Oligonucleotides for reducing PD-L1 expression

Abstract

To provide antisense oligonucleotides that are capable of reducing expression of PD-L1 in a target cell.SOLUTION: The oligonucleotides hybridize to PD-L1 mRNA. The present invention further relates to conjugates of the oligonucleotide and pharmaceutical compositions and to methods of using the oligonucleotide for treatment of: viral liver infections such as HBV, HCV and HDV; parasite infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; or liver cancer or metastases in the liver.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to oligonucleotides (oligomers) that are complementary to programmed death-ligand-1 (PD-L1) and reduce the expression of PD-L1 in the liver. The present invention also relates to methods for alleviating T cell exhaustion resulting from liver infection or cancer in the liver. Relevant infectious diseases include chronic HBV, HCV, and HDV and parasitic infections such as malaria and toxoplasmosis (e.g., caused by protozoans of the Plasmodium species, particularly P. vivax, P. malariae, and P. falciparum). [Background technology]

[0002] The costimulatory pathway consisting of the programmed death-1 (PD-1) receptor and its ligand PD-L1 (or B7-H1 or CD274) is known to directly contribute to T cell exhaustion and the resulting lack of viral control during chronic infection of the liver. The PD-1 pathway is also involved in autoimmunity, as mice disrupted in this pathway develop autoimmune disease.

[0003] Antibodies that block the interaction between PD-1 and PD-L1 have been shown to enhance T cell responses, particularly CD8+ cytotoxic T cell responses (see Barber et al 2006 Nature Vol 439 p682 and Maier et al 2007 J. Immunol.Vol 178 p 2714).

[0004] WO 2006 / 042237 describes a method for diagnosing cancer by assessing the expression of PD-L1 (B7-H1) in tumors, and suggests delivering to patients an agent that disrupts the PD-1 / PD-L1 interaction. The disrupting agent can be an antibody, antibody fragment, siRNA, or antisense oligonucleotide. Specific examples of such disrupting agents are not provided, nor is chronic liver infection described.

[0005] RNA interference-mediated inhibition of PD-L1 using double-stranded RNA (dsRNA, RNAi or siRNA) molecules has also been disclosed in, for example, WO 2005 / 007855, WO 2007 / 084865, and U.S. Patent No. 8,507,663, none of which describe targeted delivery to the liver.

[0006] Dolina et al., 2013, Molecular Therapy-Nucleic Acids, 2 e72, describes the in vivo delivery of PD-L1 targeting siRNA molecules to Kupffer cells, thereby enhancing NK and CD8+ T cell clearance in MCMV-infected mice. The paper concludes that PD-L1 targeting siRNA molecules delivered to hepatocytes are ineffective at enhancing CD8+ T cell effector function.

[0007] The siRNA approach is significantly different from the single-stranded antisense oligonucleotide approach because of their completely different biodistribution and mode of action. As described by Xu et al. 2003 Biochem. Biophys. Res. Comm. Vol 306 pp 712-717, antisense oligonucleotides and siRNAs have different target site preferences in mRNA.

[0008] WO 2016 / 138278 describes the inhibition of immune checkpoints, including PD-L1, using two or more single-stranded antisense oligonucleotides linked at the 5' end. This application does not mention hepatitis B virus (HBV) or targeted delivery to the liver. Summary of the Invention

[0009] The present invention identifies novel oligonucleotides and oligonucleotide conjugates that highly efficiently reduce PD-L1 mRNA in hepatocytes, both in parenchymal cells (e.g., hepatocytes) and in non-parenchymal cells such as Kupffer cells and liver sinusoidal endothelial cells (LSECs). The oligonucleotides and oligonucleotide conjugates reduce PD-1-mediated inhibition by reducing or silencing PD-L1, thereby promoting immune stimulation of exhausted T cells. By alleviating T cell exhaustion in chronic pathogenic infections of the liver, blood viral antigen levels are reduced and immune control is restored during chronic pathogenic infections of the liver. Natural killer (NK) cells and natural killer T (NKT) cells are also believed to be capable of activation by the oligonucleotides and oligonucleotide conjugates of the present invention.

[0010] The oligonucleotide conjugates ensure localized reduction of PD-L1 in hepatocytes, thereby reducing the risk of autoimmune side effects, such as pneumonia, non-viral hepatitis, and colitis, associated with systemic depletion of PD-L1.

[0011] The present invention relates to oligonucleotides or conjugates that target nucleic acids capable of regulating the expression of PD-L1, and to treating or preventing diseases associated with PD-L1 function. The oligonucleotides or oligonucleotide conjugates may be used, in particular, to treat diseases in which the immune response against infectious agents is exhausted.

[0012] Thus, in a first aspect, the present invention provides an oligonucleotide comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary to a PD-L1 target nucleic acid. The oligonucleotide may be an antisense oligonucleotide, preferably having a gapmer design. Preferably, the oligonucleotide is capable of inhibiting PD-L1 expression by cleavage of the target nucleic acid. Preferably, cleavage is achieved by recruitment of a nuclease.

[0013] In a further embodiment, the oligonucleotide is conjugated to at least one asialoglycoprotein receptor targeting binding moiety, e.g., a binding moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety. The binding moiety and oligonucleotide may be linked together via a linker, particularly a biocleavable linker.

[0014] In a further aspect, the present invention provides a pharmaceutical composition comprising an oligonucleotide or oligonucleotide conjugate of the invention and a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant.

[0015] In a further aspect, the invention provides an in vivo or in vitro method for reducing the level of PD-L1 expression in a target cell that expresses PD-L1 by administering to said cell an effective amount of an oligonucleotide or composition of the invention.

[0016] In a further aspect, the present invention provides an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition for use in restoring immunity to a virus or parasite.

[0017] In a further aspect, the present invention provides an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition for use as a medicament.

[0018] In a further aspect, the present invention provides a method for treating or preventing a disease, disorder or dysfunction by administering a therapeutically or prophylactically effective amount of an oligonucleotide of the invention to a subject suffering from or susceptible to said disease, disorder or dysfunction, in particular a disease selected from viral hepatitis infection or parasitic infection.

[0019] In a further embodiment, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is used in the treatment or prevention of viral hepatitis infections, such as HBV, HCV and HDV; or parasitic infections, such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; or liver cancer or liver metastases. [Brief explanation of the drawings]

[0020] [Figure 1A-B] The oligonucleotide is represented as a wavy line (AD) or "oligonucleotide" (EH) or T2(I), and the asialoglycoprotein receptor targeting binding moiety is a trivalent N-acetylgalactosamine moiety. Compounds A through D contain a di-lysine plug in the molecule to a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety without a linker. In compounds C and D, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety via a C6 linker. Compound EI contains a treble-branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 1C-E]The oligonucleotide is represented as a wavy line (AD) or "oligonucleotide" (EH) or T2(I), and the asialoglycoprotein receptor targeting binding moiety is a trivalent N-acetylgalactosamine moiety. Compounds A through D contain a di-lysine plug in the molecule to a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety without a linker. In compounds C and D, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety via a C6 linker. Compound EI contains a treble-branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 1F-H] The oligonucleotide is represented as a wavy line (AD) or "oligonucleotide" (EH) or T2(I), and the asialoglycoprotein receptor targeting binding moiety is a trivalent N-acetylgalactosamine moiety. Compounds A through D contain a di-lysine plug in the molecule to a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety without a linker. In compounds C and D, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety via a C6 linker. Compound EI contains a treble-branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 1I]The oligonucleotide is represented as a wavy line (AD) or "oligonucleotide" (EH) or T2(I), and the asialoglycoprotein receptor targeting binding moiety is a trivalent N-acetylgalactosamine moiety. Compounds A through D contain a di-lysine plug in the molecule to a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety without a linker. In compounds C and D, the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting binding moiety via a C6 linker. Compound EI contains a treble-branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 2] Figure 1 shows graphs illustrating EC50 (A) and PD-L1 knockdown as % of saline (B) for compounds tested in Example 2, relative to their location on the target nucleic acid. In this cell line, the compounds tested were THP1 (●) and Karpas (*). [Figure 3] 1 is a structural formula of a trivalent GalNAc cluster (GN2), which is useful as a binding moiety in the present invention. The wavy lines illustrate the binding sites of the cluster (e.g., to a C6 amino linker or directly to an oligonucleotide). [Figure 4] This is the structural formula of CMP ID number 766_2. [Figure 5] This is the structural formula of CMP ID number 767_2. [Figure 6] This is the structural formula of CMP ID number 768_2. [Figure 7] This is the structural formula of CMP ID number 769_2. [Figure 8] This is the structural formula of CMP ID number 770_2. [Figure 9A-C]Western blots were performed to detect PD-L1 protein expression in livers from poly(IC)-induced animals after treatment with saline and the indicated CMP ID numbers. Each blot shows the same oligonucleotide: naked oligonucleotide version vs. GalNAc-conjugated version in blot A.) CMP ID numbers 744_1 and 755_2; B) CMP ID numbers 747_1 and 758_2; C) CMP ID numbers 748_1 and 759_2; D) CMP ID numbers 752_1 and 763_2; and E) CMP ID numbers 753_1 and 764_2. The upper band is a vinculin loading control, and the lower band is PD-L1 protein. The first lane in each blot represents saline-treated mice without poly(IC) induction. These mice express very little PD-L1 protein. [Figure 9D-E] Western blots were performed to detect PD-L1 protein expression in livers from poly(IC)-induced animals after treatment with saline and the indicated CMP ID numbers. Each blot shows the same oligonucleotide: naked oligonucleotide version vs. GalNAc-conjugated version in blot A.) CMP ID numbers 744_1 and 755_2; B) CMP ID numbers 747_1 and 758_2; C) CMP ID numbers 748_1 and 759_2; D) CMP ID numbers 752_1 and 763_2; and E) CMP ID numbers 753_1 and 764_2. The upper band is a vinculin loading control, and the lower band is PD-L1 protein. The first lane in each blot represents saline-treated mice without poly(IC) induction. These mice express very little PD-L1 protein. [Figure 10]Figure 18 shows the mononuclear cell population in the liver after treatment with ● vehicle (Groups 10 and 1), ◆ DNA vaccine (Groups 11 and 2), 〇 anti-PD-L1 antibody (Group 12), ▲ naked PD-L1 antisense oligonucleotide (ASO) + DNA vaccine (Group 7), or △ GalNAc-linked PD-L1 ASO + DNA vaccine (Group 8), with individual animals per group represented and the group mean indicated by a vertical line (see Table 18). Statistical significance between the DNA vaccine group and the three treatment groups was assessed and, when present, is indicated by * between groups (*=P<0.05, ***=P<0.001, and ****=P<0.0001). A) represents the number of T cells in the liver after treatment. B) represents the fraction of CD4+ T cells. C) represents the fraction of CD8+ T cells. [Figure 11] ● Vehicle (Groups 10 and 1), ◆ DNA vaccine (Groups 11 and 2), 〇 Anti-PD-L1 antibody (Group 12), ▲ Naked PD-L1 ASO + DNA vaccine (Group 7), or △ GalNAc-conjugated PD-L1 ASO + DNA vaccine (Group 8). Individual animals per group are represented, with the group mean indicated by a vertical line (see Table 19). Statistical significance between the DNA vaccine group and the three treatment groups was assessed and, if present, is indicated by an * between groups (*=P<0.05, and ****=P<0.0001). A) Percentage of CD8+ T cells expressing PD-L1 in the liver after treatment. B) Percentage of CD4+ T cells expressing PD-L1 in the liver after treatment. C) Percentage of B cells expressing PD-L1 in the liver after treatment. [Figure 12]HBV antigen-specific CD8+ cytokine-secreting cells in the liver after treatment with ● vehicle (Groups 10 and 1), ◆ DNA vaccine (Groups 11 and 2), 〇 anti-PD-L1 antibody (Group 12), ▲ naked PD-L1 ASO + DNA vaccine (Group 7), or △ GalNAc-linked PD-L1 ASO + DNA vaccine (Group 8), with individual animals per group represented and the group mean indicated by a vertical line (see Table 20). Statistical significance between the DNA vaccine group and the three treatment groups was assessed and, if present, is indicated by an * between groups (*=P<0.05). A) Percentage of IFN-γ-secreting CD8+ T cells in the liver specific for HBV preS2+S antigen after treatment. B) Percentage of IFN-γ-secreting CD8+ T cells in the liver specific for HBV core antigen after treatment. C) Percentage of IFN-γ and TNF-α secreting CD8+ T cells in the liver specific for HBV pre-S2+S antigen after treatment. [Figure 13] HBV-DNA, HBsAg, and HBeAg in AAV / HBV mice treated with GalNAc-conjugated PD-L1 antisense CMP No. 759_2 (▼) compared with vehicle (■). The vertical line indicates the end of treatment.

[0021] definition Oligonucleotides The term "oligonucleotide" is generally defined herein 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 nucleic acid molecules or oligomers. Oligonucleotides are typically produced in a laboratory (by solid-phase chemical synthesis) and subsequently purified. When referring to the sequence of an oligonucleotide, the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides or modifications thereof is described. The oligonucleotides of the present invention are artificial and chemically synthesized, and are typically purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides or nucleotides.

[0022] antisense oligonucleotides In this specification, the term " antisense oligonucleotide " is defined as the oligonucleotide that can regulate the expression of target gene by hybridizing with target nucleic acid, particularly the continuous sequence on target nucleic acid.Antisense oligonucleotide is not essentially double-stranded, therefore it is not siRNA.The antisense oligonucleotide of the present invention is single-stranded, but is preferred.

[0023] 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 form a contiguous nucleotide sequence. In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence and, optionally, may include additional nucleotides (e.g., a nucleotide linker region that can be used to attach a functional group to the contiguous nucleotide sequence). The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0024] nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for purposes of the present invention, include both natural and non-natural nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides). Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers."

[0025] 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 introducing one or more modifications to the sugar or (nucleo)base moieties. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used herein synonymously with the term "nucleoside analogue" or modified "unit" or modified "monomer."

[0026] Modified internucleoside linkages The term "modified internucleoside linkage" is widely understood by those skilled in the art as a linkage other than a phosphodiester (PO) linkage and is defined as a linkage that covalently links two nucleosides together. A nucleotide having a modified internucleoside linkage is also referred to as a "modified nucleotide." In some embodiments, the modified internucleoside linkage increases the nuclease resistance of an oligonucleotide compared to a phosphodiester linkage. In naturally occurring oligonucleotides, the internucleoside linkage includes a phosphate group that creates a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages 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 in the oligonucleotides of the invention, for example, within the gap region of a gapmer oligonucleotide, as well as within regions of modified nucleosides.

[0027] In embodiments, the oligonucleotide contains one or more internucleoside linkages that have been modified from natural phosphodiester to, for example, a linkage that has increased resistance to nuclease attack. Methods for quantifying nuclease resistance can include incubating the oligonucleotide in serum or 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 increase 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 in the oligonucleotide or its contiguous nucleotide sequence are modified (e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, or e.g., at least 90%) of the internucleoside linkages in the oligonucleotide or its contiguous nucleotide sequence are modified. In some embodiments, all internucleoside linkages in the oligonucleotide or its contiguous nucleotide sequence are modified. It will be appreciated that in some embodiments, the nucleosides (e.g., linkages) linking the oligonucleotides of the invention to the non-nucleotide functional group can be phosphodiester, hi some embodiments, all of the internucleoside linkages in the oligonucleotide or its contiguous nucleotide sequence are nuclease-resistant internucleoside linkages.

[0028] The modified internucleoside linkage can be selected from the group consisting of phosphorothioate, diphosphorothioate, and boranophosphate. In some embodiments, the modified internucleoside linkage is compatible with recruitment of RNase H by the oligonucleotides of the invention, e.g., phosphorothioate, diphosphorothioate, or boranophosphate.

[0029] In some embodiments, the internucleoside linkage comprises a sulfur (S) (e.g., a phosphorothioate internucleoside linkage).

[0030] 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 in an oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate (e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, or e.g., at least 90% of the internucleoside linkages in an oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate). In some embodiments, all internucleoside linkages in an oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate.

[0031] In some embodiments, the oligonucleotide comprises one or more neutral internucleoside linkages, particularly internucleoside linkages selected from phosphotriester, methylphosphonate, MMI, amide-3, formacetal, or thioformacetal.

[0032] Further internucleoside linkages are disclosed in WO 2009 / 124238 (herein incorporated by reference). In some embodiments, the internucleoside linkage is selected from the linkers disclosed in WO 2007 / 031091 (herein incorporated by reference). In particular, the internucleoside linkage is selected from the linkers disclosed in WO 2007 / 031091 (herein incorporated by reference). In particular, the internucleoside linkage is selected from the linkers -OP(O)2-O-, -OP(O,S)-O-, -OP(S)2-O-, -SP(O)2-O-, -SP(O,S)-O-, -SP(S)2-O-, -OP(O)2-S-, -OP(O,S)-S-, -SP(O)2-S-, -O-PO(R H )-O-, 0-PO(OCH3)-0-, -O-PO(NR H )-O-, -O-PO(OCH2CH2S-R)-O-, -O-PO(BH3)-O-, -O-PO(NHR H )-O-, -OP(O)2-NR H -, -NR H -P(O)2-O-, -NR H -CO-O-, -NR H -CO-NR Hand / or the internucleoside linker can be selected from -O-CO-O-, -O-CO-NR H -, -NR H -CO-CH2-, -O-CH2-CO-NR H -, -O-CH2-CH2-NR H -, -CO-NR H -CH2-, -CH2-NR H CO-, -O-CH2-CH2-S-, -S-CH2-CH2-O-, -S-CH2-CH2-S-, -CH2-SO2-CH2-, -CH2-CO-NR H -, -O-CH2-CH2-NR H -CO-, -CH2-NCH3-O-CH2- (in the formula, R H is selected from hydrogen and C1-4-alkyl.

[0033] Nuclease-resistant linkages such as phosphothioate linkages are particularly useful in regions of oligonucleotides that have the ability to recruit nucleases upon duplex formation with a target nucleic acid, such as region G for gapmers, or in the unmodified nucleoside regions of the head and tail mers, although phosphorothioate linkages may also be useful in non-nuclease recruiting regions and / or affinity-enhancing regions such as regions F and F' for gapmers, or in the modified nucleoside regions of the head and tail.

[0034] However, each design region may contain internucleoside linkages other than phosphorothioate, such as phosphodiester linkages, in regions where modified nucleosides, such as locked nucleic acids (LNAs), protect against nuclease degradation. The inclusion of phosphodiester linkages, such as one or two linkages, particularly between modified nucleoside units (or typically in non-nuclease complementation regions), can modify the bioavailability and / or biodistribution of the oligonucleotide. See WO 2008 / 113832, incorporated herein by reference.

[0035] In certain embodiments, all internucleoside linkages in the oligonucleotide are phosphorothioate and / or boranophosphate linkages. Preferably, all internucleoside linkages in the oligonucleotide are phosphorothioate linkages.

[0036] Nucleic acid bases The term "nucleobase" encompasses purine (e.g., adenine and guanine) moieties and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term "nucleobase" also encompasses modified nucleobases that 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.

[0037] 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 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).

[0038] The nucleobase moieties may be represented by the letter code of each corresponding nucleobase (e.g., A, T, G, C, or U), and each letter may optionally include a modified base having an equivalent function. For example, in exemplary oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, 5-methylcytosine LNA nucleosides can be used as LNA gapmers.

[0039] 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.

[0040] Complementarity The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). Oligonucleotides may contain nucleosides with modified nucleobases. For example, 5-methylcytosine is often used instead of cytosine, so it is understood that the term "complementarity" encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, 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).

[0041] As used herein, the term "% complementary" refers to the percentage of nucleotides in a contiguous nucleotide sequence within a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to (i.e., form Watson-Crick base pairs with) a contiguous nucleotide sequence at a given position in a separate nucleic acid molecule (e.g., a target nucleic acid). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the oligonucleotide sequence from 5'-3' and 3'-5' of the target sequence), dividing this by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, non-aligned (base-paired) nucleic acid bases / nucleotides are referred to as mismatches.

[0042] The term "fully complementary" refers to 100% complementarity.

[0043] An example of an oligonucleotide (SEQ ID NO: 5) that is perfectly complementary to the target nucleic acid (SEQ ID NO: 772) is shown below. 5'gcagtagagccaatta3' (SEQ ID NO: 772) 3'cgtcatctcggttaat5' (SEQ ID NO: 5)

[0044] identity As used herein, the term "identity" refers to the proportion (number expressed as a percentage) of nucleotides in a contiguous nucleotide sequence within a nucleic acid molecule (e.g., an oligonucleotide) that are identical (i.e., capable of forming Watson-Crick base pairs with complementary nucleosides) to a contiguous nucleotide sequence at a given position in a separate nucleic acid molecule (e.g., a target nucleic acid). The percentage is calculated by counting the number of aligned bases that are identical between the two sequences, including gaps, divided by the total number of nucleotides in the oligonucleotide, and multiplied by 100. % Identity = (Match x 100) / Length of Aligned Region (with Gaps)

[0045] Hybridization As used herein, the term "hybridize" or "hybridizing" refers to two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands, thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of hybridization and is often measured by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotide becomes duplexed with the target nucleic acid. m ) are explained in relation to 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° is a more accurate representation of binding affinity, ΔG° = -RTln(K d ) where R is the gas constant and T is the absolute temperature. d) is related to the reactivity of the oligonucleotide with the target nucleic acid. Therefore, a very low ΔG° between the oligonucleotide and the target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction in which the water concentration is 1M, pH is 7, and the temperature is 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and G° is set to be less than zero to induce spontaneous reaction Δ. ΔG° can be experimentally measured, for example, by isothermal titration calorimetry (ITC), 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 commercial instruments are available for measuring ΔG°. ΔG° can be numerically estimated using nearest neighbor methods as described in SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, or using appropriately derived thermodynamic parameters as described in Sugimoto et al., 1995, Biochemistry 34: 11211-11216 and McTigue et al., 2004, Biochemistry 43: 5388-5405. To potentially modulate their intended nucleic acid targets through hybridization, oligonucleotides of the present invention hybridize to target nucleic acids with estimated ΔG° values ​​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 Gibbs free energy ΔG°. The oligonucleotides hybridize to target nucleic acids with estimated ΔG° values ​​in the range of less than -10 kcal (e.g., less than -15 kcal, less than -20 kcal, e.g., less than -25 kcal) for oligonucleotides 8 to 30 nucleotides in length. In some embodiments, the oligonucleotides hybridize to target nucleic acids with estimated ΔG° values ​​of -10 to -60 kcal, e.g., -12 to -40, e.g., -15 to -30 kcal, or -16 to -27 kcal, e.g., -18 to -25 kcal.

[0046] target nucleic acid According to the present invention, the target nucleic acid is a nucleic acid encoding a mammalian PD-L1, and may be, for example, a gene, RNA, mRNA, and pre-mRNA, mature mRNA, or cDNA sequence. Thus, the target may be referred to as a PD-L1 target nucleic acid. Oligonucleotides of the present invention may, for example, target exon regions of a mammalian PD-L1, or may, for example, target intron regions of PD-L1 pre-mRNA (see Table 1).

[0047] [Table 1]

[0048] Preferably, the target nucleic acid encodes a PD-L1 protein, in particular a mammalian PD-L1, such as human PD-L1 (see, for example, Tables 2 and 3 which describe the mRNA and pre-mRNA sequences of human, monkey and mouse PD-L1). In the context of the present invention, a pre-mRNA is also considered to be a nucleic acid encoding a protein.

[0049] In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or naturally occurring variants thereof (e.g., sequences encoding mammalian PD-L1 proteins).

[0050] When using the oligonucleotides of the invention in research or diagnostics, the target nucleic acid may be cDNA derived from DNA or RNA, or a synthetic nucleic acid.

[0051] For in vivo or in vitro use, the oligonucleotides of the invention are typically capable of inhibiting expression of a PD-L1 target nucleic acid in a cell that expresses the PD-L1 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotides of the invention is typically complementary to a PD-L1 target nucleic acid when measured over the length of the oligonucleotide, optionally excluding one or two mismatches, and optionally excluding a nucleotide-based linker region that may link the oligonucleotide to any functional group (e.g., a conjugate) or other non-complementary terminal nucleotide (e.g., region D' or D"). In some embodiments, the target nucleic acid may be RNA or DNA (e.g., messenger RNA such as mature mRNA or pre-mRNA). In some embodiments, the target nucleic acid is RNA or DNA encoding a mammalian PD-L1 protein, such as human PD-L1, which is the human PD-L1 pre-mRNA sequence, such as that disclosed as SEQ ID NO: 1, or the human mRNA sequence having NCBI Reference No. NM_014143. Further information regarding exemplary target nucleic acids is provided in Tables 2 and 3.

[0052] [Table 2]

[0053] [Table 3-1]

[0054] Target sequence As used herein, the term "target sequence" refers to a sequence of nucleotides present in a target nucleic acid, comprising a nucleobase sequence complementary to an oligonucleotide of the present invention. In some embodiments, a target sequence consists of a region on a target nucleic acid that is complementary to a contiguous nucleotide sequence of an oligonucleotide of the present invention. In some embodiments, a target sequence is longer than the complementary sequence of a single oligonucleotide, and may represent, for example, a preferred region of a target nucleic acid that can be targeted by several oligonucleotides of the present invention.

[0055] The target sequence can be a subsequence of the target nucleic acid.

[0056] In some embodiments, the subsequence is a sequence selected from the group consisting of a1 to a149 (see Table 4). In some embodiments, the subsequence is a sequence selected from the group consisting of human PD-L1 mRNA exons, for example, a PD-L1 human mRNA exon selected from the group consisting of e1, e2, e3, e4, e5, e6, and e7 (see Table 1 above).

[0057] In some embodiments, the subsequence is a sequence selected from the group consisting of human PD-L1 mRNA introns, for example a PD-L1 human mRNA intron selected from the group consisting of i1, i2, i3, i4, i5, and i6 (see Table 1 above).

[0058] The oligonucleotides of the present invention comprise a contiguous nucleotide sequence that is complementary to or hybridizes to a target nucleic acid (eg, a subsequence of a target nucleic acid, such as a target sequence described herein).

[0059] The oligonucleotide comprises a contiguous nucleotide sequence of at least 8 nucleotides that is complementary to or hybridizes to a target sequence present in a target nucleic acid molecule, where the contiguous nucleotide sequence (and thus the target sequence) consists of at least 8 contiguous nucleotides, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides (e.g., 12-25, 14-18 contiguous nucleotides, etc.).

[0060] target cell As used herein, the term "target cell" refers to a cell that expresses 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 mammalian cell, such as a rodent cell (e.g., a mouse cell or a rat cell) or a primate cell (e.g., a monkey cell or a human cell).

[0061] In a preferred embodiment, the target cell expresses PD-L1 mRNA, such as PD-L1 pre-mRNA or PD-L1 mature mRNA. The polyA tail of PD-L1 mRNA is typically ignored for antisense oligonucleotide targeting.

[0062] Naturally occurring variants The term "naturally occurring variant" refers to a variant of the PD-L1 gene, or a transcript derived from the same locus as the target nucleic acid, that may differ due to degeneracy of the genetic code resulting in multiple codons encoding the same amino acid, or the presence of pre-mRNA due to alternative splicing, or the presence of polymorphism (e.g., single nucleotide polymorphisms and allelic variants). Thus, the oligonucleotides of the invention can target the target nucleic acid and its naturally occurring variants based on the presence of sufficient complementary sequence to the oligonucleotide.

[0063] In some embodiments, the naturally occurring variant has at least 95%, such as at least 98% or at least 99%, homology to a mammalian PD-L1 target nucleic acid (e.g., a target nucleic acid selected from the group consisting of SEQ ID NOs: 1, 2, and 3). A number of single nucleotide polymorphisms are known in the PD-L1 gene, for example, as disclosed in the table below (the human pre-mRNA start / reference sequence is SEQ ID NO: 2).

[0064] [Table 3-2]

[0065] [Table 3-3]

[0066] Regulation of expression As used herein, the term "modulation of expression" should be understood as a general term for the ability of an oligonucleotide to alter the amount of PD-L1 compared to the amount of PD-L1 before administration of the oligonucleotide. Alternatively, modulation of expression can be quantified by reference to a control experiment. A control is generally understood to be an individual or target cells treated with a saline composition, an individual or target cells treated with a non-targeting oligonucleotide (mock), but can also be an individual treated according to the standard of care.

[0067] One type of modulation is the ability of the oligonucleotide to inhibit, downregulate, reduce, suppress, eliminate, interrupt, block, prevent, mitigate, decrease, avoid, or stop expression of PD-L1, for example, by degradation of mRNA or interference with transcription. Another type of modulation is the ability of the oligonucleotide to restore, increase, or enhance expression of PD-L1, which can be achieved by preventing splicing, removal, or blocking of inhibitory mechanisms such as splice site repair or microRNA suppression.

[0068] High-affinity modified nucleosides High affinity modified nucleosides, when incorporated into an oligonucleotide, increase, for example, the melting temperature (T mThe high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature of between +0.5 and +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Many high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides, as well as 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).

[0069] sugar modification Oligomers of the invention may include one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety compared to the ribose sugar moiety found in DNA and RNA.

[0070] A number of nucleosides have been created with modifications to the ribose sugar moiety, primarily for the purpose of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0071] Such modifications include those in which the ribose ring structure has been modified, for example, by substitution of a hexose ring (HNA) or bicyclic ring, typically having a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring (e.g., UNA), typically lacking 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 is replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0072] 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 present in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position. Nucleosides having modified sugar moieties also include 2'-modified nucleosides, such as 2'-substituted nucleosides. Indeed, considerable focus has been placed on the development of 2'-substituted nucleosides, and many of these have been found to exhibit beneficial properties, such as increased nucleoside tolerance and increased affinity, when incorporated into oligonucleotides.

[0073] 2'-modified nucleosides.

[0074] 2'-Sugar-modified nucleosides have a substituent other than H or -OH at the 2'-position (2'-substituted nucleosides) or contain a 2'-linked biradical, including 2'-substituted nucleosides and LNA (2'-4' biradical bridge) nucleosides. For example, 2'-modified sugars can confer increased binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, e.g., 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 diagrams of some 2'-substituted modified nucleosides.

[0075] [ka]

[0076] [ka]

[0077] Locked nucleic acid nucleosides (LNA). LNA nucleosides are modified nucleosides that contain a linker group (called a biradical or bridge) between C2' and C4'. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). In some embodiments, the modified nucleosides or LNA nucleosides of the oligomers of the invention have the general structure of Formula I or Formula II.

[0078] [ka]

[0079] In the formula, W is -O-, -S-, -N(R a )-, -C(R a R b )-, e.g., in some embodiments, -O-, B represents a nucleobase or modified nucleobase moiety, Z represents an internucleoside linkage to an adjacent nucleoside or the 5'-terminus, and Z * represents an internucleoside linkage to an adjacent nucleoside or the 3'-terminal group, and X represents -C(R a R b )-, -C(R a )=C(R b )-, -C(R a )=N-, -O-, -Si(R a )2-, -S-, -SO2-, -N(R a )-, and >C=Z. In some embodiments, X is —O—, —S—, NH—, NR a R b , -CH2-, CR a R b , -C(=CH2)-, and -C(=CR a R b )-. In some embodiments, X is —O—. Y is -C(R a R b )-, -C(R a )=C(R b )-, -C(R a )=N-, -O-, -Si(R a )2-, -S-, -SO2-, -N(R a )-, and >C=Z. In some embodiments, Y is —CH—, —C(R a R b )-, -CH2CH2-, -C(R a R b )-C(R a R b )-, -CH2CH2CH2-, -C(R a R b )C(R a R b )C(R a R b )-, -C(R a )=C(R b )- and -C(R a )=N-. In some embodiments, Y is —CH—, —CHR a -, -CHCH3-, CR a R b - or -X-Y-, together representing a divalent linker group (also called a substrate), -C(R a R b )-, -C(R a )=C(R b )-, -C(R a )=N-, -O-, -Si(R a )2-, -S-, -SO2-, -N(R a represents a divalent linker group consisting of 1, 2, 3 or 4 groups / atoms selected from the group consisting of: >C=Z; In some embodiments, -X-Y- is -X-CH2-, -X-CR2-, or -X-CR2-. a R b -, -X-CHR a-、 -XC(HCH3) -、-OY-, -O-CH2-, -S-CH2-, -NH-CH2-, -O-CHCH3-, -CH2-O-CH2, -O-CH(CH3CH3)-, -O-CH2-CH2-, OCH2-CH2-CH2-, -O-CH2OCH2-, -O-NCH2-, -C(=CH2)-CH2-, -NR a -CH2-, NO-CH2, -S-CR a R b -and-S-CHR a represents a biradical selected from the group consisting of:

[0080] In some embodiments, -XY- represents -O-CH2- or -O-CH(CH3)-, where Z is -O-, -S-, and -N(R a )- and R a is R b each independently represents hydrogen, optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -alkenyl, optionally substituted C 2-6 -alkynyl, hydroxy, optionally substituted C 1-6 -alkoxy, C 2-6 -Alkenyloxy, C 2-6 -Alkenyloxy, carboxy, C 1-6 -alkoxycarbonyl, C 1-6 -Alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino; mono- and di(C 1-6 -alkyl)amino, carbamoyl; mono- and di(C 1-6 -alkyl)-amino-carbonyl, amino-C 1-6 -Alkyl-aminocarbonyl; mono- and di(C 1-6 -alkyl)amino-C 1-6 -Alkyl-aminocarbonyl, C 1-6 -Alkylcarbonylamino, carbamide, C 1-6 -Alkanoyloxy, sulfono, C 1-6-Alkyl sulfono(sulphono)oxy, nitro, azido, sulfanyl, C 1-6 -alkylthio, halogen, wherein aryl and heteroaryl are optionally substituted, and two geminal substituents R a and R b together can represent an optionally substituted methylene (=CH2), where for all chiral centers, the asymmetric group can be found in either the R or S orientation. R 1 , R 2 , R 3 , R 5 and R 5* are independently hydrogen, optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -alkenyl, optionally substituted C 2-6 -alkynyl, hydroxy, C 1-6 -alkoxy, C 2-6 -Alkenyloxy, C 2-6 -Alkenyloxy, carboxy, C 1-6 -alkoxycarbonyl, C 1-6 -Alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino; mono- and di(C 1-6 -alkyl)amino, carbamoyl; mono- and di(C 1-6 -alkyl)-amino-carbonyl, amino-C 1-6 -Alkyl-aminocarbonyl; mono- and di(C 1-6 -alkyl)amino-C 1-6 -Alkyl-aminocarbonyl, C 1-6 -Alkylcarbonylamino, carbamide, C 1-6 -Alkanoyloxy, sulfono, C 1-6 -Alkyl sulfonyloxy, nitro, azido, sulfanyl, C 1-6-alkylthio, halogen, wherein aryl and heteroaryl can be optionally substituted, and two geminal substituents together can represent oxo, thioxo, imino, and optionally substituted methylene.

[0081] In some embodiments, R 1 , R 2 , R 3 , R 5 and R 5* independently, C 1-6 It is selected from alkyl (eg, methyl and hydrogen).

[0082] In some embodiments, R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen.

[0083] In some embodiments, R 1 , R 2 , R 3 are both hydrogen atoms, and similarly, R 5 and R 5* is hydrogen, and R 5 and R 5* The other is not hydrogen (e.g., C such as methyl) 1-6 alkyl).

[0084] In some embodiments, R a is either hydrogen or methyl. In some embodiments, when present, R b is either hydrogen or methyl.

[0085] In some embodiments, R a and R b One or both of is hydrogen.

[0086] In some embodiments, R a and R b One of the groups is hydrogen and the other is other than hydrogen.

[0087] In some embodiments, R a and R b One of the groups is methyl and the other is hydrogen.

[0088] In some embodiments, R a and R b are both methyl.

[0089] In some embodiments, the biradical -XY- is -O-CH2-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. Such LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, and WO 2004 / 046160, all of which are incorporated herein by reference, and include what are commonly referred to as β-D-oxy LNA and α-L-oxy LNA nucleosides.

[0090] In some embodiments, the biradical -XY- is -S-CH2-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. Such thioLNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which applications are incorporated herein by reference.

[0091] In some embodiments, the biradical -XY- is -NH-CH2-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5*are all hydrogen. Such amino LNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which applications are incorporated herein by reference.

[0092] In some embodiments, the biradical -XY- is -O-CH-CH- or -O-CH-CH-CH-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. Such LNA nucleosides are disclosed in WO 00 / 047599 and Morita et al, Bioorganic & Med. Chem. Lett. 12 73-76, which are incorporated herein by reference, and include those commonly referred to as 2'-O-4'C-ethylene-bridged nucleic acids (ENA).

[0093] In some embodiments, the biradical -XY- is -O-CH2-, W is O, and R 1 , R 2 and R 3 All of the above and R 5 and R 5* One of these R 5 and R 5* The other is a non-hydrogen group, such as methyl. 1-6 Such 5'-substituted LNA nucleosides are disclosed in WO 2007 / 134181, which is incorporated herein by reference.

[0094] In some embodiments, the biradical -XY- is -O-CR a R b - and R a and R b is other than hydrogen (e.g., methyl), W is O, and R 1 , R 2 and R 3All of the above and R 5 and R 5* One of these R 5 and R 5* The other is not hydrogen (e.g., C such as methyl) 1-6 Such bis-modified LNA nucleosides are disclosed in WO 2010 / 077578, which is incorporated herein by reference.

[0095] In some embodiments, the biradical -X-Y- represents a divalent linker group -O-CH(CHOCH)-(2'O-methoxyethyl bicyclic nucleoside (Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). In some embodiments, the biradical -X-Y- represents a divalent linker group -O-CH(CHCH)-(2'O- ...R a -, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. Such 6' substituted LNA nucleosides are disclosed in WO 10036698 and WO 07090071, both of which are incorporated herein by reference.

[0096] In some embodiments, the biradical -XY- is -O-CH(CHOCH)-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. Such LNA nucleosides, also known in the art as cyclic MOEs (cMOEs), are disclosed in WO 07090071.

[0097] In some embodiments, the biradical -X-Y-, within either the -R- or -S- configuration, represents the divalent linker group -O-CH(CH3)-. In some embodiments, the biradicals -X-Y- together represent the divalent linker group -O-CH2-O-CH2 (Seth at al., 2010, J. Org. Chem). In some embodiments, the biradical -X-Y- is -O-CH(CH3)-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. Such 6' methyl LNA nucleosides are also known in the art as cET nucleosides and can be either the (S)cET or (R)cET stereoisomers as disclosed in WO 07090071 (β-D) and WO 2010 / 036698 (α-L), both of which are incorporated herein by reference.

[0098] In some embodiments, the biradical -XY- is -O-CR a R b - and R a or R b are not hydrogen, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. In some embodiments, R a and R b are both methyl. Such 6' disubstituted LNA nucleosides are disclosed in WO2009006478, which is incorporated herein by reference.

[0099] In some embodiments, the biradical -X-Y- is -S-CHR a -, W is O, and R 1 , R 2 , R 3 , R5 and R 5* are all hydrogen. Such 6'-substituted thio LNA nucleosides are disclosed in WO 11156202, which is incorporated herein by reference. In some 6'-substituted thio LNA embodiments, R a is methyl.

[0100] In some embodiments, the biradical -XY- is -C(=CH)-C(R a R b )-, for example -C(=CH2)-CH2-, or -C(=CH2)-CH(CH3)-W is O and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. Such vinylcarbon LNA nucleosides are disclosed in WO 08154401 and WO 09067647, both of which are incorporated herein by reference.

[0101] In some embodiments, the biradical -XY- is -N(-OR a )-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. In some embodiments, R a is C such as methyl 1-6 In some embodiments, the biradical -X-Y- is a bivalent linker group -O-NR a In some embodiments, the biradical -XY- is a -N(R a )-, W is O, and R 1 , R2 , R 3 , R 5 and R 5* are all hydrogen. In some embodiments, R a is C 1-6 Alkyl, for example methyl.

[0102] In some embodiments, R 5 and R 5* One or both of R 5 and R 5* The other of these is C 1-6 alkyl, e.g., methyl. In such embodiments, R 1 , R 2 , R 3 may both be hydrogen, and the biradical -XY- may be -O-CH2- or -OC(HCR a )-, for example -OC(HCH3)-.

[0103] In some embodiments, the biradical is -CR a R b -O-CR a R b -, for example CH2-O-CH2-, W is O and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. In some embodiments, R a is C 1-6ア Such LNA nucleosides are also known as conformationally restricted nucleotides (CRNs) and are disclosed in WO 2013036868, which is incorporated herein by reference.

[0104] In some embodiments, the biradical is -O-CR a R b -O-CR a R b -, for example, O-CH2-O-CH2-, W is O, and R1 , R 2 , R 3 , R 5 and R 5* are all hydrogen. In some embodiments, R a is C 1-6 Alkyl, for example, methyl. Such LNA nucleosides, also known as COC nucleotides, are disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, which is incorporated herein by reference.

[0105] Unless specified, it will be appreciated that LNA nucleosides can be in the β-D or α-L stereoisomer.

[0106] Some examples of LNA nucleosides are illustrated in Scheme 1.

[0107] [ka]

[0108] As illustrated in the Examples, in some embodiments of the present invention, the LNA nucleosides in the oligonucleotides are β-D-oxy-LNA nucleosides.

[0109] Nuclease-mediated degradation Nuclease-mediated degradation refers to an oligonucleotide that can mediate the degradation of a complementary nucleotide sequence when it forms a duplex with such a sequence.

[0110] In some embodiments, oligonucleotides of the invention can function through nuclease-mediated degradation of target nucleic acids, having the ability to recruit nucleases, particularly endonucleases, preferably endonucleases (RNases), such as RNase H. Nuclease-mediated mechanisms are oligonucleotides that typically contain a region of at least five or six DNA nucleosides and are flanked on one or both sides by affinity-enhancing nucleosides, e.g., gapmers, headers, and tailmers.

[0111] RNase H activity and recruitment The 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 measuring RNase H activity that can be used to determine its ability to recruit RNase H. An oligonucleotide is typically considered to have the ability to recruit RNase H when provided with a complementary target nucleic acid sequence, and has an initial rate measured in pmol / l / min that is at least 5%, e.g., at least 10% or greater than 20% of the quantified initial rate (when using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology described in Examples 91-95 of WO 01 / 23613, which is incorporated herein by reference).

[0112] Gapmar As used herein, the term "gapmer" refers to an antisense oligonucleotide that contains a region of an RNase H-recruiting oligonucleotide (gap) flanked on the 5' and 3' sides by regions containing one or more affinity-enhancing modified nucleosides (flanks or wings). Various gapmer designs are described herein and are characterized by their ability to recruit RNase H. Headers and tailmers are oligonucleotides that lack one of the flanks, i.e., only one of the ends of the oligonucleotide contains affinity-enhancing modified nucleosides, and have the ability to recruit RNase H. In the case of headmers, the 3' flank is missing (i.e., the 5' flank contains the affinity-modified nucleoside), while tailmers lack the 5' flank (i.e., the 3' flank contains the affinity-modified nucleoside).

[0113] LNA gapmers The term LNA gapmer refers to a gapmer oligonucleotide in which at least one of the affinity-enhancing modified nucleosides is an LNA nucleoside.

[0114] Mixed Wing Gappa The term mixed wing gapmer or mixed flanking gapmer refers to an LNA gapmer in which at least one of the flanking regions comprises at least one LNA nucleoside and at least one non-LNA modified nucleoside (e.g., at least one 2'-substituted modified nucleoside such as 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleoside). In some embodiments, a mixed wing gapmer comprises only LNA nucleosides on one side (e.g., 5' or 3') and 2'-substituted modified nucleosides and optionally LNA nucleosides on the other side (3' or 5', respectively).

[0115] Gap Breaker The term "gap breaker oligonucleotide" refers to a gapmer that retains RNase H recruitment even when the gap region is disrupted with a non-RNase H-recruiting nucleoside (gap breaker nucleoside, E) resulting in fewer than five consecutive DNA nucleosides within the gap region. Non-RNase H-recruiting nucleosides are nucleosides in a 3'-terminal conformation, such as LNA, where the bridge between the C2' and C4' ends of the ribose sugar ring of the nucleoside is in a β conformation, such as β-D-oxyLNA or ScET nucleosides. The ability of gap breaker oligonucleotides to recruit RNase H is typically sequence-specific or even compound-specific (see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487). "Gap breaker" oligonucleotides are disclosed herein. This gap breaker oligonucleotide recruits RNase H, which in some instances provides more specific cleavage of the target RNA.

[0116] In some embodiments, the oligonucleotide of the invention is a gap breaker oligonucleotide, which includes a 5'-flank (F), a gap (G), and a 3'-flank (F'), where the gap is broken by a non-RNase H-replenishing nucleoside (gap blocker nucleoside, E) such that the gap contains at least three or four consecutive DNA nucleosides. In some embodiments, the gap breaker nucleoside (E) is an LNA nucleoside, the bridge between C2' and C4' of the ribose sugar ring of the nucleoside is in a β conformation and is positioned within the gap region, the gap breaker LNA nucleosides are at least 3' (5') and 3' (3') DNA nucleosides, or at least 3' (5') and 4' (3') DNA nucleosides, or at least 4' (5') and 3' (3') DNA nucleosides adjacent 5' and 3', and the oligonucleotide has the ability to recruit RNase H.

[0117] The gap breaker oligonucleotide can be represented by the following formula: FGEG-F'; especially F 1-7 -G 3-4 -E1-G 3-4 -F' 1-7 D'-FG-F', especially D' 1-3 -F 1-7 -G 3-4 -E1-G 3-4 -F' 1-7 FG-F'-D”, especially F 1-7 -G 3-4 -E1-G 3-4 -F' 1-7 -D” 1-3 D'-FG-F'-D", especially D' 1-3 -F 1-7 -G 3-4 -E1-G 3-4- -F' 1-7 -D” 1-3

[0118] Regions D' and D" are described in the "Gapmer Design" section.

[0119] In some embodiments, the gap breaker nucleoside (E) is β-D-oxyLNA or ScET, as depicted in Scheme 1, or another β-LNA nucleoside.

[0120] Conjugates As used herein, the term conjugate refers to an oligonucleotide covalently attached to a non-nucleotide moiety (linking moiety or region C or third region), also called an oligonucleotide conjugate.

[0121] When oligonucleotides of the invention are conjugated to one or more non-nucleotide moieties, the pharmacology of the oligonucleotide may be improved. This may be achieved by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments, the conjugated moiety targets the oligonucleotide to the liver. At the same time, the conjugate functions to reduce the activity of the oligonucleotide in non-target cell types, tissues, or organs (e.g., tissues or organs), blocking the target activity or activity in non-target cell types, tissues, or organs. In one embodiment of the invention, the oligonucleotide conjugates of the invention demonstrate improved inhibition of PD-L1 in target cells compared to unconjugated oligonucleotides. In another embodiment, the oligonucleotide conjugates of the invention demonstrate improved cellular distribution between the liver and other organs, such as the spleen or kidney, compared to unconjugated oligonucleotides. That is, conjugated oligonucleotides tend to migrate to the liver rather than the spleen or kidney. In another embodiment, the oligonucleotide conjugates of the invention demonstrate improved cellular uptake of conjugated oligonucleotides into the liver compared to conjugated oligonucleotides.

[0122] Suitable binding moieties are provided in International Publication Nos. WO 93 / 07883 and WO 2013 / 033230, which are incorporated herein by reference. More preferred binding moieties are those capable of binding to the asialoglycoprotein receptor (ASGPr). In particular, trivalent N-acetylgalactosamine binding moieties are suitable for binding to the ASGPr. See, for example, International Publication Nos. WO 2014 / 076196, WO 2014 / 207232, and WO 2014 / 179620, which are incorporated herein by reference. The binding moiety is essentially part of an antisense oligonucleotide conjugate that is not composed of nucleic acid.

[0123] Oligonucleotide conjugates and their synthesis have also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, STCrooke, ed., Ch. 16, Marcel Dekker, Inc., 2001, and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, which are incorporated herein by reference in their entireties.

[0124] In some embodiments, the non-nucleotide moiety (binding moiety) is selected from the group consisting of a carbohydrate, a cell surface receptor ligand, a drug substance, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0125] Linker A linkage or linker is a bond 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 binding moiety can be attached to the oligonucleotide directly or via a linking moiety (e.g., a linker or tether). The linker functions to covalently link a third region, such as a binding moiety (region C), to a first region, such as an oligonucleotide or a continuous nucleotide sequence (region A) complementary to a target nucleic acid.

[0126] In some embodiments of the invention, the conjugate or oligonucleotide conjugate of the invention optionally comprises a linker region (second region, i.e., region B and / or region Y) located between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (first region, i.e., region A) and the binding moiety (third region, i.e., region C).

[0127] Region B refers to 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. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentrations found within or similar to those encountered within mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as proteases, hydrolases, or nucleases. In one embodiment, the biocleavable linker is susceptible to cleavage by S1 nuclease. In a preferred embodiment, the nuclease-sensitive linker comprises 1 to 10 nucleosides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, more preferably 2 to 6 nucleosides, most preferably at least two consecutive phosphodiester bonds, e.g., at least three, four, or five consecutive phosphodiester bonds, or 2 to 4 linked nucleosides. The nucleosides are preferably DNA or RNA. Phosphodiester-containing biocleavable linkers are described in detail in WO 2014 / 076195, which is incorporated herein by reference.

[0128] Region Y is a linker that is not necessarily biocleavable, but primarily functions to covalently attach the binding moiety (region C or region 3) to the oligonucleotide or contiguous nucleotide sequence (region A or region 1) complementary to the target nucleic acid. The linker of region Y may comprise a chain structure or oligomer of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugates of the present invention can be configured as ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl (e.g., a C2-C36 aminoalkyl group, such as a C6-C12 aminoalkyl group). In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

[0129] treatment As used herein, the term "treatment" refers to the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Accordingly, it will be recognized that in some embodiments the treatment referred to herein may be prophylactic.

[0130] Restoring immune responses to pathogens Immune responses are classified into innate and adaptive immune responses. The innate immune system provides an immediate but nonspecific response. The adaptive immune response is activated by the innate immune system and is highly specific to a particular pathogen. When pathogen-derived antigens are presented on the surface of antigen-presenting cells, immune cells of the adaptive immune response (i.e., T lymphocytes and B lymphocytes) are activated via antigen-specific receptors, leading to the development of pathogen-specific immune responses and immunological memory. Chronic viral infections, such as HBV and HCV, are associated with T cell exhaustion, characterized by the nonresponsiveness of virus-specific T cells. T cell exhaustion has been well studied; for a review, see, for example, Yi et al. 2010 Immunology 129, 474-481. Chronic viral infections are also associated with impaired function of NK cells, an innate immune cell. Enhanced viral immune responses are important for the clearance of chronic infections. Restoration of T cell and NK cell-mediated immune responses to pathogens can be assessed by measuring proliferation, cytokine secretion and cytolytic function (Dolina et al. 2013 Molecular Therapy-Nucleic Acids, 2 e72, and Example 6 herein). DETAILED DESCRIPTION OF THE INVENTION

[0131] The present invention relates to the use of antisense oligonucleotides and their conjugates, as well as pharmaceutical compositions containing them, for restoring immune responses to pathogens that infect animals, particularly humans.The antisense oligonucleotide conjugates of the present invention are particularly useful against pathogens that infect the liver, particularly chronic liver infections such as HBV.The conjugates allow targeted distribution of oligonucleotides and prevent systemic knockdown of target nucleic acids.

[0132] Oligonucleotides of the Invention The present invention relates to oligonucleotides capable of modulating the expression of PD-L1. Modulation can be achieved by hybridizing to a target nucleic acid encoding PD-L1 or a target nucleic acid involved in the modulation of PD-L1. The target nucleic acid can be a mammalian PD-L1 sequence (e.g., a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3). The target nucleic acid can also be pre-mRNA, mRNA, or any RNA sequence expressed from a mammalian cell that supports the expression or modulation of PD-L1.

[0133] The oligonucleotides of the invention are antisense oligonucleotides that target PD-L1.

[0134] In one embodiment of the invention, the oligonucleotides of the invention are conjugated to a binding moiety, particularly an asialoglycoprotein receptor targeting binding moiety.

[0135] In some embodiments, the antisense oligonucleotides of the invention can modulate target expression by inhibiting or down-regulating the target. Such modulation preferably results in at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target's normal expression level. Such modulation preferably results in at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% inhibition of expression compared to the expression level when cells or organisms are challenged with an infectious agent or treated with an agent that simulates challenge with an infectious agent (e.g., poly I:C or LPS). In some embodiments, the oligonucleotides of the invention may be capable of inhibiting PD-L1 mRNA expression levels by at least 60% or 70% in vivo using KARPAS-299 or THP1 cells. In some embodiments, compounds of the present invention may be capable of inhibiting PD-L1 protein expression levels in vivo by at least 50% using KARPAS-299 or THP1 cells. Preferably, the Examples provide assays that can be used to measure PD-L1 RNA (e.g., Example 1). Target modulation is triggered by hybridization between a contiguous nucleotide sequence of the oligonucleotide and a target nucleic acid. In some embodiments, the oligonucleotides of the present invention contain mismatches between the oligonucleotide and the target nucleic acid. Regardless of the mismatch, hybridization to the target nucleic acid may still be sufficient to exhibit the desired modulation of PD-L1 expression. If mismatches result in reduced binding affinity, it may be advantageous to compensate by increasing the number of nucleotides in the oligonucleotide and / or by enhancing binding affinity to the target (e.g., by the presence of 2'-modified nucleosides, such as LNA, within the oligonucleotide sequence).

[0136] In some embodiments, the antisense oligonucleotides of the present invention are capable of restoring pathogen-specific T cells. In some embodiments, the oligonucleotides of the present invention are capable of increasing pathogen-specific T cells by at least 40%, 50%, 60%, or 70% when compared to untreated controls or controls treated with standard care. In one embodiment, the antisense oligonucleotides or conjugates of the present invention are capable of increasing HBV-specific T cells compared to untreated controls or controls treated with standard care. The Examples preferably provide assays that can be used to measure HBV-specific T cells (e.g., T cell proliferation, cytokine secretion, and cytolytic activity). In another embodiment, the antisense oligonucleotides or conjugates of the present invention are capable of increasing HCV-specific T cells compared to untreated controls or controls treated with standard care. In another embodiment, the antisense oligonucleotides or conjugates of the present invention are capable of increasing HDV-specific T cells compared to untreated controls or controls treated with standard care.

[0137] In some embodiments, the antisense oligonucleotides of the invention can reduce HBs antigen levels in animals or humans. In some embodiments, the oligonucleotides of the invention can reduce HBs antigen levels by at least 40%, 50%, 60%, or 70%, more preferably at least 80%, 90%, or 95%, compared to pre-treatment levels. Most preferably, the oligonucleotides of the invention can achieve HBs antigen seroconversion in animals or humans infected with HBV.

[0138] An embodiment of the present invention relates to an antisense oligonucleotide comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary to a PD-L1 target nucleic acid.

[0139] In some embodiments, the oligonucleotide comprises a contiguous sequence that is at least 90% complementary, such as at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, or 100% complementary to a region of the target nucleic acid.

[0140] In preferred embodiments, the oligonucleotides of the present invention, or their contiguous nucleotide sequences, are perfectly complementary (100% complementary) to a region of the target nucleic acid, or in some embodiments, may contain one or two mismatches between the oligonucleotide and the target nucleic acid.

[0141] In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence 10-30 nucleotides in length that is at least 90% complementary, e.g., completely (i.e., 100%) complementary, to a target nucleic acid region present in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the oligonucleotide sequence is 100% complementary to the corresponding target nucleic acid region present in SEQ ID NO:1 and SEQ ID NO:2. In some embodiments, the oligonucleotide sequence is 100% complementary to the corresponding target nucleic acid region present in SEQ ID NO:1 and SEQ ID NO:3.

[0142] In some embodiments, the oligonucleotide or oligonucleotide conjugate comprises a contiguous nucleotide sequence 10-30 nucleotides in length having at least 90% complementarity, e.g., 100% complementarity, to a corresponding region of the target nucleic acid, wherein the contiguous nucleotide sequence is complementary to a subsequence of the target nucleic acid selected from the group consisting of positions 371-3068, 5467-12107, and 15317-19511 of SEQ ID NO: 1. In further embodiments, the subsequence of the target nucleic acid is selected from the group consisting of positions 371-510, 822-1090, 1992-3068, 5467-5606, 6470-12107, 15317-15720, 15317-18083, 18881-19494, and 1881-19494 of SEQ ID NO: 1. In a preferred embodiment, the subsequence of the target nucleic acid is selected from the group consisting of positions 7300-7333, 8028-8072, 9812-9859, 11787-11873 and 15690-15735 on SEQ ID NO:1.

[0143] In some embodiments, the oligonucleotide or oligonucleotide conjugate comprises a contiguous nucleotide sequence 10-30 nucleotides in length that is at least 90% complementary, e.g., 100% complementary, to a corresponding target nucleic acid region present in SEQ ID NO:1, wherein the target nucleic acid region is selected from the group consisting of regions a1 to a449 of Table 4.

[0144] [Table 4-1]

[0145] [Table 4-2]

[0146] [Table 4-3]

[0147] [Table 4-4]

[0148] In some embodiments, the oligonucleotide or contiguous nucleotide sequence is complementary to a region of a target nucleic acid selected from the group consisting of a7, a26, a43, a119, a142, a159, a160, a163, a169, a178, a179, a180, a189, a201, a202, a204, a214, a221, a224, a226, a243, a254, a258, a269, a274, a350, a360, a364, a365, a370, a372, a381, a383, a386, a389, a400, a427, a435, and a438.

[0149] In a preferred embodiment, the oligonucleotide or contiguous nucleotide sequence is complementary to a region of the target nucleic acid region selected from the group consisting of a160, a180, a221, a269 and a360.

[0150] In some embodiments, the oligonucleotides of the invention comprise or consist of 8 to 35 nucleotides in length, such as 9 to 30, such as 10 to 22, such as 11 to 20, such as 12 to 18, such as 13 to 17, or 14 to 16 contiguous nucleotides in length. In preferred embodiments, the oligonucleotides comprise or consist of 16 to 20 nucleotides in length. Any ranges set forth herein should be understood to include the endpoints of the range. Thus, when an oligonucleotide is said to comprise 10 to 30 nucleotides, both 10 nucleotides and 30 nucleotides are included.

[0151] In some embodiments, the contiguous nucleotide sequence comprises or consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides in length. In preferred embodiments, the oligonucleotide comprises or consists of 16, 17, 18, 19, or 20 nucleotides in length.

[0152] In some embodiments, the oligonucleotide or contiguous nucleotide sequence comprises or consists of a sequence selected from the group consisting of the sequences set forth in Table 5.

[0153] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a 10-30 nucleotide length that is at least 90% identical, preferably 100% identical, to a sequence selected from the group consisting of SEQ ID NOs: 5-743 (see motif sequences listed in Table 5).

[0154] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a 10-30 nucleotide length that is at least 90% identical, preferably 100% identical, to a sequence selected from the group consisting of SEQ ID NOs: 5-743 and 771.

[0155] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence is selected from the group consisting of SEQ ID NOs: 6, 8, 9, 13, 41, 42, 58, 77, 92, 111, 128, 151, 164, 166, 169, 171, 222, 233, 245, 246, 250, 251, 252, 256, 272, 273, 287, 292, 303, 314, 318, 320, 324, 336, 342, 343, 344, 345, 346, 349, 359, 360, 374, 408, 409, 415, 417, 424, 429, 430, 458, 464, 46 6, 474, 490, 493, 512, 519, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636, 637, 638, 640, 645, 650, 651, 652, 653, 658, 659, 660, 665, 678, 679, 680, 682, 683, 684, 687, 694, 706, 716, 728, 733, 734, and 735.

[0156] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a 10-30 nucleotide length that is at least 90% identical, preferably 100% identical to SEQ ID NO:287.

[0157] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a 10-30 nucleotide length that is at least 90% identical, preferably 100% identical to SEQ ID NO:342.

[0158] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a 10-30 nucleotide length that is at least 90% identical, preferably 100% identical to SEQ ID NO:640.

[0159] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a 10-30 nucleotide length that is at least 90% identical, preferably 100% identical to SEQ ID NO:466.

[0160] In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of a 10-30 nucleotide length that is at least 90% identical, preferably 100% identical to SEQ ID NO:566.

[0161] In embodiments in which the oligonucleotide is longer than the contiguous nucleotide sequence (complementary to the target nucleic acid), the motif sequences in Table 5 form the contiguous nucleotide sequence portion of the antisense oligonucleotide of the invention. In some embodiments, the sequence of the oligonucleotide is equivalent to the contiguous nucleotide sequence (e.g., if no biocleavable linker is added).

[0162] It is understood that the consecutive nucleic acid base sequences (motif sequences) can be modified, for example, to enhance nuclease resistance and / or binding affinity to the target nucleic acid. Modifications are described in the "Definitions" and "Oligonucleotide Design" sections. Table 5 lists preferred designs for each motif sequence.

[0163] Oligonucleotide Design Oligonucleotide design refers to the pattern of nucleoside sugar modifications in an oligonucleotide sequence. The oligonucleotides of the present invention contain sugar-modified nucleosides and may also contain DNA or RNA nucleosides. In some embodiments, the oligonucleotides contain sugar-modified nucleosides and DNA nucleosides. By incorporating modified nucleosides into the oligonucleotides of the present invention, the affinity of the oligonucleotide for a target nucleic acid can be enhanced. In this case, the modified nucleosides may be referred to as affinity-enhancing modified nucleotides, and the modified nucleosides may be referred to as units.

[0164] In embodiments, the oligonucleotide comprises at least one modified nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In some embodiments, the oligonucleotide comprises 1 to 10 modified nucleosides, e.g., 2 to 8 modified nucleosides, e.g., 3 to 7 modified nucleosides, e.g., 4 to 6 modified nucleosides, e.g., 3, 4, 5, 6, or 7 modified nucleosides.

[0165] In an 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. Even more preferably, the one or more modified nucleosides are locked nucleic acids (LNA).

[0166] In further embodiments, the oligonucleotide comprises at least one modified internucleoside linkage. In preferred embodiments, all internucleoside linkages within a contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages. In some embodiments, all internucleoside linkages within a contiguous sequence of the oligonucleotide are phosphorothioate linkages.

[0167] In some embodiments, the oligonucleotide of the present invention comprises at least one LNA nucleoside, for example, 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides, for example, 2 to 6 LNA nucleosides, for example, 3 to 7 LNA nucleosides, 4 to 6 LNA nucleosides, or 3, 4, 5, 6, or 7 LNA nucleosides. In some embodiments, at least 75% of the modified nucleosides in the oligonucleotide are LNA nucleosides, for example, 80%, for example, 85%, for example, 90% of the modified nucleosides are LNA nucleosides. In even further embodiments, all modified nucleosides in the oligonucleotide are LNA nucleosides. In further embodiments, the oligonucleotide may comprise both β-D-oxy-LNA and one or more of the following LNA nucleosides (thio-LNA, amino-LNA, oxy-LNA, and / or ENA), in either the β-D or α-L configuration, or a combination thereof. In further embodiments, all LNA cytosine units are 5-methyl-cytosine. In preferred embodiments, the oligonucleotide or contiguous nucleotide sequence has at least one LNA nucleoside at the 5'-end and at least two LNA nucleosides at the 3'-end of the nucleotide sequence.

[0168] In some embodiments, oligonucleotides of the invention include at least one modified nucleoside that is a 2'-MOE-RNA nucleoside (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-MOE-RNA nucleosides). In some embodiments, at least one of the modified nucleosides is a 2'-fluoro-DNA (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro-DNA nucleosides).

[0169] In some embodiments, the oligonucleotides of the invention comprise at least one LNA nucleoside and at least one 2'-substituted modified nucleoside.

[0170] In some embodiments of the present invention, an oligonucleotide comprises both 2' sugar-modified nucleosides and DNA units. Preferably, the oligonucleotide comprises both LNA and DNA nucleosides (units). The total number of LNA and DNA units is preferably 8 to 30 (e.g., 10 to 25), preferably 12 to 22 (e.g., 12 to 18), and even more preferably 11 to 16. In some embodiments of the present invention, the nucleotide sequence (e.g., consecutive nucleotide sequence) of the oligonucleotide comprises at least one or two LNA nucleosides, with the remaining nucleosides being DNA units. In some embodiments, the oligonucleotide comprises only LNA nucleosides and naturally occurring nucleosides (RNA or DNA, most preferably DNA nucleosides), optionally with modified internucleoside linkages (e.g., phosphorothioates).

[0171] In an embodiment of the invention, the oligonucleotides of the invention have the ability to recruit RNase H.

[0172] The structural design of the oligonucleotide of the present invention can be selected from gapmers, gapbreakers, headmers and tailmers.

[0173] Gapmar Design In a preferred embodiment, the oligonucleotides of the invention have a gapmer design or structure, also referred to herein simply as "gapmers." In a gapmer structure, the oligonucleotide comprises at least three distinct structural regions: a 5' flank, a gap, and a 3' flank, i.e., FG-F' in a "5-to-3" orientation. In this design, the flanking F and F' regions (also referred to as wing regions) comprise consecutive stretches of modified nucleotides complementary to a PD-L1 target nucleic acid, and the gap region G has the ability to recruit a nuclease, preferably an endonuclease such as an RNase, e.g., RNase H, when the oligonucleotide comprising the consecutive stretch of nucleotides is duplexed with the target nucleic acid. Nucleosides capable of recruiting nucleases, particularly RNase H, can be selected from the group consisting of DNA, α-L-oxy-LNA, 2'-fluoro-ANA, and UNA. Regions F and F' adjacent to the 5' and 3' ends of region G preferably comprise non-nuclease-supplemented nucleosides (nucleosides having a 3'-end structure), more preferably one or more affinity-enhancing modified nucleosides. In some embodiments, the 3' flank comprises at least one LNA nucleoside, preferably at least two LNA nucleosides. In some embodiments, the 5' flank comprises at least one LNA nucleoside. In some embodiments, both the 5' and 3' flanking regions comprise LNA nucleosides. In some embodiments, all nucleosides in the flanking regions are LNA nucleosides. In other embodiments, the flanking regions may comprise both LNA nucleosides and other nucleosides (mixed flanks), such as DNA nucleosides and / or non-LNA modified nucleosides (e.g., 2'-substituted nucleosides). In this case, the gap is defined as a consecutive sequence of at least five RNase H-replete nucleosides (nucleosides with a 2'-endo structure, preferably DNA) flanked at the 5' and 3' ends by affinity-enhancing modified nucleosides, preferably LNA (e.g., β-D-oxy-LNA).As a result, the nucleosides in the 5' flanking region and the 3' flanking region that flank the gap region are modified nucleosides, preferably non-nuclease supplemented nucleosides.

[0174] Area F Region F (the 5' side or 5' wing) attached to the 5' end of region G comprises, contains, or consists of at least one modified nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 modified nucleosides. In some embodiments, region F comprises or consists of 1 to 7 modified nucleosides, e.g., 2 to 6 modified nucleosides, e.g., 2 to 5 modified nucleosides, e.g., 2 to 4 modified nucleosides, e.g., 1 to 3 modified nucleosides, e.g., 1, 2, 3, or 4 modified nucleosides. The F region is defined by having at least modified nucleosides at the 5' end and the 3' end of the region.

[0175] In some embodiments, the modified nucleosides in region F have a 3'-end structure.

[0176] In an embodiment, one or more of the modified nucleosides in region F are 2'-modified nucleosides. In one embodiment, all of the nucleosides in region F are 2'-modified nucleosides.

[0177] In another embodiment, region F comprises DNA and / or RNA in addition to 2'-modified nucleosides. Aspects comprising DNA and / or RNA are characterized by having 2'-modified nucleosides within the 5'- and 3'-ends (adjacent to the G region) of the F region. In one embodiment, region F comprises DNA nucleosides (e.g., 1-3 consecutive DNA nucleosides, such as 1-3 or 1-2 consecutive DNA nucleosides). Preferably, the flanking DNA nucleosides are naturally capable of recruiting RNase H. In some embodiments, the 2'-modified nucleosides of the F region and the DNA and / or RNA nucleosides alternate with 1-3 2'-modified nucleosides and 1-3 DNA and / or RNA nucleosides. Such flanks are sometimes referred to as alternating flanks. The 5' flank (region F) in an alternating-flanked oligonucleotide can be 4 to 10 nucleosides in length, such as 4 to 8, such as 4 to 6 nucleosides, such as 4, 5, 6, or 7 modified nucleosides. In some embodiments, only the 5' flank of the oligonucleotide is alternating. Specific examples of regions F having alternating nucleosides include: 2' 1-3 -N' 1-4 -2' 1-3 2' 1-2 -N' 1-2 -2' 1-2 -N' 1-2 -2' 1-2 is.

[0178] wherein 2' represents a modified nucleoside and N' is RNA or DNA. In some embodiments, all modified nucleosides within the alternating flanks are LNA and N' is DNA. In further embodiments, one or more of the 2'-modified nucleosides in region F is selected from a 2'-O-alkyl-RNA unit, a 2'-O-methyl-RNA, a 2'-amino-DNA unit, a 2'-fluoro-DNA unit, a 2'-alkoxy-RNA, an MOE unit, an LNA unit, an arabinonucleic acid (ANA) unit, and a 2'-fluoro-ANA unit.

[0179] In some embodiments, the F region comprises both LNA and 2'-substituted modified nucleosides. These are often referred to as mixed wing or mixed flanking oligonucleotides.

[0180] In one embodiment of the invention, all modified nucleosides in region F are LNA nucleosides. In a further embodiment, all nucleosides in region F are LNA nucleosides. In a further embodiment, the LNA nucleosides in region F are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and / or ENA, in either the β-D or α-L configuration or a combination thereof. In a preferred embodiment, region F comprises at least one β-D-oxy LNA unit at the 5' end of the contiguous sequence. area G

[0181] Region G (gap region) preferably comprises or consists of at least 4, such as at least 5, for example 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 consecutive nucleosides capable of recruiting the aforementioned nucleases, in particular RNase H. In further embodiments, region G comprises or consists of 5 to 12, or 6 to 10 or 7 to 9 (e.g. 8 consecutive nucleotide units capable of recruiting the aforementioned nucleases).

[0182] In some embodiments, the nucleoside units of region G capable of recruiting nucleases are selected from the group consisting of DNA, α-L-LNA, C4'-alkylated DNA (International Application PCT / EP2009 / 050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, both of which are incorporated herein by reference), and arabinose-derived nucleosides such as ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (Source: Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference). UNAs are typically unlocked nucleic acids in which the bond between C2 and C3 of the ribose has been removed to produce an unlocked "sugar" moiety.

[0183] In still further embodiments, at least one nucleoside unit in region G is a DNA nucleoside unit (e.g., 1 to 18 DNA units (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 DNA units), preferably 2 to 17 DNA units (e.g., 3 to 16 DNA units (e.g., 4 to 15 DNA units), for example, 5 to 14 DNA units (e.g., 6 to 13 DNA units (e.g., 7 to 12 DNA units (e.g., 8 to 11 DNA units), more preferably 8 to 17 DNA units, or 9 to 16 DNA units, 10 to 15 DNA units, or 11 to 13 DNA units (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 4, 16, 17 DNA units). In some embodiments, region G consists of 100% DNA units.

[0184] In further embodiments, region G may consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage. Region G may consist of at least 50% DNA, more preferably 60%, 70% or 80% DNA, and even more preferably 90% or 95% DNA.

[0185] In yet a further embodiment, at least one nucleoside unit in region G is an α-L-LNA nucleoside unit (e.g., at least one α-L-LNA (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 α-L-LNA). In a further embodiment, region G comprises at least one α-L-LNA that is α-L-oxy-LNA. In a further embodiment, region G comprises a combination of DNA and α-L-LNA nucleoside units.

[0186] In some embodiments, the nucleosides within region G have a 2'-end structure.

[0187] In some embodiments, region G can include a gap breaker nucleoside that leads to a gap breaker oligonucleotide that has the ability to recruit RNase H.

[0188] Area F' Region F' (the 3' side or 3' wing) attached to the 3' terminus of region G comprises, contains, or consists of at least one modified nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 modified nucleosides. In some embodiments, region F' comprises or consists of 1 to 7 modified nucleosides (e.g., 2 to 6 modified nucleosides (e.g., 2 to 4 modified nucleosides (e.g., 1 to 3 modified nucleosides (e.g., 1, 2, 3, or 4 modified nucleosides)). The F' region is defined by having at least modified nucleosides at the 5' terminus and the 3' terminus of the region.

[0189] In some embodiments, the modified nucleosides in region F' have a 3'-end structure.

[0190] In an embodiment, one or more of the modified nucleosides in region F' are 2'-modified nucleosides. In one embodiment, all of the nucleosides in region F' are 2'-modified nucleosides.

[0191] In an embodiment, one or more of the modified nucleosides in region F' is a 2' modified nucleoside.

[0192] In one embodiment, all nucleosides in region F' are 2'-modified nucleosides. In another embodiment, region F' includes DNA or RNA in addition to the 2'-modified nucleosides. A side containing DNA and / or RNA is characterized by having 2'-modified nucleosides in the 5'-end (adjacent to the G region) and 3'-end of the F' region. In one embodiment, region F' includes DNA nucleosides (e.g., 1 to 4 consecutive DNA nucleosides, such as 1 to 3 or 1 to 2 consecutive DNA nucleosides). The flanking DNA nucleosides are preferably naturally capable of recruiting RNase H. In some embodiments, the 2'-modified nucleosides and DNA and / or RNA nucleosides in the F' region alternate with 1 to 3 2'-modified nucleosides and 1 to 3 DNA and / or RNA nucleosides; such a side may be referred to as an alternating side. The length of the 3' flank (region F') in an alternating-flank oligonucleotide can be 4 to 10 nucleosides (e.g., 4 to 8), e.g., 4 to 6 nucleosides (e.g., 4, 5, 6, or 7 modified nucleosides). In some embodiments, only the 3' flank of the oligonucleotide is alternating. Specific examples of region F' having alternating nucleosides include: 2' 1-2 -N' 1-4 -2' 1-4 2' 1-2 -N' 1-2 -2' 1-2 -N' 1-2 -2' 1-2 is.

[0193] wherein 2' represents a modified nucleoside and N' is RNA or DNA. In some embodiments, all modified nucleosides within the alternating flanks are LNA and N' is DNA. In further embodiments, the modified nucleosides within region F' are 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.

[0194] In some embodiments, the F' region comprises both LNA and 2'-substituted modified nucleosides. These are often referred to as mixed wing or mixed flanking oligonucleotides.

[0195] In one embodiment of the invention, all modified nucleosides in region F' are LNA nucleosides. In a further embodiment, all modified nucleosides in region F' are LNA nucleosides. In a further embodiment, the LNA nucleosides in region F' are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and / or ENA, in either the β-D or α-L configuration, or a combination thereof. In a preferred embodiment, region F has at least two β-D-oxy LNA units at the 3' end of the contiguous sequence.

[0196] Areas D' and D" Regions D' and D" can be attached to the 5' end of region F or the 3' end of region F', respectively. Regions D' or D" are optional.

[0197] Region D' or D" can comprise 0 to 5, for example 1 to 5, for example 2 to 4, for example 0, 1, 2, 3, 4, or 5 additional nucleotides, which can be complementary or non-complementary to the target nucleic acid. In this regard, the oligonucleotide of the invention can, in some embodiments, comprise a contiguous nucleotide sequence that can modulate the target adjacent to the 5' and / or 3' end by additional nucleotides. Such additional nucleotides can function as a nuclease-sensitive, biocleavable linker (see definition of linker). In some embodiments, the additional 5' and / or 3' terminal nucleosides are linked with phosphodiester bonds and can be DNA or RNA. In another embodiment, the additional 5' and / or 3' terminal nucleosides are modified nucleosides that can be included, for example, to increase nuclease stability or for ease of synthesis. In one embodiment, the oligonucleotide of the invention comprises region D' and / or D" at the 5' or 3' end of the contiguous nucleotide sequence. In a further embodiment, the D' and / or D" regions consist of 1 to 5 phosphodiester-linked DNA or RNA nucleosides that are not complementary to the target nucleic acid.

[0198] The gapmer oligonucleotide of the present invention can be represented by the following formula: 5'-FG-F'-3'; especially F 1-7 -G 4-12 -F' 1-7 5'-D'-FG-F'-3', especially D' 1-3 -F 1-7 -G 4-12 -F' 1-7 5'-FG-F'-D"-3', especially F 1-7 -G 4-12 -F' 1-7 -D” 1-3 5'-D'-FG-F'-D'-3", especially D' 1-3 -F 1-7 -G 4-12 -F' 1-7 - D” 1-3

[0199] Preferred numbers and types of nucleosides within regions F, G and F', D' and D" are described above. The oligonucleotide conjugates of the present invention have region C, particularly the gapmer oligonucleotides shown above, covalently attached to either the 5' or 3' end of the oligonucleotide.

[0200] In one embodiment, an oligonucleotide conjugate of the invention comprises an oligonucleotide of the formula 5'-D'-FG-F'-3' or 5'-FG-F'-D"-3', wherein regions F and F' independently comprise 1 to 7 modified nucleosides, G is a region of 6 to 16 nucleosides capable of recruiting RNase H, and region D' or D" comprises 1 to 5 phosphodiester-linked nucleosides. Region D' or D" is preferably located at the terminus of the oligonucleotide where attachment to a binding moiety is intended.

[0201] An example of an oligonucleotide with alternating flanks can be represented by the formula: 2' 1-3 -N' 1-4 -2' 1-3 -G 6-12 -2' 1-2 -N' 1-4 -2' 1-4 2' 1-2 -N' 1-2 -2' 1-2 -N' 1-2 -2' 1-2 -G 6-12 -2' 1-2 -N' 1-2 - 2' 1-2 -N' 1-2 -2' 1-2 FG 6-12 -2' 1-2 -N' 1-4 -2' 1-4 FG 6-12 -2' 1-2 -N' 1-2 -2' 1-2 -N' 1-2 -2' 1-2 2' 1-3-N' 1-4 -2' 1-3 -G 6-12 -F' 2' 1-2 -N' 1-2 -2' 1-2 -N 1-2 -2' 1-2 -G 6-12 -F'

[0202] The sides are designated F or F' and contain only 2'-modified nucleosides, such as LNA nucleosides. The preferred numbers and types of nucleosides within the alternating regions and regions F, G and F', D' and D" are described above.

[0203] In some embodiments, the oligonucleotide is a gapmer consisting of 16, 17, 18, 19, 20, 21, or 22 nucleotides in length, wherein each F region and F' region is independently composed of 1, 2, 3, or 4 modified nucleoside units that are complementary to a PD-L1 target nucleic acid, region G is composed of 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleoside units and is capable of recruiting a nuclease when duplexed with a PD-L1 target nucleic acid, and region D' is composed of two phosphodiester-linked DNA.

[0204] In further embodiments, the oligonucleotide is a gapmer in which each F region and F′ region is independently composed of 3, 4, 5, or 6 modified nucleoside units (e.g., nucleoside units containing 2′-O-methoxyethyl-ribose sugars (2′-MOE), or nucleoside units containing 2′-fluorodeoxyribose sugars, and / or LNA units, and region G is composed of 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleoside units (e.g., DNA units), or other nuclease-supplemented nucleosides (e.g., α-L-LNA), or a mixture of DNA and nuclease-supplemented nucleosides.

[0205] In a more specific embodiment, the oligonucleotide is a gapmer, in which each of the F and F' regions consists of two LNA units, and the G region consists of 12, 13, or 14 nucleoside units, preferably DNA units. Specific gapmer designs of this nature include 2-12-2, 2-13-2, and 2-14-2.

[0206] In more specific embodiments, the oligonucleotide is a gapmer in which each F and F' region is independently composed of three LNA units, and region G is composed of 8, 9, 10, 11, 12, 13, or 14 nucleoside units, preferably DNA units. Specific gapmer designs of this nature include 3-8-3, 3-9-3, 3-10-3, 3-11-3, 3-12-3, 3-13-3, and 3-14-3.

[0207] In a more specific embodiment, the oligonucleotide is a gapmer, with each F and F' region consisting of four LNA units, and region G consisting of 8, 9, 10, 11 or 12 nucleoside units, preferably DNA units. Specific gapmer designs of this nature include 4-8-4, 4-9-4, 4-10-4, 4-11-4 and 4-12-4.

[0208] Specific gapmer designs of this nature include a gap containing 6 nucleosides and, independently, 1 to 4 modified nucleosides within the wings, e.g., FG-F' designs selected from the group consisting of 1-6-1, 1-6-2, 2-6-1, 1-6-3, 3-6-1, 1-6-4, 4-6-1, 2-6-2, 2-6-3, 3-6-2, 2-6-4, 4-6-2, 3-6-3, 3-6-4, and 4-6-3 gapmers.

[0209] Specific gapmer designs of this nature include FG-F' designs, which have a gap containing 7 nucleosides and, independently, 1 to 4 modified nucleosides in the wings, e.g., selected from the group consisting of 1-7-1, 2-7-1, 1-7-2, 1-7-3, 3-7-1, 1-7-4, 4-7-1, 2-7-2, 2-7-3, 3-7-2, 2-7-4, 4-7-2, 3-7-3, 3-7-4, 4-7-3 and 4-7-4 gapmers.

[0210] Specific gapmer designs of this nature include FG-F' designs, which have a gap having 8 nucleosides and independently 1 to 4 modified nucleosides within the wings, e.g., selected from the group consisting of 1-8-1, 1-8-2, 1-8-3, 3-8-1, 1-8-4, 4-8-1, 2-8-1, 2-8-2, 2-8-3, 3-8-2, 2-8-4, 4-8-2, 3-8-3, 3-8-4, 4-8-3, and 4-8-4 gapmers.

[0211] Specific gapmer designs of this nature include FG-F' designs, which have a gap containing 9 nucleosides and, independently, 1 to 4 modified nucleosides in the wings, e.g., selected from the group consisting of 1-9-1, 2-9-1, 1-9-2, 1-9-3, 3-9-1, 1-9-4, 4-9-1, 2-9-2, 2-9-3, 3-9-2, 2-9-4, 4-9-2, 3-9-3, 3-9-4, 4-9-3 and 4-9-4 gapmers.

[0212] Specific gapmer designs of this nature include gaps containing 10 nucleosides, such as FG-F' designs selected from the group consisting of 1-10-1, 2-10-1, 1-10-2, 1-10-3, 3-10-1, 1-10-4, 4-10-1, 2-10-2, 2-10-3, 3-10-2, 2-10-4, 4-10-2, 3-10-3, 3-10-4, 4-10-3 and 4-10-4 gapmers.

[0213] Specific gapmer designs of this nature include gaps containing 11 nucleosides, such as FG-F' designs selected from the group consisting of 1-11-1, 2-11-1, 1-11-2, 1-11-3, 3-11-1, 1-11-4, 4-11-1, 2-11-2, 2-11-3, 3-11-2, 2-11-4, 4-11-2, 3-11-3, 3-11-4, 4-11-3 and 4-11-4 gapmers.

[0214] Specific gapmer designs of this nature include gaps containing 12 nucleosides, such as FG-F' designs selected from the group consisting of 1-12-1, 2-12-1, 1-12-2, 1-12-3, 3-12-1, 1-12-4, 4-12-1, 2-12-2, 2-12-3, 3-12-2, 2-12-4, 4-12-2, 3-12-3, 3-12-4, 4-12-3 and 4-12-4 gapmers.

[0215] Specific gapmer designs of this nature include gaps containing 13 nucleosides, such as FG-F' designs selected from the group consisting of 1-13-1, 2-13-1, 1-13-2, 1-13-3, 3-13-1, 1-13-4, 4-13-1, 2-13-2, 2-13-3, 3-13-2, 2-13-4, 4-13-2, 3-13-3, 3-13-4, 4-13-3 and 4-13-4 gapmers.

[0216] Specific gapmer designs of this nature include gaps containing 14 nucleosides, such as FG-F' designs selected from the group consisting of 1-14-1, 2-14-1, 1-14-2, 1-14-3, 3-14-1, 1-14-4, 4-14-1, 2-14-2, 2-14-3, 3-14-2, 2-14-4, 4-14-2, 3-14-3, 3-14-4, 4-14-3 and 4-14-4 gapmers.

[0217] Specific gapmer designs of this nature include gaps containing 15 nucleosides, such as the FG-F' designs selected from the group consisting of 1-15-1, 2-15-1, 1-15-2, 1-15-3, 3-15-1, 1-15-4, 4-15-1, 2-15-2, 2-15-3, 3-15-2, 2-15-4, 4-15-2 and 3-15-3 gapmers.

[0218] Specific gapmer designs of this nature include gaps containing 16 nucleosides, such as the FG-F' designs selected from the group consisting of 1-16-1, 2-16-1, 1-16-2, 1-16-3, 3-16-1, 1-16-4, 4-16-1, 2-16-2, 2-16-3, 3-16-2, 2-16-4, 4-16-2 and 3-16-3 gapmers.

[0219] Specific gapmer designs of this nature include gaps containing 17 nucleosides, such as the FG-F' designs selected from the group consisting of 1-17-1, 2-17-1, 1-17-2, 1-17-3, 3-17-1, 1-17-4, 4-17-1, 2-17-2, 2-17-3 and 3-17-2 gapmers.

[0220] In all instances, the FG-F' design further comprises regions D' and / or D", which may have 1, 2, or 3 nucleoside units (e.g., DNA units (e.g., 2 phosphodiester-linked DNA units). The nucleosides of regions F and F' are preferably modified nucleosides, and the nucleotides of region G are preferably unmodified nucleosides.

[0221] In each design, the preferred modified nucleoside is LNA.

[0222] In another embodiment, all of the internucleoside linkages in the gap in a gapmer are phosphorothioate and / or boranophosphate linkages. In another embodiment, all of the internucleoside linkages in the flanking regions (F and F' regions) in a gapmer are phosphorothioate and / or boranophosphate linkages. In another preferred embodiment, all of the internucleoside linkages in the D' and D" regions in a gapmer are phosphodiester linkages.

[0223] With respect to the specific gapmers disclosed herein, where a cytosine (C) moiety is annotated as 5-methyl-cytosine, in various embodiments, one or more of the Cs present in the oligonucleotide may be unmodified C moieties.

[0224] In certain embodiments, gapmers are so-called shortmers as described in WO 2008 / 113832, which is incorporated herein by reference.

[0225] Further gapmer designs are disclosed in WO 2004 / 046160, WO 2007 / 146511, which are incorporated by reference.

[0226] In certain embodiments of the invention, the oligonucleotide is selected from the group consisting of oligonucleotide compounds having CMP-ID numbers: 5_1 to 743_1 and 771_1.

[0227] In one embodiment of the invention, the oligonucleotides are selected from the group consisting of CMP-ID numbers 6_1, 8_1, 9_1, 13_1, 41_1, 42_1, 58_1, 77_1, 92_1, 111_1, 128_1, 151_1, 164_1, 166_1, 169_1, 171_1, 222_1, 233_1, 245_1, 246_1, 250_1, 251_1, 252_1, 253_1, 254_1, 255_1, 256_1, 257_1, 258_1, 259_1, 260_1, 261_1, 262_1, 263_1, 264_1, 265_1, 266_1, 267_1, 268_1, 269_1, 270_1, 271_1, 272_1, 273_1, 274_1, 275_1, 276_1, 277_1, 278_1, 279_1, 280_1, 281_1, 282_1, 283_1, 284_1, 285_1, 286_1, 287_1, 288_1, 289_1, 290_1, 300_1, 301_1, 302_1, 303_1, 304_1, 305_1, 306_1, 307_1, 308_1, 309_1, 31 1, 256_1, 272_1, 273_1, 287_1, 292_1, 303_1, 314_1, 318_1, 320_1, 324_1, 336_1, 342_1, 343_1, 344_1, 345_1, 346_1, 349_1, 359_1, 360_1, 374_1, 408_1, 409_1, 415_1, 417_1, 424_1, 429_1, 430_1, 458_1, 464_1, 466_1, 474_1, 490_1, 493_1, 512_1, 519_1, 519_1, 529_1, 533_1, 534_1, 547_1, 566_1, 567_1, 578_1, 582_1, 601_1, 619_1, 620_1, 636_1, 637_1, 638_1, 640_1, 645_1, 65 0_1, 651_1, 652_1, 653_1, 658_1, 659_1, 660_1, 665_1, 678_1, 679_1, 680_1, 682_1, 683_1, 684_1, 687_1, 694_1, 706_1, 716_1, 728_1, 733_1, 734_1, and 735_1.

[0228] In one preferred embodiment of the invention, the oligonucleotide has CMP-ID number: 287_1.

[0229] In another preferred embodiment of the invention, the oligonucleotide is CMP-ID number: 342_1.

[0230] In another preferred embodiment of the present invention, the oligonucleotide has CMP-ID number: 640_1.

[0231] In another preferred embodiment of the invention, the oligonucleotide has CMP-ID number: 466_1.

[0232] In another preferred embodiment of the invention, the oligonucleotide has CMP-ID number: 566_1.

[0233] In further embodiments of the present invention, the contiguous nucleotide sequences of the oligonucleotide motifs and oligonucleotide compounds of the present invention comprise a contiguous nucleotide sequence (e.g., region D') at the 5' end of two to four additional phosphodiester-linked nucleosides. In one embodiment, the nucleosides serve as a biocleavable linker (see the "Biocleavable Linkers" section). In a preferred embodiment, a cA (cytidine-adenosine) dinucleotide is linked via a phosphodiester bond to the 5' end of the contiguous nucleotide sequence (i.e., any one of the motif sequences or oligonucleotide compounds listed in Table 5). In a preferred embodiment, the double-stranded nucleotide is not complementary to the target sequence at the position where the remainder of the contiguous nucleotide is complementary.

[0234] In some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence is selected from the group consisting of motif sequences of SEQ ID NOs: 766, 767, 768, 769 and 770 nucleotides.

[0235] In some embodiments of the invention, the oligonucleotide is selected from the group consisting of oligonucleotide compounds having CMP-ID numbers 766_1, 767_1, 768_1, 769_1 and 770_1.

[0236] Carbohydrate-binding moiety Carbohydrate-binding moieties include, but are not limited to, galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6-phosphate. Carbohydrate conjugates can be used to enhance delivery or activity in a range of tissues, such as the liver and / or muscle. See, for example, EP 1495769; WO 99 / 65925; Yang et al., Bioconjug Chem (2009) 20(2): 213-21; Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10): 1401-17.

[0237] In some embodiments, the carbohydrate-binding moiety is multivalent, e.g., two, three, or four identical or non-identical carbohydrate moieties can be covalently attached to the oligonucleotide, optionally via a linker or linkers. In some embodiments, the invention provides a conjugate comprising an oligonucleotide of the invention and a carbohydrate-binding moiety.

[0238] In some embodiments, the binding moiety is or comprises mannose or mannose-6-phosphate, which is particularly useful for targeting muscle cells (see, e.g., U.S. Patent Application Publication No. 2012 / 122801).

[0239] Binding moieties capable of binding to the asialoglycoprotein receptor (ASGPr) are particularly useful for targeting hepatocytes within the liver. In some embodiments, the present invention provides oligonucleotide conjugates comprising an oligonucleotide of the present invention and an asialoglycoprotein receptor-targeting binding moiety. The asialoglycoprotein receptor-targeting binding moiety comprises one or more carbohydrate moieties capable of binding to the asialoglycoprotein receptor (ASPGr-binding carbohydrate moiety) with an affinity equal to or greater than that of galactose. The affinity of numerous galactose derivatives for the asialoglycoprotein receptor has been studied (see, e.g., Jobst, ST and Drickamer, K. JB.C. 1996, 271, 6686). Thus, these affinities are readily determined using methods typical in the art.

[0240] One aspect of the invention is an antisense oligonucleotide conjugate comprising: a) an oligonucleotide (Region A) comprising a contiguous nucleotide sequence 10-30 nucleotides in length that has at least 90% complementarity to a PD-L1 target nucleic acid; and b) at least one asialoglycoprotein receptor targeting binding moiety (Region C) covalently attached to the oligonucleotide in a). The oligonucleotide or contiguous nucleotide sequence can be as described in any of the sections "Oligonucleotides of the Invention," "Oligonucleotide Design," and "Gapmer Design."

[0241] In some embodiments, the asialoglycoprotein receptor-targeting binding moiety comprises at least one ASPGr-binding carbohydrate moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine. In some embodiments, the asialoglycoprotein receptor-targeting binding moiety is monovalent, divalent, trivalent, or tetravalent (i.e., comprises one, two, three, or four terminal carbohydrate moieties capable of binding to the asialoglycoprotein receptor). The asialoglycoprotein receptor-targeting binding moiety is bivalent, and even more preferably trivalent. In a preferred embodiment, the asialoglycoprotein receptor-targeting binding moiety comprises one to three N-acetylgalactosamine (GalNAc) moieties (also referred to as GalNAc conjugates). In some embodiments, the oligonucleotide conjugate comprises an asialoglycoprotein receptor targeting binding moiety that is a trivalent N-acetylgalactosamine (GalNAc) moiety. GalNAc conjugates are available in a variety of formats, including phosphodiester, methylphosphonate, and PNA antisense oligonucleotides (see, e.g., U.S. Pat. No. 5,994,517, and Hangeland et al., Bioconjug Chem. 1995 Nov-Dec; 6(6): 695-701, Biessen et al. 1999 Biochem J. 340, 783-792, and Maier et al. 2003 Bioconjug Chem 14, 18-29), siRNA (e.g., WO 2009 / 126933, WO 2012 / 089352, and WO 2012 / 083046), and LNA and 2'-MOE modified nucleosides (WO 2014 / 076196, WO 2014 / 207232, and WO 2014 / 179620), all of which are incorporated herein by reference.

[0242] To generate an asialoglycoprotein receptor-targeting binding moiety, an ASPGr-binding carbohydrate moiety (preferably GalNAc) is attached to the branched molecule via the C-1 carbon of the sugar. The ASPGr-binding carbohydrate moiety is preferably linked to the branched molecule via a spacer. A preferred spacer is a flexible, hydrophilic spacer (US Patent 5,885,968; Biessen et al., J. Med. Chem. 1995, Vol. 39, pp. 1538-1546). A preferred flexible, hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3 spacer (three ethylene units). The branched molecule can be any small molecule that allows for the attachment of two or three terminal ASPGr-binding carbohydrate moieties and further allows for the attachment of the branched point to an oligonucleotide. A typical branched molecule is a dilysine. The dilysine molecule contains three amine groups to which three ASPGr-binding carbohydrate moieties can be attached and a carboxyl-reactive group to which the dilysine can be attached to an oligonucleotide. Alternative branching molecules may be doublers or treblers, such as those supplied by Glen Research. In some embodiments, the branching molecule may be 1,3-bis-[5-(4,4'-pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (Glen Research catalog number: 10-1920-xx), tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (Glen Research catalog number: 10-1920-xx).

[0033] The compound may be selected from the group consisting of tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 1-[5-(4,4'-dimethoxytrityloxy)pentylamido]-3-[5-fluoromethoxycarbonyloxy-pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research Catalog Number: 10-1925-xx).The production of various GalNAc-linked moieties is described in International Publication No. WO 2014 / 179620 and International Application No. PCT / EP2015 / 073331, which are incorporated herein by reference. One or more linkers may be inserted between the branched molecule and the oligonucleotide. In a preferred embodiment, the linker is a biocleavable linker. The linker can be selected from those described in the "Linkers" section and its subsections.

[0243] The asialoglycoprotein receptor targeting binding moiety, particularly the GalNAc binding moiety, can be attached to the 3' or 5' end of the oligonucleotide using methods known in the art. In a preferred embodiment, the asialoglycoprotein receptor targeting binding moiety is linked to the 5' end of the oligonucleotide.

[0244] Pharmacokinetic modifiers relevant to siRNA delivery are described in WO 2012 / 083046 (incorporated herein by reference). In some embodiments, the carbohydrate-binding moiety comprises a pharmacokinetic modifier selected from the group consisting of a hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16-20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl and cholesterol. In a preferred embodiment, the pharmacokinetic modifier containing the carbohydrate-binding moiety is a GalNAc conjugate.

[0245] The carbohydrate-binding moiety preferably comprises one to three terminal ASPGr-linked carbohydrate moieties, preferably N-acetylgalactosamine moieties. In some embodiments, the carbohydrate-binding moiety comprises three ASPGr-linked carbohydrate moieties, preferably N-acetylgalactosamine moieties, linked to a branched molecule via a spacer. The spacer molecule can be 8 to 30 atoms in length. A preferred carbohydrate-binding moiety comprises three terminal GalNAc moieties linked to a di-lysine branched molecule via a PEG spacer. A preferred PEG spacer is a 3PEG spacer. A suitable asialoglycoprotein receptor-targeting binding moiety is shown in Figure 1. A suitable asialoglycoprotein receptor-targeting binding moiety is shown in Figure 3.

[0246] Other GalNAc-linked moieties include, for example, small peptides with an attached GalNAc moiety, such as Tyr-Glu-Glu-(aminohexylGalNAc)3 (YEE(ahGalNAc)3); glycotripeptides that bind to the asialoglycoprotein receptor on hepatocytes (see, e.g., Duff, et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose moieties (L3G4; Biessen, et al., Cardovasc. Med., 1999, 214); and cholan-based galactose moieties (e.g., the carbohydrate recognition motif of the asialoglycoprotein receptor).

[0247] In some embodiments of the invention, the antisense oligonucleotide conjugate is selected from the group consisting of the following CPM ID Nos: 766_2, 767_2, 768_2, 769_2 and 770_2.

[0248] In a preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound depicted in FIG.

[0249] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound depicted in FIG.

[0250] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound depicted in FIG.

[0251] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound depicted in FIG.

[0252] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound depicted in FIG.

[0253] Linker Biocleavable linker (region B) The use of conjugates is often associated with enhanced pharmacokinetic or pharmacodynamic properties. However, the presence of a linking moiety can interfere with the intended activity of the target oligonucleotide, for example, through steric hindrance that prevents hybridization or nuclease recruitment (e.g., RNase H). The use of a physiologically labile linkage (biocleavable linker) between the oligonucleotide (region A or region 1) and the linking moiety (region C or region 3) allows for improved properties due to the presence of the linking moiety, while ensuring that the linking group does not interfere with the effective activity of the oligonucleotide once in the target tissue.

[0254] Physiologically labile bonds spontaneously form when a molecule containing a pH-labile bond reaches the appropriate intracellular or extracellular environment. For example, a pH-labile bond may be cleaved when the molecule enters an acidified endosome. Thus, a pH-labile bond can be considered an endosomally cleavable bond. An enzyme-cleavable bond may be cleaved when exposed to an enzyme, such as an enzyme present in an endosome, lysosome, or cytoplasm. A disulfide bond may be cleaved when the molecule enters the highly reducing environment of the cytoplasm. Thus, a disulfide can be considered a cytoplasmic cleavable bond. As used herein, a pH-labile bond is a bond that is selectively broken under acidic conditions (pH<7). Because cellular endosomes and lysosomes have a pH below 7, such bonds may also be referred to as endosomally labile bonds.

[0255] For biocleavable linkers associated with binding moieties for targeted delivery, it is preferred that the cleavage rate observed in the target tissue (e.g., muscle, liver, kidney, or tumor) be greater than that observed in serum. Suitable methods for quantifying the percent cleavage level in serum or target tissue relative to cleavage by S1 nuclease are described in the "Materials and Methods" section. In some embodiments, the biocleavable linker (also referred to as a physiologically labile linker, or nuclease-sensitive linker or region B) is at least about 20%, such as at least about 30%, such as at least about 40%, at least about 50%, such as at least about 60%, such as at least about 70%, for example at least about 75% cleaved compared to a standard.

[0256] In some embodiments, the oligonucleotide conjugates of the invention comprise three regions: i) a first region (Region A) comprising 10-25 contiguous nucleotides complementary to a target nucleic acid; ii) a second region (Region B) comprising a biocleavable linker; and iii) a third region (Region C) comprising a binding moiety, such as an asialoglycoprotein receptor targeting binding moiety, wherein the third region is covalently linked to the second region, which is covalently linked to the first region.

[0257] In one embodiment of the present invention, the oligonucleotide conjugate comprises a biocleavable linker (region B) between the contiguous nucleotide sequence (region A) and the asialoglycoprotein receptor targeting binding moiety (region C).

[0258] In some embodiments, the biocleavable linker can be located at either the 5' and / or 3' end of the adjacent nucleotide complementary to the target nucleic acid (region A). In a preferred embodiment, the biocleavable linker is at the 5' end.

[0259] In some embodiments, the cleavable linker is susceptible to, for example, a nuclease that may be expressed in the target cell. In some embodiments, the biocleavable linker is composed of 2 to 5 consecutive phosphodiester bonds. The linker may also be a short region (e.g., 1 to 10, as detailed in the definition of linker) of phosphodiester-linked nucleosides. In some embodiments, the nucleosides in biocleavable linker region B are (optionally and independently) selected from the group consisting of DNA and RNA or modifications thereof that do not interfere with nuclease cleavage. Modifications of DNA and RNA nucleosides that do not interfere with nuclease cleavage can be non-naturally occurring nucleobases. Certain sugar-modified nucleosides, such as α-L-oxy-LNA, can also enable nuclease cleavage. In some embodiments, all nucleosides in region B (optionally and independently) contain either a 2'-OH ribose sugar (RNA) or a 2'-H sugar (i.e., RNA or DNA). In preferred embodiments, at least two consecutive nucleosides in region B are DNA or RNA nucleosides (e.g., at least 3, 4, or 5 consecutive DNA or RNA nucleotides). In even more preferred embodiments, the nucleosides in region B are preferably DNA nucleosides, such that region B consists of 1 to 5 or 1 to 4 (e.g., 2, 3, or 4 consecutive phosphodiester-linked DNA nucleosides). In preferred embodiments, region B is so short that RNase H is not recruited. In some embodiments, region B contains 3 or fewer or 4 or fewer consecutive phosphodiester-linked DNA and / or RNA nucleosides (e.g., DNA nucleosides).

[0260] When region B is composed of phosphodiester-linked nucleosides, regions A and B may together form an oligonucleotide linked to region C. In this context, region A can be distinguished from region B, and region A comprises at least one target nucleic acid (e.g., an LNA or nucleoside having a 2'-substituted sugar moiety) and two modified nucleosides with enhanced binding affinity to region A, which can themselves regulate expression of the target nucleic acid in relevant cell lines. Moreover, when region A comprises DNA or RNA nucleosides, these nucleosides are linked with nuclease-resistant internucleoside linkages, such as phosphorothioate or boranophosphate. On the other hand, region B comprises phosphate linkages between DNA or RNA nucleosides. In some embodiments, region B is not complementary to the target nucleic acid or contains at least a 50% mismatch.

[0261] In some embodiments, region B is not complementary to the target nucleic acid sequence or to consecutive nucleotides in region A that are complementary to the target nucleic acid.

[0262] In some embodiments, region B is complementary to the target nucleic acid sequence. In this regard, region A and region B together may form a single contiguous sequence that is complementary to the target sequence.

[0263] In some embodiments of the present invention, the internucleoside linkages between a first region (region A) and a second region (region B) may be considered part of the second region.

[0264] In some embodiments, the base sequence of Region B is selected to provide optimal endonuclease cleavage sites based on the predominant endonuclease cleavage enzymes present in the target tissue or cellular or subcellular compartment. In this regard, by isolating cellular extracts from target and non-target tissues, endonuclease cleavage sequences for use in Region B can be selected based on preferential cleavage activity in desired target cells (e.g., liver / hepatocytes) compared to non-target cells (e.g., kidney). In this regard, the efficacy of the compound for target downregulation can be optimized for the desired tissue / cell.

[0265] In some embodiments, region B comprises dinucleotides 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 can be substituted with U, and the internucleoside linkages are preferably phosphodiester linkages. In some embodiments, region B comprises the sequences 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 trinucleotides, wherein C may be 5-methylcytosine and / or T can be substituted for U, and wherein the internucleoside linkages are preferably phosphodiester linkages. In some embodiments, region B has the sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TA TX, TACX, TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX , CTTX, CCAX, CCTX, CCCX, CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX, and GGGX trinucleotides, where X can be selected from the group consisting of A, T, U, G, C and analogs thereof, C can be 5-methylcytosine, and / or T can be substituted with U. Preferably, the internucleoside linkage is a phosphodiester linkage.When referring to the (naturally occurring) nucleobases A, T, U, G, C, it is recognized that these can be substituted with nucleobase analogs that function as equivalent natural nucleobases (e.g., base pair with a complementary nucleoside).

[0266] Other linkers (region Y) The linker may have at least two functional groups (one attached to the oligonucleotide and the other attached to the binding moiety). Exemplary linker functional groups may be electrophilic for reacting with a nucleophilic group on the oligonucleotide or the binding moiety, or may be nucleophilic for reacting with an electrophilic group. In some embodiments, linker functional groups include, but are not limited to, phosphorothioate, phosphate, phosphite, unsaturation (e.g., double or triple bond), and the like. Some exemplary linkers (region Y) include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-caproic acid) (LCSMCC), succinimidyl m-maleimido-benzoylate (MBS), succinimidyl Examples of suitable linkers include aryl-Ne-maleimido-caproic acid (EMCS), succinimidyl 6-(beta-maleimido-propionamido)hexanoate (SMPH), succinimidyl N-(α-maleimidoacetate) (AMAS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), β-alanine (β-ALA), phenylglycine (PHG), 4-aminocyclohexanoic acid (ACHC), β-(cyclopropyl)alanine (β-CYPR), aminododecanoic acid (ADC), arylene diol, polyethylene glycol, amino acids, and the like. In some embodiments, the linker (region Y) comprises an aminoalkyl group, such as a C2-C36 aminoalkyl group, e.g., a C6-C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group. Aminoalkyl groups can be added to oligonucleotides (region A or region AB) as part of standard oligonucleotide synthesis, for example, using protected aminoalkyl phosphoramidites.The linking group between the aminoalkyl and the oligonucleotide can be, for example, phosphorothioate or phosphodiester, or one of the other nucleoside linking groups mentioned herein. The aminoalkyl group is covalently attached to the 5' or 3' end of the oligonucleotide. Commercially available aminoalkyl linkers are available, for example, 5'-amino-modifying enzyme C6, a 3'-amino-modifying reagent for linking at the 3' end of an oligonucleotide and for linking at the 5' end of an oligonucleotide. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds, or similar, were utilized by Krieg, et al., Antisense Research and Development 1991, 1, 161, to link fluorescein to the 5' end of an oligonucleotide. A wide variety of additional linker groups are known in the art and may be useful for attaching binding moieties to oligonucleotides. A review of many useful linker groups can be found, for example, in Antisense Research and Applications, ST Crooke and B. Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, pp. 303-350. Other compounds, such as acridine, are linked to the 3'-terminal phosphate group of oligonucleotides via a polymethylene bond (Asseline, et al., Proc. Natl. Acad. Sci. USA 1984, 81, 3297). All of the above groups can be used as a single linker (region Y) or in combination with one or more additional linkers (region Y-Y' or region YB or BY).

[0267] Linkers and their use in preparing oligonucleotide conjugates are described in detail in WO 96 / 11205, WO 98 / 52614, U.S. Pat. Nos. 4,948,882, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,580,731, 5,486,603, 5,608,046, 4,587,044, 4,667,025, and 5,254,469. Nos. 5,245,022, 5,112,963, 5,391,723, 5,510475, 5,512,667, 5,574,142, 5,684,142, 5,770,716, 6,096,875, 6,335,432 and 6,335,437, and throughout the art in WO 2012 / 083046, each of which is incorporated by reference in its entirety.

[0268] Manufacturing method In a further aspect, the present invention provides a method for producing an oligonucleotide of the invention, the method comprising reacting nucleotide units to form covalently linked consecutive nucleotide units contained within the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al., 1987, Methods in Enzymology, vol. 154, pages 287-313). In a further embodiment, the method further comprises reacting the consecutive nucleotide sequence with a binding moiety (ligand). In a further aspect, there is provided a method for producing a composition of the invention, the method comprising mixing an oligonucleotide or a linked oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or auxiliary.

[0269] Pharmaceutical Composition In a further aspect, the present invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). Pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate-buffered saline. In some embodiments, the oligonucleotide is used in a pharmaceutically acceptable diluent at a concentration of 50-300 μM.

[0270] Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a general review of drug delivery methods, see, for example, Langer (Science 249; 1527-1533, 1990). Further suitable and preferred examples of pharmaceutically acceptable diluents, carriers, and adjuvants are provided in WO 2007 / 031091 (incorporated herein by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also described in WO 2007 / 031091.

[0271] The oligonucleotides or oligonucleotide conjugates of the present invention can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The composition and method for the preparation of pharmaceutical compositions depend on many criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0272] These compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, and the lyophilized formulation may be combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8 (e.g., 7 to 7.5). Compositions obtained in solid form may be packaged in multiple single-dose units, each containing a fixed amount of the agent, such as a sealed package of tablets or capsules. Solid forms of the compositions may also be packaged in flexible-volume containers, such as squeeze tubes designed for topically applicable creams or ointments.

[0273] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is a prodrug, particularly for oligonucleotide conjugates, where the linking moiety is cleaved from the oligonucleotide when the prodrug is delivered to the active moiety (e.g., a target cell).

[0274] Purpose The oligonucleotides or oligonucleotide conjugates of the present invention can be used, for example, as diagnostic, therapeutic and prophylactic research reagents.

[0275] Studies have used such oligonucleotides or oligonucleotide conjugates to specifically modulate the synthesis of PD-L1 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or evaluation of its usefulness as a target for therapeutic intervention. Target modulation is typically achieved by degrading or inhibiting the mRNA that produces the protein, thereby preventing protein formation, or by degrading or inhibiting a regulator of the gene or mRNA that produces the protein.

[0276] When using the oligonucleotides of the invention in research or diagnostics, the target nucleic acid may be cDNA derived from DNA or RNA, or a synthetic nucleic acid.

[0277] The invention provides an in vivo or in vitro method for modulating PD-L1 expression in a target cell expressing PD-L1, said method comprising administering to said cell an effective amount of an oligonucleotide or oligonucleotide conjugate of the invention.

[0278] In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells may be in vitro cell cultures or in vivo cells forming part of a mammalian tissue. In a preferred embodiment, the target cells are present in the liver. Hepatocyte target cells can be selected from parenchymal cells (e.g., hepatocytes) and non-parenchymal cells such as Kupffer cells, LSECs, stellate cells (or Ito cells), cholangiocytes, and liver-associated leukocytes (including T cells and NK cells). In some embodiments, the target cells are antigen-presenting cells. Antigen-presenting cells present foreign antigens on their surface complexed with major histocompatibility complex (MHC) class I or class II. In some embodiments, antigen-presenting cells express MHC class II (i.e., professional antigen-presenting cells such as dendritic cells, macrophages, and B cells).

[0279] For diagnostic purposes, oligonucleotides can be used to detect and quantitate PD-L1 expression in cells and tissues by Northern blotting, in situ hybridization, or similar techniques.

[0280] The oligonucleotides or oligonucleotide conjugates of the invention, or pharmaceutical compositions thereof, can be administered to animals or humans suspected of having a disease or disorder that can be alleviated or treated by reducing the expression of PD-L1, particularly by reducing the expression of PD-L1 in liver target cells.

[0281] The present invention provides a method for treating or preventing a disease, comprising administering to a subject suffering from or prone to suffering from the disease a therapeutically or prophylactically effective amount of an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention.

[0282] The present invention also relates to the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention for use as a medicament.

[0283] The oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions according to the invention are typically administered in effective amounts.

[0284] The present invention also provides the use of the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention as described herein for the manufacture of a medicament for treating a disease or disorder, in one embodiment, the disease is a) viral hepatitis infection, such as HBV, HCV, and HDV; b) parasitic infection, such as malaria, toxoplasmosis, leishmaniasis, and trypanosomiasis; and c) liver cancer or liver metastasis.

[0285] In one embodiment, the present invention relates to an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition for use in treating a disease or disorder selected from a viral infection or a parasitic infection. In a further embodiment, the disease is selected from a) viral hepatitis infections such as HBV, HCV and HDV; b) parasitic infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; and c) liver cancer or liver metastasis.

[0286] The diseases or disorders referred to herein are associated with immune exhaustion. In particular, the diseases or disorders are associated with exhaustion of virus-specific T cell responses. In some embodiments, the diseases or disorders can be alleviated or treated by reducing PD-L1 expression.

[0287] The method of the present invention is preferably used for the purpose of treating or preventing diseases associated with immune exhaustion.

[0288] In one embodiment of the present invention, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is used to restore the immune response against liver cancer or liver metastasis.

[0289] In one embodiment of the present invention, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention is used to restore an immune response to a pathogen. In some embodiments, the pathogen can be found in the liver. The pathogen can be a virus or a parasite, particularly one described herein. In a preferred embodiment, the pathogen is HBV.

[0290] The present invention further relates to the use of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the restoration of immunity against a viral or parasitic infection as described herein.

[0291] The oligonucleotide or oligonucleotide conjugate, or pharmaceutical composition of the present invention can be used to treat viral infections, particularly viral infections in the liver that affect the PD-1 pathway (see, for example, Kapoor and Kottilil, 2014, Future Virol, Vol. 9, pp. 565-585; and Salem and El-Badawy, 2015, World J Hepatol, Vol. 7, pp. 2449-2458). The viral hepatitis infection can be selected from the group consisting of hepatitis viruses, particularly HBV, HCV, and HDV, particularly chronic forms of these infections. In one embodiment, the oligonucleotide or oligonucleotide conjugate, or pharmaceutical composition of the present invention is used to treat HBV, particularly chronic HBV. Indicators of chronic HBV infection are a high level of viral load (HBV DNA) and higher levels of empty HBs antigen particles in the circulation (100-fold excess over viral particles).

[0292] The oligonucleotide or oligonucleotide conjugate of the present invention can also be used to treat viral hepatitis infections that occur as co-infections with HIV.Other viral infections that can be treated with the oligonucleotide or oligonucleotide conjugate or pharmaceutical composition of the present invention include LCMV (lymphocytic choriomeningitis virus), as well as HIV, HSV-1, HSV-2, and other herpes viruses as single infections.These viruses are not hepatotrophic, but may be sensitive to PDL1 downregulation.

[0293] In some embodiments, restoration of immunity or immune response includes improved T cell and / or NK cell responses and / or alleviation of T cell depletion, particularly restoration of HBV-specific T cell responses, HCV-specific T cell responses, and / or HDV-specific T cell responses. Improved T cell responses can be assessed, for example, as an increase in T cells (particularly an increase in CD8+ and / or CD4+ T cells) in the liver compared to a control (e.g., pre-treatment levels or levels in vehicle-treated subjects). In further embodiments, virus-specific CD8+ T cells are restored or increased compared to a control, particularly HBV-specific CD8+ T cells, HCV-specific CD8+ T cells, or HDV-specific CD8+ T cells are restored or increased compared to a control. In a preferred embodiment, the method comprises administering an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition to HBV s antigen (HBsAg)-specific CD8+ T cells and / or HBV e antigen (HBeAg)-specific CD8+ T cells and / or HBV core antigen (HBcAg)-specific CD8+ T cells. Preferably, the HBV antigen-specific CD8+ T cells produce one or more cytokines, such as interferon gamma (IFN-γ) or tumor necrosis factor alpha (TNF-α). The increase in CD8+ T cells is particularly observed in the liver. The increase described herein should be statistically significant compared to a control. Preferably, the increase is at least 20%, e.g., 25%, e.g., 50%, e.g., 75%, compared to a control. In another embodiment, natural killer (NK) cells and / or natural killer T (NKT) cells are activated by the oligonucleotide or oligonucleotide conjugate of the present invention.

[0294] The oligonucleotide or oligonucleotide conjugate, or pharmaceutical composition of the present invention can be used to treat parasitic infections, particularly parasitic infections that affect the PD-1 pathway (see, for example, Bhadra et al. 2012 J Infect Dis vol 206 pp. 125-134; Bhadra et al. 2011 Proc Natl Acad Sci USA Vol. 108 pp. 9196-9201; Esch et al. J Immunol vol 191 pp 5542-5550; Freeman and Sharpe 2012 Nat Immunol Vol 13 pp. 113-115; Gutierrez et al. 2011 Infect Immun Vol 79 pp. 1873-1881; Joshi et al. 2009 PLoS Pathog Vol 5 e1000431; Liang et al. 2006 Eur J Immunol Vol. 36 pp 58-64; Wykes et al. 2014 Front Microbiol Vol 5 pp 249). The parasitic infection can be selected from the group consisting of malaria, toxoplasmosis, leishmaniasis and trypanosomiasis. Malaria infection is caused by protozoa of the genus Plasmodium, particularly the species P. vivax, P. malariae and P. falciparum. Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii. Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. Trypanosomiasis is caused by protozoa of the genus Trypanosoma. Tropical chaga disease is caused by the species Trypanosoma cruzi, and sleeping sickness is caused by the species Trypanosoma brucei.

[0295] In some embodiments, restoration of immunity involves restoration of parasite-specific T cell and NK cell responses, particularly Plasmodium-specific T cell responses, Toxoplasma gondii-specific T cell and NK cell responses, Leishmania-specific T cell and NK cell responses, Trypanosoma cruzi-specific T cell and NK cell responses, or Trypanosoma brucei-specific T cell and NK cell responses. In further embodiments, parasite-specific CD8+ T cell and NK cell responses are restored.

[0296] Administration The oligonucleotides or pharmaceutical compositions of the invention can be administered topically (to the skin, by inhalation, to the eye or ear) or enterally (such as orally or through the gastrointestinal tract) or parenterally (intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly or intrathecally).

[0297] In a preferred embodiment, the oligonucleotide or pharmaceutical composition of the invention is administered by a parenteral route, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intrathecal or intracranial, intracerebral or intracavitary, and intravitreal administration. In one embodiment, the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment, the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

[0298] In some embodiments, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention is administered at a dose of 0.1 to 15 mg / kg (e.g., 0.1 to 10 mg / kg, e.g., 0.2 to 10 mg / kg, e.g., 0.25 to 10 mg / kg, e.g., 0.1 to 5 mg / kg, e.g., 0.2 to 5 mg / kg, e.g., 0.25 to 5 mg / kg. Administration may be once a week, once every two weeks, once every three weeks, or once a month.

[0299] Combination therapy In some embodiments, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention is used in combination therapy with other therapeutic agents, which may be, for example, the standard of care for the above-mentioned diseases or disorders.

[0300] For the treatment of chronic HBV infection, a combination of antiviral drugs and immune system modulators is recommended. Antiviral drugs effective against HBV include nucleoside analogs. There are five nucleoside analogs approved for treatment.

[0301] Antiviral drugs for HBV, namely lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread), telbivudine (Tyzeka), and entecavir (Baraclude), are effective in suppressing viral replication but have no effect on HBsAg levels. Other antiviral drugs include ribavirin and HBV antibody therapy (monoclonal or polyclonal). Immune system modulators can be, for example, interferon α-2a and pegylated interferon α-2a (Pegasys), or TLR7 agonists (e.g., GS-9620), or therapeutic vaccines. IFN-α treatment has been shown to be very ineffective in reducing viral load, but this treatment results in a modest (less than 10% after 48 weeks of treatment) reduction in HBsAg, albeit very inefficiently.

[0302] The oligonucleotides or oligonucleotide conjugates of the invention can be used in combination with other antiviral agents effective against HBV, such as the antisense oligonucleotides described in WO 2012 / 145697 and WO 2014 / 179629, or the siRNA molecules described in WO 2005 / 014806, WO 2012 / 024170, WO 2012 / 2055362, WO 2013 / 003520 and WO 2013 / 159109.

[0303] When the oligonucleotides or oligonucleotide conjugates of the invention are administered in combination therapy with other agents, they can be administered sequentially or simultaneously to an individual. Alternatively, the pharmaceutical compositions of the invention can consist of a combination of the oligonucleotides or oligonucleotide conjugates of the invention in association with a pharmaceutically acceptable excipient as described herein and another therapeutic or prophylactic agent known in the art.

[0304] The following embodiments of the present invention can be used in conjunction with other embodiments described herein.

[0305] 1. An antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length that can reduce the expression of PD-L1.

[0306] 2. The oligonucleotide of embodiment 1, wherein the contiguous nucleotide sequence is at least 90% complementary to a PD-L1 target nucleic acid.

[0307] 3. The oligonucleotide of embodiment 1 or 2, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and / or SEQ ID NO:3.

[0308] 4. An oligonucleotide described in embodiments 1 to 3, wherein the contiguous nucleotide sequence is complementary to a region within positions 1 and 15720 on SEQ ID NO:1.

[0309] 5. The oligonucleotide of embodiments 1 to 4, wherein the oligonucleotide is capable of hybridizing to a target nucleic acid selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and / or SEQ ID NO:3, and has a ΔG° of less than 10 kcal.

[0310] 6. An oligonucleotide described in embodiments 1 to 5, wherein the contiguous nucleotide sequence is complementary to a subsequence of the target nucleic acid, and the subsequence is selected from the group consisting of positions 371-3068, 5467-12107, 15317-15720, 15317-18083, 15317-19511 and 18881-19494 on SEQ ID NO:1.

[0311] 7. The oligonucleotide of embodiment 6, wherein the subsequence is selected from the group consisting of positions 7300-7333, 8028-8072, 9812-9859, 11787-11873 and 15690-15735 on SEQ ID NO:1.

[0312] 8. The oligonucleotide of any one of embodiments 2 to 7, wherein the target nucleic acid is RNA.

[0313] 9. The oligonucleotide of embodiment 8, wherein the RNA is mRNA.

[0314] 10. The oligonucleotide of embodiment 9, wherein the mRNA is a precursor mRNA or a mature mRNA.

[0315] 11. The oligonucleotide according to embodiments 1 to 10, wherein the contiguous nucleotide sequence comprises or consists of at least 14 contiguous nucleotides, in particular 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides.

[0316] 12. The oligonucleotide according to any one of embodiments 1 to 10, wherein the contiguous nucleotide sequence comprises or consists of 16 to 20 nucleotides.

[0317] 13. The oligonucleotide according to any one of embodiments 1 to 10, wherein the oligonucleotide comprises or consists of a length of 14 to 35 nucleotides.

[0318] 14. The oligonucleotide of embodiment 13, wherein the oligonucleotide comprises or consists of a length of 18 to 22 nucleotides.

[0319] 15. The oligonucleotide of any one of embodiments 1 to 14, wherein the oligonucleotide or the contiguous nucleotide sequence is single-stranded.

[0320] 16. The oligonucleotide of embodiments 1 to 15, wherein the contiguous nucleotide sequence is complementary to a subsequence of a target nucleic acid, and the subsequence is selected from the group consisting of A7, A26, A43, A119, A142, A159, A160, A163, A169, A178, A179, A180, A189, A201, A202, A204, A214, A221, A224, A226, A243, A254, A258, A269, A274, A350, A360, A364, A365, A370, A372, A381, A383, A386, A389, A400, A427, A435 and A438.

[0321] 17. The oligonucleotide of embodiment 16, wherein the subsequence is selected from the group consisting of A221, A360, A180, A160 and A269.

[0322] 18. The oligonucleotide of embodiments 1 to 17, wherein the oligonucleotide is not an siRNA and is not self-complementary.

[0323] 19. The oligonucleotide of any one of embodiments 1 to 18, wherein the contiguous nucleotide sequence comprises or consists of a sequence selected from SEQ ID NOs: 5 to 743 or 771.

[0324] 20. The contiguous nucleotide sequence is SEQ ID NO:6, 8, 9, 13, 41, 42, 58, 77, 92, 111, 128, 151, 164, 166, 169, 171, 222, 233, 245, 246, 250, 251, 252, 256, 272, 273, 287, 292, 303, 314, 318, 320, 324, 336, 342, 343, 344, 345, 346, 349, 359, 360, 374, 408, 409, 415, 417, 424, 429, 430, 458, 464, 466, 47 20. The oligonucleotide of embodiment 1, comprising or consisting of a sequence selected from: 4, 490, 493, 512, 519, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636, 637, 638, 640, 645, 650, 651, 652, 653, 658, 659, 660, 665, 678, 679, 680, 682, 683, 684, 687, 694, 706, 716, 728, 733, 734, and 735.

[0325] 21. The oligonucleotide of embodiments 1 to 20, wherein the contiguous nucleotide sequence comprises or consists of a sequence selected from SEQ ID NOs: 466, 640, 342, 287 and 566.

[0326] 22. An oligonucleotide described in embodiments 1 to 21, wherein the consecutive nucleotide sequence has 0 to 3 mismatches compared to a target nucleic acid, and the consecutive nucleotides are complementary to the target nucleic acid.

[0327] 23. The oligonucleotide of embodiment 22, wherein the consecutive nucleotide sequence has one mismatch compared to the target nucleic acid.

[0328] 24. The oligonucleotide of embodiment 22, wherein the consecutive nucleotide sequence has two mismatches compared to the target nucleic acid.

[0329] 25. The oligonucleotide of embodiment 22, wherein the contiguous nucleotide sequence is perfectly complementary to the target nucleic acid sequence.

[0330] 26. The oligonucleotide of embodiments 1 to 25, comprising one or more modified nucleosides.

[0331] 27. The oligonucleotide of embodiment 26, wherein one or more modified nucleosides are high-affinity modified nucleosides.

[0332] 28. The oligonucleotide of embodiment 26 or 27, wherein one or more modified nucleosides are 2' sugar-modified nucleosides.

[0333] 29. The oligonucleotide of embodiment 28, wherein one or more 2' sugar-modified nucleosides are 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.

[0334] 30. The oligonucleotide of embodiment 28, wherein one or more modified nucleosides are LNA nucleosides.

[0335] 31. The oligonucleotide of embodiment 30, wherein the modified LNA nucleoside is oxy-LNA.

[0336] 32. The oligonucleotide of embodiment 31, wherein the modified nucleoside is β-D-oxy-LNA.

[0337] 33. The oligonucleotide of embodiment 30, wherein the modified nucleoside is thioLNA.

[0338] 34. The oligonucleotide of embodiment 30, wherein the modified nucleoside is amino-LNA.

[0339] 35. The oligonucleotide of embodiment 30, wherein the modified nucleoside is cET.

[0340] 36. The oligonucleotide of embodiment 30, wherein the modified nucleoside is ENA.

[0341] 37. The oligonucleotide of embodiment 30, wherein the modified LNA nucleoside is selected from α-L-oxy-LNA, β-D-amino-LNA, α-L-amino-LNA, β-D-thio-LNA, α-L-amino-LNA, thio-LNA, (S)cET, (R)cETβ-D-ENA and α-L-ENA.

[0342] 38. The oligonucleotide of embodiments 30 to 37, wherein, in addition to the modified LNA nucleoside, at least one 2'-substituted modified nucleoside is present.

[0343] 39. The oligonucleotide of embodiment 38, wherein the 2'-substituted modified nucleoside is selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-DNA, and 2'-fluoro-ANA.

[0344] 40. The oligonucleotide of any one of embodiments 1 to 39, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

[0345] 41. The oligonucleotide of embodiment 40, wherein the modified internucleoside linkages are nuclease-resistant.

[0346] 42. The oligonucleotide of embodiment 40 or 41, wherein at least 50% of the internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate internucleoside linkages or boranophosphate internucleoside linkages.

[0347] 43. The oligonucleotide of embodiment 40 or 41, wherein all internucleoside linkages in the consecutive nucleotide sequence are phosphorothioate internucleoside linkages.

[0348] 44. The oligonucleotide of embodiments 1 to 43, wherein the oligonucleotide has the ability to recruit RNase H.

[0349] 45. The oligonucleotide of embodiment 44, wherein the oligonucleotide is a gapmer.

[0350] 46. ​​The oligonucleotide of embodiment 44 or 45, wherein the oligonucleotide is a gapmer of formula 5'-FG-F'-3', wherein regions F and F' independently comprise or consist of 1 to 7 modified nucleosides, and G is a region of 6 to 16 nucleosides that has the ability to recruit RNase H.

[0351] 47. The oligonucleotide of embodiment 44 or 45, wherein the gapmer has the formula 5'-D'-FG-F'-3' or 5'-FG-F'-D"-3', wherein regions F and F' independently comprise 1 to 7 modified nucleosides, G is a region of 6 to 16 nucleosides that has the ability to recruit RNase H, and region D' or D" comprises 1 to 5 phosphodiester linked nucleosides.

[0352] 48. The oligonucleotide of embodiment 47, wherein D' or D" is optional.

[0353] 49. The oligonucleotide of embodiment 47, wherein region D' consists of two phosphodiester-linked nucleosides.

[0354] 50. The oligonucleotide of embodiment 49, wherein the phosphodiester-linked nucleoside is ca (cytidine-adenosine).

[0355] 51. The oligonucleotide of embodiment 46 or 47, wherein the modified nucleoside is a 2' sugar-modified nucleoside and is 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.

[0356] 52. The oligonucleotide of embodiments 46 to 51, wherein one or more of the modified nucleosides in the F and F' regions is an LNA nucleoside.

[0357] 53. The oligonucleotide of embodiment 52, wherein all modified nucleosides in the F and F' regions are LNA nucleosides.

[0358] 54. The oligonucleotide of embodiment 53, wherein the F and F' regions consist of LNA nucleosides.

[0359] 55. The oligonucleotide of embodiments 52 to 54, wherein all modified nucleosides in the F and F' regions are oxy-LNA nucleosides.

[0360] 56. The oligonucleotide of embodiment 52, wherein at least one of regions F or F' further comprises at least one 2'-substituted modified nucleoside 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, and 2'-fluoro-DNA.

[0361] 57. The oligonucleotide of any one of embodiments 46 to 56, wherein the RNase H-repleting nucleosides in region G are independently selected from DNA, α-L-LNA, C4'-alkylated DNA, ANA and 2'F-ANA and UNA.

[0362] 58. The oligonucleotide of embodiment 57, wherein the nucleosides in region G are DNA and / or α-L-LNA nucleosides.

[0363] 59. The oligonucleotide of embodiment 57 or 58, wherein region G consists of at least 75% DNA nucleosides.

[0364] 60. The oligonucleotide of embodiment 1 to 59, wherein the oligonucleotide is selected from any one of CMP ID numbers: 5_1 to 743_1 and 771_1 (Table 5).

[0365] 61. Oligonucleotides are CMP ID numbers: 6_1, 8_1, 9_1, 13_1, 41_1, 42_1, 58_1, 77_1, 92_1, 111_1, 128_1, 151_1, 164_1, 166_1, 169_1, 171_1, 222_1, 233_1, 245_1, 246_1, 250_1, 251_1, 252_1, 256_1, 272_1, 273_1, 287_ 1, 292_1, 303_1, 314_1, 318_1, 320_1, 324_1, 336_1, 342_1, 343_1, 344_1, 345_1, 346_1, 349_1, 359_1, 360_1, 374_1, 408_1, 409_1, 415_1, 417_1, 424_1, 429_1, 430_1, 458_1, 464_1, 4 66_1, 474_1, 490_1, 493_1, 512_1, 519_1, 519_1, 529_1, 533_1, 534_1, 547_1, 566_1, 567_1, 578_1, 582_1, 601_1, 619_1, 620_1, 636_1, 637_1, 638_1, 640_1, 645_1, 650_1, 651_1, 652_ 61. The oligonucleotide of embodiment 1, selected from the group consisting of: 1, 653_1, 658_1, 659_1, 660_1, 665_1, 678_1, 679_1, 680_1, 682_1, 683_1, 684_1, 687_1, 694_1, 706_1, 716_1, 728_1, 733_1, 734_1, and 735_1.

[0366] 62. The oligonucleotide described in embodiments 1 to 61, wherein the oligonucleotide is selected from the group consisting of CMP ID numbers: 287_1, 342_1, 466_1, 640_1, 566_1, 766_1, 767_1, 768_1, 769_1 and 770_1.

[0367] 63. a. an oligonucleotide according to any one of claims 1 to 62 (region A); b. at least one binding moiety (region C) covalently attached to said oligonucleotide; An antisense oligonucleotide conjugate comprising:

[0368] 64. The oligonucleotide conjugate of embodiment 63, wherein the binding moiety is selected from a carbohydrate, a cell surface receptor ligand, a drug substance, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin, a vitamin, a viral protein, or a combination thereof.

[0369] 65. The oligonucleotide conjugate of embodiment 63 or 64, wherein the binding moiety is a carbohydrate containing moiety.

[0370] 66. The oligonucleotide conjugate of embodiment 65, wherein the carbohydrate-binding moiety comprises at least one asialoglycoprotein receptor targeting moiety and is covalently attached to the oligonucleotide of any one of claims 1 to 62.

[0371] 67. The oligonucleotide conjugate of embodiment 66, wherein the asialoglycoprotein receptor targeting binding moiety comprises at least one carbohydrate moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine.

[0372] 68. The oligonucleotide conjugate of embodiment 66 or 67, wherein the asialoglycoprotein receptor targeting binding moiety is monovalent, bivalent, trivalent, or tetravalent.

[0373] 69. The oligomeric conjugate described in embodiment 68, wherein the asialoglycoprotein receptor targeting binding moiety consists of two to four terminal GalNAc moieties and a PEG spacer connecting each GalNAc moiety to the branched molecule.

[0374] 70. The oligonucleotide conjugate of embodiments 66 to 69, wherein the asialoglycoprotein receptor targeting binding moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety.

[0375] 71. The oligonucleotide conjugate of embodiments 66 to 70, wherein the linking moiety is selected from any one of the trivalent GalNAc moieties in Figure 1.

[0376] 72. The oligonucleotide conjugate of embodiment 71, wherein the binding moiety is a trivalent GalNAc moiety as shown in Figure 3.

[0377] 73. The oligonucleotide conjugate of embodiments 63 to 72, wherein a linker is present between the oligonucleotide or consecutive oligonucleotide sequence and the binding moiety.

[0378] 74. The oligonucleotide conjugate of embodiment 73, wherein the linker is a physiologically labile linker (region B).

[0379] 75. The oligonucleotide conjugate of embodiment 74, wherein the physiologically labile linker is a nuclease-sensitive linker.

[0380] 76. The oligonucleotide conjugate of embodiment 74 or 75, wherein the physiologically labile linker is composed of 2 to 5 consecutive phosphodiester bonds.

[0381] 77. The oligonucleotide conjugate according to embodiment 76, wherein the physiologically labile linker is equal to region D' or D" present in embodiments 47 to 50.

[0382] 78. An oligonucleotide conjugate described in any one of embodiments 63 to 77, wherein the oligonucleotide conjugate is selected from CMP ID numbers: 766_2, 767_2, 768_2, 769_2 and 770_2.

[0383] 79. The oligonucleotide conjugate of embodiment 78, wherein the oligonucleotide conjugate is selected from the oligonucleotide conjugates depicted in Figures 4, 5, 6, 7, and 8.

[0384] 80. The oligonucleotide conjugate of embodiments 63 to 76, which exhibits improved inhibition of PD-L1 in target cells, or improved cellular distribution between the liver and spleen, or improved cellular uptake of the conjugated oligonucleotide into the liver, compared to the unconjugated oligonucleotide.

[0385] 81. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 1 to 62, or a conjugate according to any one of embodiments 63 to 80, and a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant.

[0386] 82. A method for producing an oligonucleotide according to any one of embodiments 1 to 62, comprising reacting nucleotide units, thereby forming covalently linked consecutive nucleotide units contained within the oligonucleotide.

[0387] 83. The method of embodiment 82, further comprising reacting the contiguous nucleotide sequence with a non-nucleotide binding moiety.

[0388] 84. A method for producing the composition described in embodiment 81, comprising mixing the oligonucleotide with a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant.

[0389] 85. An in vivo or in vitro method for modulating PD-L1 expression in a target cell expressing PD-L1, said method comprising administering to said cell an effective amount of an oligonucleotide described in embodiments 1 to 62, or a conjugate described in embodiments 63 to 80, or a pharmaceutical composition described in embodiment 81.

[0390] 86. A method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide described in embodiments 1 to 62, or a conjugate described in embodiments 63 to 80, or a pharmaceutical composition described in embodiment 81 to a subject suffering from or susceptible to the disease.

[0391] 87. A method for restoring immunity against a virus or parasite, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide conjugate described in embodiments 63 to 80, or an oligonucleotide described in embodiments 1 to 62, or a pharmaceutical composition described in embodiment 81 to a subject infected with the virus or parasite.

[0392] 88. The method of embodiment 87, wherein the restoration of immunity is an increase in CD8+ T cells specific for one or more HBV antigens in the liver compared to a control.

[0393] 89. An oligonucleotide according to any one of embodiments 1 to 62, or a conjugate according to any one of embodiments 63 to 80, or a pharmaceutical composition according to embodiment 81, for use as a medicament for the treatment or prevention of a disease in a subject.

[0394] 90. Use of an oligonucleotide according to embodiments 1 to 62 or a conjugate according to embodiments 63 to 80 for the preparation of a medicament for the treatment or prevention of a disease in a subject.

[0395] 91. An oligonucleotide according to embodiments 1 to 62, or a conjugate according to embodiments 63 to 80, or a pharmaceutical composition according to embodiment 81, for use in restoring immunity to viruses or parasites.

[0396] 92. The use described in embodiment 91, wherein the restoration of immunity is an increase in CD8+ T cells specific for one or more HBV antigens in the liver compared to a control.

[0397] 93. The use according to embodiment 92, wherein the HBV antigen is HBs antigen.

[0398] 94. The method, oligonucleotide or use according to embodiments 86 to 93, wherein the disease is associated with the in vivo activity of PD-L1.

[0399] 95. The method, oligonucleotide or use according to embodiments 86 to 94, wherein the disease is associated with increased expression of PD-L1 on antigen-presenting cells.

[0400] 96. The method, oligonucleotide or use according to embodiment 95, wherein PD-L1 is reduced by at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% compared to the expression in the absence of or before treatment with the oligonucleotide according to embodiments 1 to 62, or the conjugate according to embodiments 63 to 80, or the pharmaceutical composition according to embodiment 81.

[0401] 97. The method, oligonucleotide or use according to embodiments 86 to 95, wherein the disease is selected from a viral hepatitis infection or a parasitic infection.

[0402] 98. The method, oligonucleotide, or use according to embodiment 98, wherein the viral infection is HBV, HCV, or HDV.

[0403] 99. The method, oligonucleotide, or use of embodiments 86 to 95, wherein the disease is chronic HBV.

[0404] 100. The method, oligonucleotide, or use of embodiment 98, wherein the parasitic infection is malaria, toxoplasmosis, leishmaniasis, or trypanosomiasis.

[0405] 101. The method, oligonucleotide, or use of embodiments 86 to 100, wherein the subject is a mammal.

[0406] 102. The method, oligonucleotide, or use of embodiment 101, wherein the mammal is a human. [Example]

[0407] Materials and Methods Motif sequences and oligonucleotide compounds

[0408] [Table 5-1]

[0409] [Table 5-2]

[0410] [Table 5-3]

[0411] [Table 5-4]

[0412] [Table 5-5]

[0413] [Table 5-6]

[0414] Table 5-7

[0415] Table 5-8

[0416] Table 5-9

[0417] Table 5-10

[0418] Table 5-11

[0419] Table 5-12

[0420] Table 5-13

[0421] Table 5-14

[0422] Table 5-15

[0423] Table 5-16

[0424] [Table 5-17]

[0425] [Table 5-18]

[0426] [Table 5-19]

[0427] The motif sequence represents the consecutive sequence of nucleobases present in the oligonucleotide.

[0428] The design refers to gapmer design FG-F'. Each number represents the number of consecutive modified nucleosides. It includes a 2'-modified nucleoside (first number = 5' flank), followed by the number of DNA nucleosides (second number = gap region), followed by the number of modified nucleosides (e.g., 2'-modified nucleosides (third number = 3' flank) preceded or followed by an additional repeated region of DNA and LNA that is not part of the contiguous sequence complementary to the target nucleic acid.

[0429] The oligonucleotide compounds represent specific designs of motif sequences. Capital letters represent β-D-oxy LNA nucleosides, and lowercase letters represent DNA nucleosides. All LNA C's are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages.

[0430] [Table 6]

[0431] The motif sequence represents the consecutive sequence of nucleobases present in the oligonucleotide.

[0432] The design refers to gapmer design FG-F'. Each number represents the number of consecutive modified nucleosides. It includes a 2'-modified nucleoside (first number = 5' flank), followed by the number of DNA nucleosides (second number = gap region), followed by the number of modified nucleosides (e.g., 2'-modified nucleosides (third number = 3' flank) preceded or followed by an additional repeated region of DNA and LNA that is not part of the contiguous sequence complementary to the target nucleic acid.

[0433] The oligonucleotide compounds represent specific designs of motif sequences. Capital letters represent β-D-oxy LNA nucleosides, and lowercase letters represent DNA nucleosides. All LNA C's are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages.

[0434] [Table 7]

[0435] Uppercase letters represent β-D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides. All LNA C's are 5-methylcytosine, the subscript o represents a phosphodiester internucleoside linkage, and other internucleoside linkages are phosphorothioate internucleoside linkages unless otherwise specified.

[0436] [Table 8]

[0437] GN2 represents the trivalent GalNAc group shown in Figure 3, C6 represents a six-carbon aminoalkyl group, uppercase letters represent β-D-oxy LNA nucleosides, and lowercase letters represent DNA nucleosides. All LNA C's are 5-methylcytosine, the subscript o represents a phosphodiester nucleoside linkage, and unless otherwise specified, the internucleoside linkages are phosphorothioate internucleoside linkages. Chemical diagrams representing some molecules are shown in Figures 4 through 8.

[0438] AAV / HBV mouse model Pasteur Model: HLA-A2.1 / HLA-DR1 transgenic H-2 class I / class II knockout mice (herein referred to as HLA-A2 / DR1 mice) were generated and bred at the Institut Pasteur. These mice represent an in vivo experimental model for studying human immune function without any interference with mouse MHC responses (Pajot et al. 2004 Eur J Immunol. 34(11): 3060-9).

[0439] These studies used adeno-associated virus (AAV) vectors, AAV serotype 2 / 8 carrying a replication-competent HBV DNA genome. AAV-HBV vectors (batch GVPN#6163) were injected at 5 × 10 11 The diluted solution was diluted with sterile phosphate-buffered saline (PBS) to reach a titer of 5 x 10 mg / mL. 100 μL of this diluted solution (dose / mouse: 5 x 10 10 AAV2 / 8-HBV (vg) was intravenously (iv) injected into the tail vein of mice. Intact virus particles containing HBV DNA were detected in the blood of HBV-carrier mice. HBcAg, along with the circulating HBV proteins HBeAg and HBsAg, was detected in the liver for up to one year. In all AAV2 / 8-HBV-transduced mice, serum HBsAg, HBeAg, and HBV DNA persisted for at least one year (Dion et al. 2013 J Virol 87: 5554-5563).

[0440] Shanghai Model: In this model, mice infected with a recombinant adeno-associated virus (AAV) carrying the HBV genome (AAV / HBV) maintain stable viremia and antigenemia for more than 30 weeks (Dan Yang, et al. 2014 Cellular & Molecular Immunology 11, 71-78).

[0441] Specific pathogen-free male C57BL / 6 mice (4–6 weeks old) were purchased from SLAC (Shanghai Laboratory Animal Center of the Chinese Academy of Sciences) and housed in individually ventilated cages in the animal care facility. Animal care and use guidelines as dictated by the WuXi IACUC (Institutional Animal Care and Use Committee, WUXI IACUC protocol number R20131126-Mouse) were adhered to. Mice were allowed to acclimate to the new environment for 3 days and then classified according to the experimental design.

[0442] The recombinant AAV-HBV was diluted in PBS (200 μL per injection). This recombinant virus carries 1 to 3 copies of the HBV genome (genotype D, serotype ayw).

[0443] On day 0, all mice were injected with 200 μL of AAV-HBV into the tail vein. On days 6, 13, and 20 after AAV injection, all mice were bled submandibularly (0.1 ml blood / mouse) and serum was collected. On day 22 after injection, mice with stable viremia were ready for oligonucleotide treatment. Oligonucleotides could be unconjugated or GalNAc-conjugated.

[0444] DNA vaccines Plasmid DNA was endotoxin-free and produced at Plasmid-Factory (Germany). pCMV-S2.S encodes the pre-S2 and S domains of HBsAg (genotype D), and its expression is controlled by the cytomegalovirus immediate-early gene promoter (Michel et al. 1995 Proc Natl Acad Sci USA 92: 5307-5311). pCMV-HBc encodes the HBV capsid carrying the hepatitis core (HBc) antigen (Dion et al. 2013 J Virol 87: 5554-5563).

[0445] DNA vaccine treatment was performed as described herein. Five days before vaccination, mice were injected intramuscularly with cardiotoxin (CaTx, Latoxan ref. L81-02, 50 μl / muscle). CaTx depolarizes muscle fibers, inducing cell degeneration. New muscle fibers appear five days after injection, improving the transfection efficacy of DNA vaccines. pCMV-S2.S ayw and pCMVCore were mixed at equal volumes (1 mg / ml each) and administered to each mouse via bilateral intramuscular injections into the cardiotoxin-treated tibialis anterior muscles under anesthesia (100 μL of 12.5 mg / mL ketamine, 1.25 mg / mL xylazine) as previously described by Michel et al. (1995) Proc Natl Acad Sci USA 92:5307-5311.

[0446] Anti-PD-L1 antibody This was a mouse anti-mouse PD-L1 IgG1 antibody clone 6E11 produced internally at Genetech, a surrogate antibody that blocks atezolizumab and has similar in vitro blocking activity to atezolizumab produced internally at Roche. The antibody was administered by intraperitoneal (ip) injection at a dose of 12.5 μg / g.

[0447] Oligonucleotide synthesis Oligonucleotide synthesis is generally known in the art. Below are applicable protocols. The oligonucleotides of the present invention may be prepared by methods that vary slightly in terms of the equipment, supports, and concentrations used.

[0448] Oligonucleotides are synthesized on a uridine universal support at a 1 μmol scale using the phosphoramidite method on an Oligomaker 48. At the end of synthesis, the oligonucleotides are cleaved from the solid support using aqueous ammonia at 60 °C for 5-16 h. Oligonucleotides are purified by reverse-phase HPLC (RP-HPLC) or solid-phase extraction and characterized by UPLC, and molecular weights are further confirmed by ESI-MS.

[0449] Oligonucleotide extension: Coupling of β-cyanoethyl phosphoramidites (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), or LNA-T) was carried out using a 0.1 M solution of 5'-O-DMT-protected amidite in acetonitrile and a 0.25 M solution of DCI (4,5-dicyanoimidazole) in acetonitrile as activators. For the final cycle, a phosphoramidite bearing the desired modification can be used. This is a C6 linker for attaching the linking group, or linking group. Thiolation for the introduction of phosphorothioate bonds is carried out using xanthan gum (0.01 M in acetonitrile / pyridine 9:1). Phosphodiester bonds can be introduced using 0.02 M iodine in THF / pyridine / water (7:2:1). The remaining reagents are those typically used in the synthesis of oligonucleotides.

[0450] For post-solid-phase synthesis conjugation, commercially available C6 amino linker phosphoramidites can be used in the last cycle of solid-phase synthesis, allowing isolation of the amino-linked, deprotected oligonucleotide after deprotection and cleavage from the solid support. Standard synthetic methods are used to introduce the conjugate via activation of functional groups.

[0451] Alternatively, as described in International Application PCT / EP2015 / 073331 or European Patent Application Publication No. 15194811.4, phosphoramidites of GalNAc- or GalNAc-groups can be used to add the linking moiety to the oligonucleotide while it is still on the solid support.

[0452] Purification by RP-HPLC: The crude compound is purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μ 150 x 10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile are used as buffers at a flow rate of 5 mL / min. The collected fractions are lyophilized to give the purified compound, typically as a white solid.

[0453] Abbreviation: DCI: 4,5-dicyanoimidazole DCM: dichloromethane DMF: dimethylformamide DMT: 4,4'-dimethoxytrityl THF: tetrahydrofuran Bz: benzoyl Ibu: Isobutyryl RP-HPLC: reversed-phase high-performance liquid chromatography

[0454] T m Assay The oligonucleotide and RNA target (phosphate-linked, PO) duplexes were diluted to 3 mM in 500 ml of RNase-free water and diluted with 500 ml of 2xT m The resulting mixture is mixed with a buffer solution (200 mM NaCl, 0.2 mM EDTA, 20 mM sodium phosphate, pH 7.0). The solution is heated to 95°C for 3 minutes and then allowed to anneal at room temperature for 30 minutes. The duplex melting temperature (T mThe temperature is increased from 20°C to 95°C, then decreased to 25°C, and the absorbance is recorded at 260 nm. The first derivative and both the melting and annealing maxima are used to determine the duplex T m Evaluate.

[0455] Tissue-specific in vivo linker cleavage assay FAM-labeled oligonucleotides bearing biocleavable linkers to be tested (e.g., DNA phosphodiester linkers (PO linkers)) are cleaved in vitro using homogenates of relevant tissues (e.g., liver or kidney) and serum.

[0456] Tissue and serum samples were collected from suitable animals (e.g., mice, monkeys, pigs, or rats) and homogenized in homogenization buffer (0.5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjusted with 1 N NaOH)). Tissue homogenates and serum were spiked with oligonucleotides to a concentration of 200 μg / g tissue. Samples were incubated at 37°C for 24 hours, after which the samples were extracted with phenol-chloroform. AIE HPLC analysis of this solution was performed on a Dionex Ultimate 3000 using a Dionex DNApac p-100 column and a gradient of 10 mM to 1 M sodium perchlorate (pH 7.5). The content of cleaved and uncleaved oligonucleotides was quantified relative to standards using both a fluorescence detector (615 nm) and a UV detector (260 nm).

[0457] S1 nuclease sensitivity assay FAM-labeled oligonucleotides with S1 nuclease-sensitive linkers (eg, DNA phosphodiester linkers (PO linkers)) are cleaved in vitro in S1 nuclease extracts or serum.

[0458] In vitro cleavage of 100 μM oligonucleotides was performed with S1 nuclease in nuclease buffer (60 U / 100 μL) for 20 and 120 min. Enzyme activity was terminated by adding EDTA to the buffer. AIE HPLC analysis of this solution was performed on a Dionex Ultimate 3000 using a Dionex DNApac p-100 column and a gradient of 10 mM to 1 M sodium perchlorate (pH 7.5). The content of cleaved and uncleaved oligonucleotides was quantified against standards using both a fluorescence detector (615 nm) and a UV detector (260 nm).

[0459] Preparation of liver mononuclear cells Hepatocytes from AAV / HBV mice were prepared using the method described by Tupin et al. (2006) Methods Enzymol 417: 185-201, with minor modifications. After euthanasia, the livers of mice were perfused with 10 ml of sterile PBS via the portal vein using a syringe with a G25 needle. When the organs were pale, they were harvested in Hank's Balanced Salt Solution (HBSS) (GIBCO® HBSS, 24020) + 5% fetal calf serum (FCS). The harvested liver was gently pressed through a 100 μm cell strainer (BD Falcon, 352360) to suspend the cells in 30 ml of HBSS + 5% FCS. The cell suspension was centrifuged at 50 g for 5 minutes, and the supernatant was centrifuged at 289 g for 10 minutes at 4°C. After centrifugation, the supernatant was discarded, and the pellet was resuspended in 35% isotonic Percoll solution (GE Healthcare Percoll #17-0891-01, diluted in RPMI 1640 (GIBCO, 31870)) at room temperature in 15 ml portions and transferred to a 15 ml tube. The cells were further centrifuged at 1360 g for 25 minutes at room temperature. The supernatant was discarded by aspiration, and the pellet containing the mononuclear cells was washed twice with HBSS + 5% FCS.

[0460] Cells were cultured in complete medium consisting of alpha minimal essential medium (Gibco, 22571) supplemented with 10% FCS (Hyclone, #SH30066, lot APG21570), 100 U / mL penicillin + 100 μg / mL streptomycin + 0.3 mg / mL L-glutamine (Gibco, 10378), 1× non-essential amino acids (Gibco, 11140), 10 mM Hepes (Gibco, 15630), 1 mM sodium pyruvate (Gibco, 11360), and 50 μM β-mercaptoethanol (LKB, 1830).

[0461] Cell surface labeling Cells were seeded into U-bottom 96-well plates and washed with PBS FACS (PBS containing 1% bovine serum albumin and 0.01% sodium azide). Cells were then incubated with 5 μL of PBS FACS containing rat anti-mouse CD16 / CD32 antibody and the viability marker LD Fixable Yellow (Thermofisher, L34959) for 10 min in the dark at 4°C. Cells were then stained for 20 min in the dark at 4°C with 25 μL of PBS FACS containing monoclonal antibodies (MAbs) against NK P46 BV421 (rat MAb anti-mouse NK P46, Biolegend, 137612) and F4 / 80 (rat MAb anti-mouse F4 / 80 FITC, BD Biolegend, 123108), and two complementary surface markers: PDL1 (rat MAb anti-mouse PD1 PE, BD Biosciences, 551892) and PDL1 (rat MAb anti-mouse PDL1 BV711, Biolegend, 124319).

[0462] Intracellular cytokine staining (ICS) assay ICS assays were performed on both splenocytes and liver mononuclear cells. Cells were seeded into U-bottom 96-well plates. The plates containing cells were incubated overnight at 37°C with complete medium alone as a negative control or in combination with the peptides listed in Table 9 at a concentration of 2 μg / ml. After 1 hour of incubation, 2 μg / ml of Brefeldin A (Sigma, B6542) was added.

[0463] After overnight culture, the cells were washed with PBS FACS and incubated with 5 μL of PBS FACS containing rat anti-mouse CD16 / CD32 antibody and the viability marker LD Fixative Yellow (Thermofisher, L34959) for 10 min in the dark at 4°C. The cells were then stained with 25 μL of PBS FACS containing MAbs for 20 min in the dark at 4°C. The mixture consisted of monoclonal antibodies against CD3 (hamster MAb anti-mouse CD3-PerCP, BD Biosciences, 553067), CD8 (rat MAb anti-mouse CD8-APC-H7, BD Biosciences, 560182), CD4 (mouse CD4-PE-Cy7, BD Biosciences, 552775), and NK cells (rat MAb anti-mouse NK P46 BV421, Biolegend, 137612). After several washes, cells were fixed and permeabilized with Cytofix / Cytoperm for 20 minutes in the dark at room temperature, then washed with Perm / Wash solution (BD Biosciences, 554714) at 4°C.

[0464] Intracellular cytokines were stained for 30 min at 4°C in the dark with antibodies against IFNγ (rat Mab anti-mouse IFNγ-APC, clone XMG1.2, BD Biosciences, 554413) and tumor necrosis factor alpha (TNFα) (rat Mab anti-mouse TNFα-FITC, clone MP6-XT22; 1 / 250 (BD Biosciences, 554418)). Cells were washed with Perm / Wash and resuspended in PBS containing 1% formaldehyde prior to flow cytometric analysis using a MACSQuant Analyzer.

[0465] Viable CD3+CD8+CD4- and CD3+CD8-CD4+ cells were gated and presented on dot plots. Two gate regions were defined for each cytokine-positive cell. The number of events detected within these gates was divided by the total number of events in the parent population to calculate the T cell response rate. For each mouse, the percentage obtained with medium alone was considered background and subtracted from the percentage obtained with peptide stimulation.

[0466] A positivity threshold was defined according to the experimental background (i.e., the mean percentage of stained cells obtained per group in the medium alone condition plus two standard deviations). Only percentages of cytokines representing at least five events were considered positive.

[0467] [Table 9]

[0468] Example 1: In vitro efficacy testing A gene walk was performed across the entire human PD-L1 transcript using primarily 16- to 20-mer gapmers. Efficacy testing was performed in vitro in the human leukemia-monocytic cell line THP1 and the human non-Hodgkin's lymphoma cell line (KARPAS-299).

[0469] cell lineage THP1 and Karpas-299 cell lines were originally purchased from the European Collection of Authenticated Cell Cultures (ECACC) and maintained in a humidified incubator at 37°C with 5% CO2 as recommended by the supplier.

[0470] Effect of oligonucleotides THP-1 cells (3.104 in RPMI-GLutamax, 10% FBS, 1% Pen-Strep (Thermo Fisher Scientific)) were cultured in 96-well round-bottom plates for 6 days in a final volume of 100 μl / well. Oligonucleotides were screened at a single concentration (20 μM) and at a dose range of 25 μM to 0.004 μM (1:3 dilution in water). Total mRNA was extracted using the MagNA Pure 96 Cellular RNA Large Volume Kit (MagNA Pure 96 System (Roche Diagnostics)) according to the manufacturer's instructions. For gene expression analysis, predesigned TaqMan primers (Thermo Fisher Scientific) targeting human PDL1 and ACTB, used as endogenous controls, were used in a QuantStudio machine (Applied Biosystems) with the TaqMan RNA-to-ct1-Step Kit (Thermo Fisher Scientific). RT-qPCR was performed using a Scientific. Relative PD-L1 mRNA expression levels were calculated using the 2ΔΔC(T) (2(-Delta Delta C(T))) method and the percentage of inhibition as a percentage compared to the control sample (untreated cells).

[0471] Karpas-299 cells were cultured in RPMI 1640, 2 mM glutamine, and 20% FBS (Sigma). The cells were seeded at 10,000 cells / well in 96-well plates and incubated for 24 hours before the addition of oligonucleotides dissolved in PBS. The final oligonucleotide concentrations were a single 5 μM dose in a final culture volume of 100 μl / well, or a dose-response range of 50 μM, 15.8 μM, 5.0 μM, 1.58 μM, 0.5 μM, 0.158 μM, 0.05 μM, and 0.0158 μM in a 100 μL culture volume. Three days after addition of the oligonucleotide compounds, cells were harvested, and RNA was extracted using the PureLink Pro 96 RNA Purification Kit (Ambion) according to the manufacturer's instructions. cDNA was synthesized using M-MLT reverse transcriptase, RETROscript, RNase inhibitor (Ambion), and a 100 mM dNTP set (Invitrogen, PCR Grade). For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2X) (Ambion) in a duplex setup with TaqMan primer assays for PD-L1 (Applied Biosystems; Hs01125299_m1) and TBP (Applied Biosystems; 4325803). Relative PD-L1 mRNA expression levels, as % of the control sample (cells treated in PBS), are shown in Table 10.

[0472] [Table 10-1]

[0473] [Table 10-2]

[0474] [Table 10-3]

[0475] Table 10-4

[0476] Table 10-5

[0477] Table 10-6

[0478] Table 10-7

[0479] Table 10-8

[0480] Table 10-9

[0481] Table 10-10

[0482] Table 10-11

[0483] Table 10-12

[0484] Table 10-13

[0485] [Table 10-14]

[0486] [Table 10-15]

[0487] [Table 10-16]

[0488] [Table 10-17]

[0489] [Table 10-18]

[0490] [Table 10-19] [Table 10-20] [Table 10-21]

[0491] Example 2 - In vitro efficacy testing in dose response curves Selected oligonucleotides from Table 10 were tested in KARPAS-299 cells using half-log serial dilutions in PBS (50μM, 15.8μM, 5.0μM, 1.58μM, 0.5μM, 0.158μM, μM, 0.05μM, 0.0158μM oligonucleotide) and evaluated in the in vitro efficacy assay described in Example 1. The IC50 and maximum inhibition (residual % PD-L1 expression) of the oligonucleotides were evaluated.

[0492] EC50 calculations were performed in GraphPad Prism 6. Table 11 shows the IC50 and maximum PD-L1 knockdown levels as % of control (PBS) treated cells.

[0493] [Table 11-1]

[0494] [Table 11-2]

[0495] [Table 11-3]

[0496] Selected oligonucleotides from Table 6 were tested in THP-1 cells using a 1:3 serial dilution in water from 25 μM to 0.004 μM in the in vitro efficacy assay described in Example 1. The IC50 and maximum inhibition (residual PD-L1 expression) were assessed for the oligonucleotides.

[0497] EC50 calculations were performed in GraphPad Prism 6. IC50 and maximum PD-L1 knockdown levels are shown in Table 12 as % of control (PBS) treated cells.

[0498] [Table 12]

[0499] The results in Tables 7 and 8 are also shown in Figure 2 with respect to their positions targeting the PD-L1 pre-mRNA of SEQ ID NO:1.

[0500] This indicates that almost all compounds had EC50 values ​​below 1 μM and target knockdown below 25% of PD-L1 expression levels in control cells (treated with saline).

[0501] Example 3 – In vitro potency and efficacy and in vivo PD-L1 reduction in poly(I:C)-induced mice using naked and GalNAc-conjugated PD-L1 antisense oligonucleotides Efficacy and efficacy testing was performed in vitro in dose-response studies in MCP-11 cells using the oligonucleotides in Table 6. The same oligonucleotides, as well as GalNAc-linked versions (CMP ID numbers 755_2 to 765_2 in Table 8), were tested in vivo for their ability to reduce PD-L1 mRNA and protein expression in poly(I:C)-induced C57BL / 6J female mice.

[0502] In vitro assays MCP-11 cells (originally purchased from ATCC) suspended in DMEM (Sigma catalog no. D0819) supplemented with 10% horse serum, 2 mM L-glutamine, 0.025 mg / ml gentamicin, and 1 mM sodium pyruvate were added to oligonucleotides (10 μl) in 96-well round-bottom plates at a density of 8000 cells / well and cultured for 3 days in a final volume of 200 μl / well in a humidified incubator at 37°C with 5% CO. Oligonucleotides were screened at a dose range of concentrations (50 μM, 15.8 μM, 5.0 μM, 1.58 μM, 0.5 μM, 0.158 μM, 0.05 μM, and 0.0158 μM).

[0503] Total mRNA was extracted using the PureLink Pro 96 RNA Purification Kit (Ambion) according to the manufacturer's instructions, and cDNA was synthesized using M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion), and a 100 mM dNTP set (Invitrogen, PCR Grade) according to the manufacturer's instructions. For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2X) (Ambion) in a duplex setup with TaqMan primer assays for PD-L1 (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and Gusb (Thermo Fisher Scientific; VIC-MGB-PL Mm01197698-m1). Table 9 shows the relative PD-L1 mRNA expression levels as a percentage of the residual PD-L1 expression in the PBS control sample (PBS-treated cells). EC50 calculations were performed using GraphPad Prism 6. Table 13 shows the EC50 and maximum PD-L1 knockdown levels as % of control (PBS) cells.

[0504] In vivo assay C57BL / 6J female mice (20-23 g; 5 mice per group) were subcutaneously injected with 5 mg / kg of unconjugated oligonucleotides against mouse PD-L1 or 2.8 mg / kg of GalNAc-conjugated oligonucleotides against mouse PD-L1. Three days later, the mice were intravenously injected with 10 mg / kg poly(I:C) (LWM, Invivogen). Five hours after poly(I:C) injection, the mice were sacrificed, and liver samples were either placed in RNAlater (Thermo Fisher Scientific) for RNA extraction or frozen on dry ice for protein extraction.

[0505] Total mRNA was extracted from homogenized liver samples using the PureLink Pro 96 RNA Purification Kit (Ambion) according to the manufacturer's instructions. cDNA was synthesized using M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion), and 100 mM dNTPs (Invitrogen, PCR Grade) according to the manufacturer's instructions. For gene expression analysis, qPCR was performed using TaqMan® Fast Advanced Master Mix (2X) (Ambion) in a duplex setup with TaqMan primer assays for PD-L1 mRNA (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1). Table 13 shows the relative expression levels of PD-L1 mRNA as a % of control samples from saline and poly(I:C) injected mice.

[0506] Liver homogenates were prepared by homogenizing 2 ml of liver sample per 100 mg of tissue in T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) mixed with 1x Stop Protease Inhibitor Cocktail, EDTA-free (Thermo Fisher Scientific). Protein concentrations in the liver homogenates were measured using the Coomassie Plus (Bradford) Assay Reagent (Thermo Scientific) according to the manufacturer's instructions. Liver homogenates (40 μg of protein) were separated on a 4-12% Bis-Tris Plus polyacrylamide gel (Thermo Fisher Scientific) in 1x MOPS running buffer using the iBLOT dry blotting system (Thermo Fisher Scientific) according to the manufacturer's instructions, and transferred to a nitrocellulose membrane. Each blot was cut horizontally into two sections at the 64 kDa band. Membranes were blocked in TBS containing 5% skim milk and 0.05% Tween 20 and then incubated overnight at 4°C with rabbit monoclonal anti-vinculin (Abcam catalog no. ab129002) diluted 1:10,000 (upper membrane) or goat polyclonal anti-mPD-L1 (R&D Systems catalog no. AF1019) diluted 1:1,000 (lower membrane) in TBS containing 5% skim milk and 0.05% Tween 20. Membranes were washed in TBS containing 0.05% Tween 20 and then exposed to HRP-conjugated swine anti-rabbit IgG (DAKO) diluted 1:3,000 or HRP-conjugated rabbit anti-goat IgG (DAKO) diluted 1:2,000 in TBS containing 5% skim milk and 0.05% Tween 20 for 1 hour at room temperature. After washing the membrane, reactivity was detected using ECL Select (Amersham GE Healthcare). For each oligonucleotide-treated group, the intensity of the PD-L1 band relative to the vinculin band was assessed, compared with the PD-L1 / vinculin band intensity in mice injected with saline and poly(I:C) (control).The results are shown in Table 13 and Western blots using pairs of naked and conjugated oligonucleotides are shown in Figures 9A-9E.

[0507] [Table 13]

[0508] As can be seen from the data in Table 13, it is clear that GalNAc conjugation of oligonucleotides improves in vivo PD-L1 reduction. mRNA reduction generally correlates with PD-L1 protein reduction. With the exception of CMP ID No: 754_1, the lower in vitro EC50 values ​​generally reflect better in vivo PD-L1 mRNA reduction once the oligonucleotides are GalNAc conjugated.

[0509] Example 4 - In vivo PK / PD in sorted hepatocytes and non-parenchymal cells from poly(I:C)-induced mice We investigated the distribution of naked and GalNAc-conjugated oligonucleotides and the reduction of PD-L1 mRNA in hepatocytes and non-parenchymal cells isolated from poly(I:C)-induced mice.

[0510] C57BL / 6J female mice were subcutaneously injected with 5 mg / kg of unconjugated oligonucleotide (748_1) or 7 mg / kg of GalNAc-conjugated oligonucleotide (759_2) targeting mouse PD-L1 mRNA (n=3 per group). Two days later, mice were intraperitoneally injected with 15 mg / kg poly(I:C) (LWM, InvivoGen). Mice were anesthetized 18–20 hours after poly(I:C) injection, and the livers were perfused with Hank's balanced salt solution containing 15 mM Hepes and 0.38 mM EGTA at a flow rate of 7 ml / min for 5 minutes. The liver was then washed for 12 minutes with a collagenase solution (Hank's Balanced Salt Solution) containing 0.17 mg / ml collagenase type 2 (Worthington 4176), 0.03% BSA, 3.2 mM CaCl2, and 1.6 g / L NaHCO3. After perfusion, the liver was removed, the liver capsule was opened, and the liver suspension was filtered through a 70 μm cell strainer using Williams' E medium. An aliquot of the cell suspension (=mixed hepatocytes) was removed for further analysis. The remaining cell suspension was centrifuged at 50 × g for 3 minutes. The supernatant was collected for later purification of non-parenchymal cells. The pellet was resuspended in 25 ml of William's E medium (Sigma catalog number W1878, supplemented with 1x Pen / Strep, 2 mM L-glutamine, and 10% FBS (ATCC #30-2030)), mixed with 25 ml of William's E medium containing 90% Percoll and hepatocytes, and centrifuged at 50 × g for 10 minutes to precipitate the hepatocytes. After washing twice with William's E medium, the precipitated hepatocytes were resuspended in William's E medium. The supernatant containing the unorganized cells was centrifuged at 500 × g for 7 minutes, and the cells were resuspended in 4 ml of RPMI medium and centrifuged at 1800 × g for 30 minutes through two Percoll layers (25% and 50% Percoll). After the unorganized cells between the two Percoll layers were collected, the cells were washed and resuspended in RPMI medium.

[0511] Total mRNA was extracted from purified hepatocytes, non-parenchymal cells, and whole liver suspension (unfractionated hepatocytes) using the PureLink Pro 96 RNA Purification Kit (Ambion) according to the manufacturer's instructions. cDNA was synthesized using M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion), and 100 mM dNTP set (Invitrogen, PCR Grade) according to the manufacturer's instructions. For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2X) (Ambion) in a duplex setup with TaqMan primer assays for PD-L1 (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1). Table 10 shows the relative expression levels of PD-L1 mRNA as a % of control samples from saline and poly(I:C) injected mice.

[0512] Oligonucleotide content was analyzed using an ELISA with a biotinylated capture probe with the sequence 5'-TACCGT-s-Bio-3' and a digoxigenin-conjugated detection probe with the sequence 5'-DIG-C12-S1-CCTGTG-3'. The probe consisted exclusively of LNA with a phosphodiester backbone. Liver samples (approximately 50 mg) were homogenized in 1.4 mL of MagNa pure lysis buffer (Roche catalog number 03604721001) in a 2 mL Eppendorf tube containing one 5 mm diameter stainless steel bead. Samples were homogenized using a Retsch MM400 homogenizer (Merck Eurolab) until a homogenous lysate was obtained. Samples were incubated at room temperature for 30 minutes. Standards were generated by spiking a defined concentration of an unconjugated antisense oligonucleotide compound (CMP ID number 748_1) into untreated liver samples and processing these as samples. The spike-in concentration is chosen to match the expected sample oligo content (within about 10-fold).

[0513] The homogenized samples were diluted a minimum of 10-fold in 5x SSCT buffer (containing 750 mM NaCl, 75 mM sodium citrate, and 0.05% (v / v) Tween-20, pH 7.0) and serially diluted 6x 2-fold using capture-detection solution (35 nM capture probe and 35 nM detection probe in 5x SSCT buffer) and incubated at room temperature for 30 minutes. Samples were transferred to a 96-well streptavidin-coated plate (Nunc catalog number 436014) at 100 μL per well. The plate was incubated at room temperature for 1 hour with gentle agitation. After washing three times with 2x SSCT buffer, 100 μL of freshly prepared anti-DIG-AP Fab fragment (Roche Applied Science, catalog no. 11093274910) diluted 1:4000 in phosphate-buffered saline (PBST) containing 0.05% (v / v) Tween-20 (pH 7.2) was added to each well and incubated for 1 hour at room temperature with gentle agitation. After washing three times with 2x SSCT buffer, 100 μL of alkaline phosphatase (AP) substrate solution (Blue Phos substrate, KPL product code 50-88-00, freshly prepared) was added. After a 30-minute incubation with gentle agitation, the color intensity was measured spectrophotometrically at 615 nm. Raw data were exported from the reader (Gen5 2.0 software) to Excel format and further analyzed in Excel. Calibration curves were generated using GraphPad Prism 6 software and a logistic 4PL regression model.

[0514] [Table 14]

[0515] The results demonstrate that naked (CMP ID No. 748_1) and conjugated (CMP ID No. 759_2) oligonucleotides effectively reduce PD-L1 mRNA in whole hepatocytes. In isolated hepatocytes, the effect of the conjugated oligonucleotide was approximately five-fold more potent than that of the naked oligonucleotide, and the naked oligonucleotide was two-fold more potent than the GalNAc-conjugated oligonucleotide in non-parenchymal cells. The reduction of PD-L1 mRNA expression in hepatocytes and non-parenchymal cells correlates to some extent with the oligonucleotide content in these cell types.

[0516] Example 5 –In vivo PD-L1 knockdown in AAV / HBV mice using naked and GalNAc-conjugated PD-L1 antisense oligonucleotides In this study, AAV / HBV mice were treated with naked or GalNAc-conjugated PD-L1 antisense oligonucleotides, and PD-L1 mRNA expression levels and HBV gene expression in the liver were assessed.

[0517] At week 1, 5-8 week old female HLA-A2 / DR1 mice (5 animals per group) were pre-treated with vehicle (saline), naked PD-L1 antisense oligonucleotide (CMP ID No. 752_1 (5 mg / kg sc)), and GalNAc (5 mg / kg sc) PD-L1 antisense oligonucleotide (CMP ID No. 763_2 at 7 mg / kg subcutaneously). These doses correspond to equimolar concentrations of oligonucleotides. Mice were treated with 5 x 10 10 Mice were transduced with 1000 mg of AAV-HBV. For details, see the description of the AAV / HBV mouse model in the Materials and Methods section. Mice received four additional subcutaneous injections of PD-L1 oligonucleotide or vehicle (saline) at weekly intervals from week 1 to week 4 after AAV-HBV transduction.

[0518] Blood samples were taken one week before transduction and one week after each injection.

[0519] Two weeks after the last injection, mice were sacrificed and their livers were removed after perfusion with PBS. The livers were cut into smaller pieces and directly frozen.

[0520] To measure HBV gene expression, DNA was extracted from serum on a Qiagen Biorobot using the QIAamp One-For-All Nucleic Acid Kit, catalog number 965672, serum was diluted 1:20 in PBS, and a total of 100 μl was eluted in 200 μl of Buffer AL, followed by elution of DNA from the kit in 100 μl increments.

[0521] For real-time qPCR, TaqMan Gene Expression Master Mix (catalog no. 4369016, Applied Biosystems) was used with a primer mixture containing 100 μM each of the following primers F3_core, R3_core, and P3_core (Integrated DNA Technologies) at a ratio of 1:1:0.5. Forward (F3_core): CTG TGC CTT GGG TGG CTT T (SEQ ID NO: 784) Reverse (R3_core): AAG GAA AGA AGT CAG AAG GCA AAA (SEQ ID NO: 785) Probe (P3_core): 56-FAM-AGC TCC AAA / ZEN / TTC TTT ATA AGG GTC GAT GTC CAT G-3IABkFQ (SEQ ID NO: 786)

[0522] A standard curve using HBV plasmid (genotype D, GTD) was prepared using the initial 1 × 10 9 A 10-fold dilution was prepared from copies / μl down to 1 copy / μl and used at 5 μl per reaction.

[0523] qPCR was performed by adding 10 μl of gene expression master mix, 4.5 μl of water, 0.5 μl of primer mix, and 5 μl of sample or standard solution per reaction.

[0524] For analysis, the standard curve was used to calculate the number of copies / ml / well. The results are shown in Table 15.

[0525] PD-L1 mRNA expression levels were measured by qPCR.

[0526] mRNA was extracted from frozen liver pieces and added to a 2 ml tube containing ceramic beads (Lysing Matrix D tubes, 116913500, mpbio) and 1 ml of Trizol.

[0527] Liver pieces were homogenized using a Precellys Tissue Disruptor. 200 μl of chloroform was added to the homogenate, vortexed, and centrifuged at 10,000 rpm for 20 minutes at 4°C. The clear phase (approximately 500 μl) containing RNA was transferred to a new tube, and the same volume of 70% EtOH was added. After thorough mixing, the solution was transferred to an RNeasy spin column, and RNA was further extracted according to the RNeasy Kit manual, RNeasy Mini Kit, catalog number 74104, Qiagen (includes RNA Digestion RNase-Free DNase Set, catalog number 79254). The final RNA concentration in 50 μl of HO was measured and adjusted to 100 ng / μl for all samples.

[0528] qPCP was performed on 7.5 μl of RNA using the Taqman RNA-to-ct 1-step Kit (catalog no. 4392938, Thermo Fisher Scientific) according to the manufacturer's instructions. The primer mix used included PD-L1_1-3 (primer numbers Mm00452054_m1, Mm03048247_m1, and Mm03048248_m1) and endogenous controls (ATCB Mm00607939_s1, CANX Mm00500330_m1, YWHAZ Mm03950126_s1, and GUSB Mm01197698_m1).

[0529] Data were analyzed using the 2^-ddct method. The dct value was calculated using the mean of all four endogenous controls. PD-L1 expression relative to the mean of endogenous controls and saline (%)

[0530] [Table 15]

[0531] These results indicate that both naked and GalNAc-conjugated oligonucleotides were able to reduce PD-L1 mRNA expression in the livers of AAV / HBV mice, with GalNAc-conjugated oligonucleotides being slightly more effective, and both oligonucleotides also slightly reduced HBV DNA in the serum.

[0532] Example 6 – In vivo effects on T cell responses in AAV / HBV mice In this study, AAV / HBV mice obtained from Pasteur were treated with antibodies or antisense oligonucleotides targeting PD-L1. The antisense oligonucleotides were either naked or GalNAc-conjugated. To ensure efficient T cell priming by antigen-presenting cells during treatment, animals were immunized with DNA vaccines against HBsAg and HBcAg (see Materials and Methods). We assessed how this treatment affected cell populations in the liver and spleen, as well as PD-L1 expression in these populations, and whether HBV-specific T cell responses could be identified.

[0533] Treatment Protocol: Female HLA-A2 / DR1 mice were treated according to the following protocol: This study was conducted in two separate sub-studies that differed slightly in dosing regimens as shown in Tables 16 and 17 below.

[0534] DNA vaccines and anti-PD-L1 antibodies were administered as described in the Materials and Methods section. The antisense oligonucleotides used were CMP ID#748_1 (naked) at 5 mg / kg and CMP ID#759_2 (GalNAc-linked) at 7 mg / kg when administered subcutaneously (sc).

[0535] [Table 16]

[0536] [Table 17]

[0537] At the time of sacrifice, spleen and liver mononuclear cells from each mouse from each group were collected and depleted of red blood cells (Lysing Buffer, BD biosciences, 555899). Specific preparations of liver mononuclear cells were required, as described in the Materials and Methods section.

[0538] Cell populations: Cell populations in the liver were analyzed by surface labeling of liver mononuclear cells (see Materials and Methods) using cytometry.

[0539] No significant changes were observed in the frequencies of NK cells in the spleens and livers of treated mice compared to control groups (i.e., vehicle and DNA-immunized groups). As shown in Table 18, in the liver, the groups treated with naked PD-L1 oligonucleotide (CMP ID No.: 748_1) and GalNAc-conjugated PD-L1 oligonucleotide (CMP ID No.: 759_2) had significantly increased T cell numbers compared to either control group (i.e., vehicle and DNA-immunized groups). This is also shown in Figure 10A. This increase was due to an increase in both CD4+ and CD8+ T cell populations (Table 18, Figures 10B and 10C, respectively).

[0540] [Table 18]

[0541] PD-L1 expression level : At the time of sacrifice, PD-L1 protein expression was assessed in macrophages, B cells, and T cells from the spleen and liver. The presence of PD-L1 antibody in the surface-labeled antibody cocktail (see Materials and Methods) allowed quantification of PD-L1-expressing cells using cytometry.

[0542] No significant differences were observed between treatments in the percentage of PD-L1 expression in macrophages, B cells, or CD4+ T cells in the spleen. The percentage of CD8+ T cells expressing PD-L1 was lower in mice treated with naked PD-L1 oligonucleotide (CMP ID #748_1) and GalNAc-conjugated PD-L1 oligonucleotide (CMP ID #759_2) compared to other treatments (data not shown).

[0543] In the liver, PD-L1 was primarily expressed on CD8+ T cells. In the control groups (Figure 11A, which shows the combined vehicle-vaccinated and DNA-vaccinated groups, respectively), the mean frequencies were 32% and 41%. Treatment with naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide reduced the frequency of CD8+ T cells expressing PD-L1 (see Table 19 and Figure 11A). These cell types expressed significantly less PD-L1 than CD8+ T cells, but significant differences in the percentage of cells expressing PD-L1 were also observed in B cells and CD4+ T cells after ASO treatment (see Table 19, Figures 11B and 11C). Furthermore, reductions in PD-L1 expression were evident in all cell types following treatment with anti-PD-L1 antibody. However, this reduction was due to partial blockage of the PD-L1 epitope via the therapeutic anti-PD-L1 antibody, thereby preventing the PD-L1 detection antibody in the surface-labeled antibody mixture from binding to PD-L1. Therefore, the apparent downregulation of PD-L1 via the therapeutic anti-PD-L1 antibody may be the result of epitope competition between the therapeutic and detection antibodies.

[0544] [Table 19]

[0545] HBV-specific T cell response: Intracellular cytokine staining assays (see Materials and Methods) that detect IFNγ and TNFα production were used to detect NK cells, CD4+ T cells, and CD8+ T cells that produce inflammatory cytokines.

[0546] At the time of sacrifice, NK cells, IFNγ- and TNFα-secreting CD4+ T cells were not detected in the spleen (frequency <0.1%). IFNγ-producing CD8+ T cells targeting two HBV antigens were detected in mice treated with naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide, as well as in mice in this study that received the DNA vaccine alone (data not shown).

[0547] At the time of sacrifice, IFNγ-producing NK cells were not detected in the livers of DNA-immunized HBV carrier mice, whereas IFNγ-secreting CD4+ T cells specific for Core or S2+S were detected in the livers of some DNA-immunized mice (<0.4%, data not shown). IFNγ-producing HBV S2+S-specific CD8+ T cells were detected in the majority of DNA-immunized mice. The frequency of IFNγ-secreting CD8+ T cells increased in mice treated with a combination of DNA vaccine and naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide, whereas treatment with anti-PD-L1 antibody showed no clear additive effect to DNA vaccination (Figure 12). IFNγ-producing CD8+ T cells targeting envelope and core antigens were detected in most DNA-immunized groups (except for anti-PD-L1 antibody) (Figure 12B). Most S2-S-specific T cells produced both IFNγ and TNFα (Figure 12C). The results are also shown in Table 20.

[0548] [Table 20]

[0549] Example 7 - In vivo effects of AAV / HBV on HBV antigen and HBV DNA in mouse serum In this study, AAV / HBV mice obtained from Shanghai (see Materials and Methods) were treated with GalNAc-conjugated PD-L1 antisense oligonucleotide CMP ID number 759_2.

[0550] The effect of treatment on serum HBe and HBs antigen and HBV DNA levels was assessed compared to vehicle-treated animals.

[0551] Treatment Protocol: As described in the "Materials and Methods" section of the Shanghai model, this study used male C57BL / 6 mice infected with recombinant adeno-associated virus (AAV) carrying the HBV genome (AAV / HBV). Mice (6 mice per group) were injected once weekly for 8 weeks with either 5 mg / kg of the antisense oligonucleotide CMP ID No. 759_2 or vehicle (saline). The injections were administered subcutaneously (sc). Blood samples were collected weekly during treatment and 6 weeks post-treatment. HBV DNA, HBsAg, and HBeAg levels were measured by serological testing, as described below. Results from the first 10 weeks are shown in Table 21 and Figure 13. Because the study was still ongoing at the time of filing, data from the remaining 4 weeks were not available.

[0552] HBsAg and HBeAg detection: Serum HBsAg and HBeAg levels in infected AAV-HBV mice were measured using HBsAg chemiluminescence immunoassay (CLIA) and HBeAg CLIA kits (Autobio diagnostics Co. Ltd., Zhengzhou, China, catalog numbers CL0310-2 and CL0312-2, respectively) according to the manufacturer's protocol. Briefly, 50 μl of serum was transferred to each antibody-coated microtiter plate, and 50 μl of enzyme-linked reagent was added. After incubating the plate on a shaker at room temperature for 60 minutes, all wells were washed six times with wash buffer using an automated washer. 25 μl of substrate A, followed by 25 μl of substrate B, was added to each well. After incubating the plate at room temperature for 10 minutes, luminescence was measured using an Envision luminescence reader. HBsAg is given in units of IU / ml, where 1 ng of HBsAg = 1.14 IU. HBeAg is given in units of NCU / ml of serum.

[0553] HBV DNA extraction and qPCR: First, mouse serum was diluted 10-fold (1:10) with phosphate-buffered saline (PBS). DNA was extracted using a MagNA Pure 96 (Roche) robot. 50 μl of diluted serum was mixed with 200 μl of MagNA Pure 96 external lysis buffer (Roche, catalog number 06374913001) in a treatment cartridge and incubated for 10 minutes. DNA was then extracted using the "MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit" (Roche, catalog number 06543588001) and the "Viral NA Plasma SV external lysis 2.0" protocol. The DNA elution volume was 50 μl.

[0554] The extracted HBV DNA was quantified using a Taqman qPCR machine (ViiA7, Life Technologies). Each DNA sample was tested in duplicate by PCR. 5 μl of DNA sample was added to 15 μl of PCR master mix containing 10 μl of TaqMan Gene Expression Master Mix (Applied Biosystems, catalog number 4369016), 0.5 μl of PrimeTime XL qPCR Primer / Probe (IDT), and 4.5 μl of distilled water in a 384-well plate. PCR was performed using the following settings: UDG incubation (2 min, 50°C), enzyme activation (10 min, 95°C), and 40 PCR cycles (15 s denaturation at 95°C, and 1 min annealing and extension at 60°C). DNA copy number was calculated based on the HBV plasmid DNA calibration line by ViiA7 software. t was calculated from the values.

[0555] Sequences of TaqMan primers and probes (IDT): Forward core primer (F3_core): CTG TGC CTT GGG TGG CTT T (SEQ ID NO: 784) Reverse primer (R3_core): AAG GAA AGA AGT CAG AAG GCA AAA (SEQ ID NO: 785) Taqman probe (P3_core): 56-FAM / AGC TCC AAA / ZEN / TTC TTT ATA AGG GTC GAT GTC CAT G / 3IABkFQ (SEQ ID NO: 786).

[0556] [Table 21]

[0557] This study shows that GalNAc-linked PD-L1 antisense oligonucleotide CMP No. 759_2 has significant effects on reducing HBV-DNA, HBsAg, and HBeAg levels after 6 weeks of treatment, effects that persist for at least 2 weeks after treatment.

[0558] Example 8 – In vivo PD-L1 knockdown in human primary hepatocytes using GalNAc-conjugated PD-L1 oligonucleotides The ability of GalNAc-conjugated PD-L1 antisense oligonucleotide compounds to reduce PD-L1 transcripts in primary human hepatocytes was investigated using genomics.

[0559] cell culture Cryopreserved human hepatocytes were cultured at 5 × 10 in WME supplemented with 10% fetal bovine serum, penicillin (100 U / ml), streptomycin (0.1 mg / ml), and L-glutamine (0.292 mg / ml). 6 Dilute to 2 x 10 cells / ml 5Cells were seeded into collagen-coated 24-well plates (Becton Dickinson AG, Allschwil, Switzerland) at a density of 100 cells / well. Cells were pre-cultured for 4 hours to allow them to attach to the cell culture plate before treatment with oligonucleotides at a final concentration of 100 μM. The oligonucleotides used were as shown in Tables 21 and 8, and the vehicle was PBS. The seeding medium was replaced with 315 μl of serum-free WME supplemented with penicillin (100 U / ml), streptomycin (0.1 mg / ml), and L-glutamine (0.292 mg / ml), and 35 μl of 1 mM oligonucleotide stock solution in PBS was added to the cell culture and left on the cells for 24 or 66 hours.

[0560] Library preparation Transcript expression profiling was performed using stranded mRNA chemistry with a sequencing strategy of 2 x 51 bp paired-end reads and 30 M / sample (Q squared EA). Cells were lysed and randomized by adding 350 μl of Qiagen RLT buffer to the wells.

[0561] mRNA was purified using the Qiagen RNeasy Mini Kit. mRNA was quantified and integrity was assessed using an Agilent Bioanalyzer. Initial quality assessment of the isolated RNA indicated that all samples met the 100 ng input quality metric with a RIN score >7.0.

[0562] Starting with 100 ng of total RNA, sequencing libraries were generated for all samples using Illumina TruSeq Stranded mRNA Library Preparation. The final cDNA libraries were analyzed for size distribution and quantified using an Agilent Bioanalyzer (DNA1000 Kit), quantified by qPCR (KAPA Library Quant Kit), and normalized to 2 nM in preparation for sequencing. Using the Standard Cluster Generation Kit v5, the cDNA libraries were bound to the flow cell surface, and cBots were isothermally attached to amplify the attached cDNA constructs to clonal populations of approximately 1000 copies each. DNA sequences were determined by sequencing-by-synthesis using the TruSeq SBS Kit.

[0563] Data Processing Illumina paired-end sequencing reads of 2 × 51 bp in length were mapped to the human reference genome hg19 using the GSNAP short read alignment program. SAM-format alignments were converted to sorted alignment BAM-format files using the SAMTOOLS program. The gene read count for PD-L1 was estimated based on exon annotations from NCBI RefSeq specified in the GTF file corresponding to hg19. A normalization step, taking into account the different library sizes of each sample, was applied using the DESeq2R package.

[0564] The reduction of PD-L1 transcripts following incubation with GalNAc-linked PD-L1 antisense oligonucleotide compounds is shown in Table 22.

[0565] [Table 22-1]

[0566] Compared to vehicle-treated samples, all five GalNAc-conjugated antisense compounds showed significant reductions in PD-L1 transcripts after 24 and 66 hours of incubation.

[0567] Example 9 –Binding and EC50 of naked PD-L1 antisense oligonucleotides in HBV-infected ASGPR-HepaRG cells The efficacy of two naked PD-L1 antisense oligonucleotides and an equivalent GalNAc-conjugated PD-L1 antisense oligonucleotide in HBV-infected ASGPR-HepaRG cells was compared.

[0568] cell lineage HepaRG cells (Biopredic International, Saint-Gregoire, France) were cultured in William's E medium supplemented with 10% HepaRG growth supplement (Biopredic). A HepaRG cell line stably overexpressing human ASGPR1 and ASGPR2 was generated from this cell line using a lentiviral method. Lentivirus encoding human ASGPR1 and ASGPR2 under the control of a CMV promoter and puromycin resistance gene (CLV-CMV-ASGPR1-T2a_ASGPR2-IRES-Puro) was transduced into HepaRG cells for growth at a multiplicity of infection (MOI) of 300. Transduced cells were selected with 1 μg / ml puromycin for 11 days and then maintained in the same concentration of antibiotic to ensure stable transgene expression. ASGPR1 / 2 overexpression was confirmed at both the mRNA level by RT-qPCR (ASGPR1: 8560-fold vs. non-transduced, ASGPR2: 2389-fold vs. non-transduced) and the protein level by flow cytometry analysis.

[0569] Cells were differentiated using 1.8% DMSO for at least two weeks prior to infection. HBV genotype D was extracted from HepG2.2.15 cell culture supernatant and concentrated using PEG precipitation. To evaluate the activity of test compounds against HBV, differentiated ASGPR-HepaRG cells in 96-well plates were infected with HBV at a multiplicity of infection (MOI) of 20–30 for 20 hours. The cells were then washed four times with PBS to remove the HBV inoculum.

[0570] Oligonucleotide efficacy The following oligonucleotides

[0571] [Table 22-2]

[0572] was added to HBV-infected ASGPR-HepaRG cells at days 7 and 10 postinfection using serial dilutions from 25 μM to 0.4 nM (1:4 diluted in PBS). Cells were harvested at day 13 postinfection.

[0573] Total mRNA was extracted using the MagNA Pure 96 Cellular RNA Large Volume Kit (MagNA Pure 96 System (Roche Diagnostics)) according to the manufacturer's instructions. RT-qPCR for gene expression analysis was performed as described in Example 5.

[0574] Data were analyzed using the 2^-ddct method. Actin B was used as an endogenous control to calculate dct values. PD-L1 expression levels are relative to the endogenous control and saline vehicle.

[0575] EC50 calculations were performed in GraphPad Prism6 and are shown in Table 23.

[0576] [Table 23]

[0577] These data clearly show that GalNAc-conjugated forms of PD-L1 antisense oligonucleotides significantly improve EC50 values.

[0578] Example 10 - Stimulated T cell function in PBMCs from chronic HBV patients We investigated whether naked PD-L1 antisense compounds could enhance T cell function in chronically infected HBV (CHB) patients after ex vivo HBV antigen stimulation of peripheral blood mononuclear cells (PBMCs).

[0579] Frozen PBMCs from three patients with chronic HBV infection were thawed and seeded at a density of 200,000 cells / well in 100 μl of medium (RPMI 1640 + GlutaMax + 8% human serum + 25 mM Hepes + 1% PenStrep). The following day, cells were stimulated with 1 μM PepMix HBV large envelope protein or 1 μM PepMix HBV core protein, with or without 5 μM CMP ID No. 466_1 or CMP ID No. 640_1, in 100 μl of medium containing 100 pg / ml IL-12 and 5 ng / ml IL-7 (see Table 9). Concanavalin stimulation was applied only on day 8. Four days later, PD-L1 antisense oligonucleotide treatment was renewed with medium containing 50 IU IL-2. Eight days after the initial stimulation, cells were restimulated with PepMix or 5 μg / ml concanavalin A plus PD-L1 antisense oligonucleotide for 24 hours, followed by the addition of 0.1 μl Brefeldin A, 0.1 μl Monensin, and 3 μl anti-human CD-107 (APC) for the final 5 hours of stimulation.

[0580] After 24 hours, cells were washed with staining buffer (PBS + 1% BSA + 0.09% sodium azide + EDTA), and surface staining [anti-human CD3 (BV605), anti-human CD4 (FITC), anti-human CD8 (BV711), anti-human PDL1 (BV421), anti-human PD1 (PerCP-Cy5.5), and live and dead stain (BV510) (BD Biosciences)] was applied for 30 minutes at 4°C. Cells were fixed for 15 minutes in BD fixation buffer at 4°C. The next morning, cells were permeabilized with BD Perm / Wash Buffer for 15 minutes at 4°C, and intracellular staining [anti-human INF (PE)] was performed for 30 minutes at 4°C. After washing with Perm / Wash Buffer, cells were lysed in 250 μl of staining buffer.

[0581] FACS measurements were performed on a BD Fortessa (BD Biosciences). For analysis, the total cell population was first gated on live cells (live and dead stain, BV510) and then on CD3+ (BV605) cells. CD3+ cells were then graphed as CD107a+ (APC) versus IFN+ (PE).

[0582] The results are shown in Table 24.

[0583] [Table 24]

[0584] These data demonstrate that antigen stimulation alone can induce T cell activation (an increase in the percentage of CD3+ cells expressing INF and / or CD107a) in PBMCs from CHB patients (n=3). Addition of PD-L1 antisense oligonucleotides CMP 466_1 or 640_1 further increased CD3+ T cell responses. This increase was primarily observed in the HBV envelope-stimulated group. [1] a. an oligonucleotide (region A) comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary to a PD-L1 target nucleic acid; b. at least one asialoglycoprotein receptor targeting binding moiety (region C) covalently attached to the oligonucleotide of (a); An antisense oligonucleotide conjugate comprising: [2] The oligonucleotide conjugate according to Item 1, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. [3] The oligonucleotide conjugate according to item 1 or 2, wherein the contiguous nucleotide sequence is complementary to a subsequence of a target nucleic acid, and the subsequence is selected from the group consisting of positions 371-3068, 5467-12107, and 15317-19511 of SEQ ID NO: 1. [4] The oligonucleotide conjugate according to any one of items 1 to 3, wherein the contiguous nucleotide sequence is complementary to a subsequence of a target nucleic acid, and the subsequence is selected from the group consisting of A221, A360, A180, A160, and A269. [5] The oligonucleotide conjugate according to any one of items 1 to 4, wherein the oligonucleotide comprises a sequence selected from SEQ ID NOs: 466, 640, 342, 287, and 566. [6] The oligonucleotide conjugate according to any one of items 1 to 5, wherein the contiguous nucleotide sequence comprises one or more modified nucleosides (e.g., one or more 2' sugar-modified nucleosides). [7] The oligonucleotide conjugate of item 6, wherein the one or more 2' sugar-modified nucleosides are 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. [8] The oligonucleotide conjugate of any one of items 6 and 7, wherein all of the modified nucleosides are LNA nucleosides. [9] The oligonucleotide conjugate of any one of items 1 to 8, wherein the contiguous nucleotide sequence comprises at least one modified internucleoside linkage (e.g., at least one phosphorothioate internucleoside linkage).

[10] The oligonucleotide conjugate of any one of items 1 to 9, wherein the oligonucleotide is a gapmer.

[11] The oligonucleotide conjugate of item 10, wherein the gapmer has the formula 5'-D'-FG-F'-3' or 5'-FG-F'-D"-3', wherein regions F and F' independently contain 1 to 7 modified nucleosides, G is a region of 6 to 16 nucleosides having the ability to recruit RNase H, and region D' or D" is optional and contains 0 to 5 phosphodiester linked nucleosides.

[12] The oligonucleotide conjugate of any one of items 1 to 11, wherein the asialoglycoprotein receptor targeting binding moiety comprises at least one carbohydrate moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine.

[13] The oligonucleotide conjugate of any one of paragraphs 1 to 12, wherein the asialoglycoprotein receptor targeting binding moiety is monovalent, bivalent, trivalent, or tetravalent.

[14] The oligonucleotide conjugate of any one of items 1 to 13, wherein the asialoglycoprotein receptor targeting binding moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety.

[15] The oligonucleotide conjugate of any one of items 1 to 14, wherein the asialoglycoprotein receptor targeting binding moiety is the trivalent GalNAc moiety of Figure 3.

[16] The oligonucleotide conjugate of any one of items 1 to 15, wherein the oligonucleotide conjugate is selected from CMP ID numbers: 766_2, 767_2, 768_2, 769_2, and 770_2.

[17] An oligonucleotide or an antisense oligonucleotide comprising the consecutive nucleotide sequence of any one of items 1 to 11.

[18] A pharmaceutical composition comprising the oligonucleotide conjugate according to any one of items 1 to 16 or the oligonucleotide according to item 17, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[19] An in vivo or in vitro method for modulating PD-L1 expression in a target cell that expresses PD-L1, the method comprising administering to the cell an effective amount of the oligonucleotide conjugate of any one of items 1 to 16, or the oligonucleotide of item 17.

[20] The oligonucleotide conjugate according to any one of items 1 to 16, the oligonucleotide according to item 17, or the pharmaceutical composition according to item 18, for use in restoring an immune response to a virus or parasite.

[21] The use of item 20, wherein the restored immune response is an increase in CD8+ T cells specific to one or more HBV antigens in the liver compared to a control.

[22] The oligonucleotide conjugate according to any one of items 1 to 16, the oligonucleotide according to item 17, or the pharmaceutical composition according to item 18, for use as a medicament.

[23] The oligonucleotide conjugate according to any one of items 1 to 16, or the oligonucleotide according to item 17, or the pharmaceutical composition according to item 18, for use in the treatment or prevention of viral hepatitis infections such as HBV, HCV and HDV; parasitic infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; or liver cancer or liver metastasis.

[24] Use of an oligonucleotide conjugate according to any one of items 1 to 16, or an oligonucleotide according to item 17, or a pharmaceutical composition according to item 18, for the preparation of a medicament for the treatment or prevention of viral hepatitis infections such as HBV, HCV and HDV; parasitic infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; or liver cancer or liver metastasis.

Claims

1. Formula: GN2-C6 o c o a o An antisense oligonucleotide conjugate of the formula: CTAattgtagtagtaCTC, wherein C6 represents a 6-carbon —NH-alkylene group, 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 internucleoside linkage, and unless otherwise specified, all internucleoside linkages are phosphorothioate internucleoside linkages, and GN2 has the formula: 【Chemistry 1】 4. An antisense oligonucleotide conjugate representing a trivalent GalNAc cluster represented by the formula:

2. The antisense oligonucleotide conjugate of claim 1, wherein the oligonucleotide conjugate is CMP ID NO: 769_2.

3. The following formula: 【Chemistry 2】 Antisense oligonucleotide conjugates of.

4. A pharmaceutical composition comprising the antisense oligonucleotide conjugate of any one of claims 1 to 3 and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

5. 5. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable diluent is sterile phosphate buffered saline.

6. 6. The pharmaceutical composition of claim 4 or claim 5, wherein the pharmaceutically acceptable salt is sodium.

7. 6. The pharmaceutical composition of claim 4 or claim 5, wherein the pharmaceutically acceptable salt is potassium.

8. An in vitro method for modulating PD-L1 expression in a target cell expressing PD-L1, the method comprising administering to the cell an effective amount of the antisense oligonucleotide conjugate or pharmaceutical composition of any one of claims 1 to 7.

9. 8. The antisense oligonucleotide conjugate or pharmaceutical composition of any one of claims 1 to 7 for use in restoring an immune response to a virus.

10. The antisense oligonucleotide conjugate or pharmaceutical composition for use according to claim 9, wherein the virus is HBV.

11. The antisense oligonucleotide conjugate or pharmaceutical composition of any one of claims 1 to 7 for use in restoring an immune response to a parasite.

12. The antisense oligonucleotide conjugate or pharmaceutical composition for use according to any one of claims 9 to 11, wherein the restored immune response is an increase in CD8+ T cells in the liver specific for one or more HBV antigens compared to a control.

13. The antisense oligonucleotide conjugate or pharmaceutical composition according to any one of claims 1 to 7 for use as a medicament.

14. 8. The antisense oligonucleotide conjugate or pharmaceutical composition of any one of claims 1 to 7 for use in the treatment or prevention of HBV infection.

15. Use of the antisense oligonucleotide conjugate or pharmaceutical composition of any one of claims 1 to 7 for the preparation of a medicament for the treatment or prevention of HBV infection.

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