprising an additional, different HBV therapeutic agent, preferably wherein said additional, different HBV therapeutic agent is a TLR7 agonist, interferon-alpha, an anti-HBV antibody, an antibody that inhibits PD1 signaling, or a nucleotide analogue.

10. A composition comprising the pharmaceutical combination of claim 1.

11. 10. A kit of parts comprising an RNAi oligonucleotide targeting HBV according to claim 1 for treating hepatitis B virus infection and instructions for administering it together with an anti-PDL1 antisense oligonucleotide, i) the anti-PDL1 antisense oligonucleotide referred to in the instructions is the anti-PDL1 antisense oligonucleotide of claim 1; and / or ii) the kit comprises an RNAi oligonucleotide targeting HBV and an anti-PDL1 antisense oligonucleotide; where: the RNAi oligonucleotide is an oligonucleotide comprising a sense strand that forms a duplex region with an antisense strand, the sense strand consists of the sequence GACAAAAUCCUCACAAUAAGCAGCCGAAAGGCUGC (SEQ ID NO: 9) containing 2'-fluoro modified nucleotides at positions 3, 8-10, 12, 13, and 17, 2'-O-methyl modified nucleotides at positions 1, 2, 4-7, 11, 14-16, 18-26, and 31-36, and a phosphorothioate linkage between the nucleotide at position 1 and 2, wherein each nucleotide of the -GAAA- sequence on the sense strand is conjugated to a monovalent GalNac moiety; the antisense strand consists of the sequence UUAUUGUGAGGAUUUUUGUCGG (SEQ ID NO: 6) containing 2'-fluoro modified nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 14, 16, and 19, 2'-O-methyl modified nucleotides at positions 1, 4, 6, 9, 11, 13, 15, 17, 18, and 20-22, and phosphorothioate linkages between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 3 and 4, between the nucleotides at positions 20 and 21, and between the nucleotides at positions 21 and 22; the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a methoxyphosphonate (MOP); Preferably, the -GAAA- sequence has the following structure: 【Chemistry 4】 Including, Preferably, the 5'-nucleotide of the antisense strand has the following structure: 【Transformation 5】 or a pharmaceutically acceptable salt thereof, the anti-PDL1 antisense oligonucleotide comprises the sequence CCTATTTAACATCAGAC (SEQ ID NO: 11); Preferably, the anti-PDL1 antisense oligonucleotide has the formula GN2-C6 o c o a o CCtatttaacatcAGAC wherein C6 represents an aminoalkyl group having 6 carbons, uppercase letters represent β-D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, the subscript o represents a phosphodiester nucleoside linkage, and unless otherwise indicated, all internucleoside linkages are phosphorothioate internucleoside linkages, and GN2 represents a trivalent GalNAc cluster as follows: 【Transformation 6】 represents Additionally, the wavy line in the trivalent GalNAc cluster indicates the conjugation site of the trivalent GalNAc cluster to the C6 aminoalkyl group. or a pharmaceutically acceptable salt thereof, and / or iii) the RNAi oligonucleotide targeting HBV is formulated for subcutaneous injection, and the anti-PDL1 antisense oligonucleotide is formulated for subcutaneous administration; A kit of the above parts.

12. 12. The pharmaceutical combination, composition or kit according to any one of claims 1 to 11, wherein the RNAi oligonucleotide targeting HBV and / or the anti-PDL1 antisense oligonucleotide are in the form of a transgene engineered to express the oligonucleotide in a cell.

13. A pharmaceutical combination, composition or kit according to any one of claims 1 to 11 for use in medicine.

14. A pharmaceutical combination, composition or kit according to any one of claims 1 to 11 for use in the treatment of hepatitis B virus infection.

15. the single dose or initial dose of the RNAi oligonucleotide targeting HBV is administered prior to the administration of the single dose or initial dose of the anti-PDL1 antisense oligonucleotide; Preferably, the single dose or initial dose of the anti-PDL1 antisense oligonucleotide is administered at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, or more than 8 weeks after the single dose or initial dose of the RNAi oligonucleotide targeting HBV. A pharmaceutical combination, composition or kit for use according to claim 13.

16. the RNAi oligonucleotide targeting HBV is administered weekly and is administered at least twice; the anti-PDL1 antisense oligonucleotide is administered weekly and is administered at least twice or at least five times; A pharmaceutical combination, composition or kit for use according to claim 13.

17. 14. The pharmaceutical combination, composition or kit for use according to claim 13, wherein said pharmaceutical combination is administered for 48 weeks.

18. the RNAi oligonucleotide targeting HBV is administered at a dose of at least about 0.1 mg / kg, or at least about 0.5 mg / kg, or at least about 1 mg / kg, or at least about 1.5 mg / kg, or at least about 2 mg / kg, or at least about 3 mg / kg, or at least about 6 mg / kg, or at least about 9 mg / kg; and / or the anti-PDL1 antisense oligonucleotide is administered at a dose of at least about 0.1 mg / kg to about 35 mg / kg, or at least about 0.5 mg / kg, or at least about 1 mg / kg, or at least about 1.5 mg / kg, or at least about 2 mg / kg, or at least about 3 mg / kg, or at least about 6 mg / kg, or at least about 7 mg / kg, or at a dose of about 7 mg / kg to about 35 mg / kg, preferably the anti-PDL1 antisense oligonucleotide is administered at a maximum of five doses of about 3 mg / kg, and the doses are administered at least once every two weeks; A pharmaceutical combination, composition or kit for use according to claim 13.

19. 14. The pharmaceutical combination, composition or kit for use according to claim 13, wherein two or more, preferably at least five, doses of anti-PDL1 antisense oligonucleotide are administered once a week, wherein the first dose of anti-PDL1 antisense oligonucleotide is administered at least about 7 days after the dose of the RNAi oligonucleotide targeting HBV; and the dose of the anti-PDL1 antisense oligonucleotide is at least about 3 mg / kg.

20. 14. The pharmaceutical combination, composition or kit for use according to claim 13, wherein the RNAi oligonucleotide targeting HBV is in a dosage form for subcutaneous administration and the anti-PDL1 antisense oligonucleotide is in a dosage form for subcutaneous administration.

Images