Oligonucleotides for reducing PD-L1 expression

Abstract

This invention relates to an antisense oligonucleotide capable of reducing PD-L1 expression in target cells. [Solution] Oligonucleotides hybridize to PD-L1 mRNA. The present invention further relates to oligonucleotide conjugates and pharmaceutical compositions, as well as methods for treating viral hepatitis infections such as HBV, HCV and HDV; parasitic infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; and liver cancer or metastasis in the liver using oligonucleotides.

Description

[Technical Field]

[0001] The present invention relates to oligonucleotides (oligomers) that are complementary to programmed death ligand-1 (PD-L1) and reduce PD-L1 expression in the liver. The present invention also relates to a method for alleviating T cell exhaustion caused by liver infection or cancer in the liver. Relevant infections include chronic HBV, HCV, HDV, and parasitic infections, such as malaria and toxoplasmosis (caused by protozoa of Plasmodium falciparum, particularly Plasmodium vivax, Plasmodium malariae, and Plasmodium 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, resulting in a lack of viral control during chronic liver infections. The PD-1 pathway is also involved in autoimmunity when mice with disruption in this pathway develop autoimmune diseases.

[0003] It has been shown that antibodies blocking the interaction between PD-1 and PD-L1 enhance the T cell response, particularly the response of CD8+ cytotoxic T cells (see Barber et al 2006 Nature Vol 439 p682 and Maier et al 2007 J. Immunol. Vol 178 p 2714).

[0004] International Publication No. 2006 / 042237 describes a diagnostic method for cancer by evaluating the expression of PD-L1(B7-H1) in tumors, and suggests delivering drugs that interfere with the PD-1 / PD-L1 interaction to patients. The interfering agents can be antibodies, antibody fragments, siRNA, or antisense oligonucleotides. There are no specific examples of such interfering agents, nor is there any mention of chronic liver infection.

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

[0006] Dolina et al. (2013, Molecular Therapy-Nucleic Acids, 2 e72) describe delivering siRNA-targeting PD-L1 to Kupffer cells in vivo, thereby enhancing NK and CD8+ T cell clearance in MCMV-infected mice. This paper concludes that siRNA-targeting PD-L1 delivered to hepatocytes is not effective in enhancing CD8+ T cell effector function.

[0007] The siRNA method differs considerably from the single-stranded antisense oligonucleotide method because their in vivo distribution and mode of action are completely different. As described by Xu et al 2003 Biochem.Biophys.Res.Comm.Vol 306 pp 712-717, antisense oligonucleotides and siRNAs have different priorities for targeting sites in mRNA.

[0008] International Publication No. 2016 / 138278 describes the inhibition of immune checkpoints, including PD-L1, using two or more single-stranded antisense oligonucleotides ligated to the 5' end. This application does not refer to hepatitis B virus (HBV) or targeted delivery to the liver. [Overview of the project]

[0009] This invention identifies novel oligonucleotides and oligonucleotide conjugates that highly efficiently reduce intracellular PD-L1 mRNA in both parenchymal cells (e.g., hepatocytes) and hepatocytes, as well as non-parenchymal cells such as Kupffer cells and sinusoidal endothelial cells (LSECs) of the liver. By reducing or silencing PD-L1, the oligonucleotides and oligonucleotide conjugates reduce PD-1-mediated inhibition, thereby promoting immune stimulation of exhausted T cells. By mitigating T cell exhaustion in chronic pathogenic liver infection, a decrease in serum viral antigen levels and restoration of immune regulation during chronic pathogenic liver infection are achieved. Natural killer (NK) cells and natural killer T (NKT) cells are also said to be activatable by the oligonucleotides and oligonucleotide conjugates of this invention.

[0010] Oligonucleotide conjugates reliably achieve local reduction of PD-L1 in hepatocytes, thereby reducing the risk of autoimmune side effects such as pneumonia, nonviral hepatitis, and colitis associated with systemic depletion of PD-L1.

[0011] This invention relates to oligonucleotides or conjugates that target nucleic acids capable of regulating PD-L1 expression, and to the treatment or prevention of diseases related to PD-L1 function. Oligonucleotides or oligonucleotide conjugates may be particularly used to treat diseases in which the immune response against infectious agents is exhausted.

[0012] Accordingly, in a first embodiment, the present invention provides an oligonucleotide comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, wherein the complementarity to the PD-L1 target nucleic acid is at least 90%. The oligonucleotide may be an antisense oligonucleotide, preferably having a gapmer design. Preferably, the oligonucleotide is capable of inhibiting PD-L1 expression by cleaving the target nucleic acid. Cleavage is preferably achieved by nuclease supplementation.

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

[0014] In a further embodiment, the present invention provides pharmaceutical compositions comprising oligonucleotides or oligonucleotide conjugates of the present invention, as well as pharmaceutically acceptable diluents, carriers, salts and / or auxiliaries.

[0015] In a further embodiment, the present invention provides an in vivo or in vitro method for reducing the expression level of PD-L1 in target cells expressing PD-L1 by administering an effective amount of the oligonucleotide or composition of the present invention to the cells.

[0016] In a further embodiment, the present invention provides oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions for use in restoring immunity against viruses or parasites.

[0017] In a further embodiment, the present invention provides oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions for use as pharmaceuticals.

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

[0019] In a further embodiment, the oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the present invention are used for 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 metastasis. [Brief explanation of the drawing]

[0020] [Figure 1A-B] Oligonucleotides are represented as wavy lines (AD), “oligonucleotide” (EH), or T2(I), illustrating exemplary antisense oligonucleotide conjugates where the asialocrypoprotein receptor targeting binding site is a trivalent N-acetylgalactosamine moiety. Compounds A-D contain a dilysine plug in the molecule for a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site without a linker. In compounds C and D, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site via a C6 linker. Compound EI contains a trebler branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 1C-E]Oligonucleotides are represented as wavy lines (AD), “oligonucleotide” (EH), or T2(I), illustrating exemplary antisense oligonucleotide conjugates where the asialocrypoprotein receptor targeting binding site is a trivalent N-acetylgalactosamine moiety. Compounds A-D contain a dilysine plug in the molecule for a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site without a linker. In compounds C and D, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site via a C6 linker. Compound EI contains a trebler branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 1F-H] Oligonucleotides are represented as wavy lines (AD), “oligonucleotide” (EH), or T2(I), illustrating exemplary antisense oligonucleotide conjugates where the asialocrypoprotein receptor targeting binding site is a trivalent N-acetylgalactosamine moiety. Compounds A-D contain a dilysine plug in the molecule for a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site without a linker. In compounds C and D, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site via a C6 linker. Compound EI contains a trebler branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 1I]Oligonucleotides are represented as wavy lines (AD), “oligonucleotide” (EH), or T2(I), illustrating exemplary antisense oligonucleotide conjugates where the asialocrypoprotein receptor targeting binding site is a trivalent N-acetylgalactosamine moiety. Compounds A-D contain a dilysine plug in the molecule for a PEG3 spacer and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site without a linker. In compounds C and D, the oligonucleotide attaches directly to the asialocrypoprotein receptor targeting binding site via a C6 linker. Compound EI contains a trebler branched molecule, a spacer of variable length and structure, and three terminal GalNAc carbohydrate moieties. [Figure 2] This graph shows the EC50 (A) and PD-L1 knockdown levels in % of physiological saline (B) for the compounds tested in Example 2, relative to their positions on the target nucleic acid. In this cell lineage, the compounds tested were THP1 (●) and Karpas (*). [Figure 3] This is the structural formula of a trivalent GalNAc colony (GN2). GN2 is useful as a binding site in the present invention. The wavy lines illustrate the binding sites in the population (e.g., to a C6 aminolinker, or directly to an oligonucleotide). [Figure 4] This is the structural formula for CMP ID number 766_2. [Figure 5] This is the structural formula for CMP ID number 767_2. [Figure 6] This is the structural formula for CMP ID number 768_2. [Figure 7] This is the structural formula for CMP ID number 769_2. [Figure 8] This is the structural formula for CMP ID number 770_2. [Figure 9A-C]After treatment with saline and the indicated CMP ID number, PD-L1 protein expression in the livers of poly(IC)-induced animals was detected by Western blotting. Each blot shows the same oligonucleotide, i.e., blot A) bare oligonucleotide version vs. GalNAc-bound version) 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 the vinculin-loading control, and the lower band is the PD-L1 protein. The first lane in each blot shows saline-treated mice without poly(IC) induction. These mice express only very small amounts of PD-L protein. [Figure 9D-E] After treatment with saline and the indicated CMP ID number, PD-L1 protein expression in the livers of poly(IC)-induced animals was detected by Western blotting. Each blot shows the same oligonucleotide, i.e., blot A) bare oligonucleotide version vs. GalNAc-bound version) 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 the vinculin-loading control, and the lower band is the PD-L1 protein. The first lane in each blot shows saline-treated mice without poly(IC) induction. These mice express only very small amounts of PD-L protein. [Figure 10]● 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-conjugated PD-L1 ASO + DNA vaccine (Group 8) The table shows the mononuclear cell population in the liver after treatment with either these, as well as the individual animals in each group. The mean for each group is shown by a vertical line (see Table 18). Statistical significance between the DNA vaccine group and the three treatment groups has been evaluated and, where present, is indicated by an asterisk (*) between the 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 CD4+ T cell fraction. C) represents the CD8+ T cell fraction. [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) - the regulation of PD-L1-positive cells in the liver after treatment. Individual animals in each group are shown, and the group average is shown by a vertical line (see Table 19). Statistical significance between the DNA vaccine group and the three treatment groups has been evaluated and is indicated by * between groups where it exists (*=P<0.05, and ****=P<0.0001). A) represents the percentage of CD8+ T cells expressing PD-L1 in the liver after treatment. B) represents the percentage of CD4+ T cells expressing PD-L1 in the liver after treatment. C) represents the percentage of B cells expressing PD-L1 in the liver after treatment. [Figure 12]● 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) The graphs show the HBV antigen-specific CD8+ cytokine-secreting cells in the liver after treatment with these vaccines, with the average for each group shown by a vertical line (see Table 20). Statistical significance between the DNA vaccine group and the three treatment groups has been evaluated and, where present, is indicated by an asterisk (* = P < 0.05). A) represents the percentage of IFN-γ-secreting CD8+ T cells in the liver that are specific to the HBV pre-S2+S antigen after treatment. B) represents the percentage of IFN-γ-secreting CD8+ T cells in the liver that are specific to the HBV core antigen after treatment. C) represents the percentage of IFN-γ and TNF-α secreting CD8+ T cells in the liver that are specific to the HBV pre-S2+S antigen after treatment. [Figure 13] Comparison of HBV-DNA, HBs antigen, and HBe antigen in AAV / HBV mice treated with GalNAc-conjugated PD-L1 antisense CMP number: 759_2 (▼) and the vehicle (■). Vertical lines indicate the end of treatment.

[0021] definition Oligonucleotides In this specification, the term “oligonucleotide” is generally defined as a molecule commonly understood by those skilled in the art, comprising two or more covalently bonded nucleosides. Such covalently bonded nucleosides may also be nucleic acid molecules or oligomers. Oligonucleotides are typically prepared in the laboratory (by solid-phase chemical synthesis) and subsequently purified. When referring to the sequence of an oligonucleotide, the sequence or order of the nucleic acid base portion of the covalently bonded nucleotide or nucleoside, or its 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 an oligonucleotide capable of regulating the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid. Antisense oligonucleotides are not inherently double-stranded and therefore are not siRNAs. The antisense oligonucleotides of the present invention are single-stranded, but preferred.

[0023] Sequential nucleotide sequence The term “continuous nucleotide sequence” refers to a region of oligonucleotide complementary to the target nucleic acid. This term is used herein synonymously with the terms “continuous nucleic acid base sequence” and “oligonucleotide motif sequence.” In some embodiments, all nucleotides of the oligonucleotide constitute a continuous nucleotide sequence. In some embodiments, the oligonucleotide includes adjacent nucleotide sequences and may optionally include further nucleotides (e.g., nucleotide linker regions that can be used to attach functional groups to the continuous nucleotide sequence). The nucleotide linker regions may or may not be complementary to the target nucleic acid.

[0024] nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of this invention, this includes both natural and unnatural nucleotides. In nature, nucleotides such as DNA and RNA nucleotides consist of a ribose sugar moiety, a nucleic acid base moiety, and one or more phosphate groups (which are not present in nucleosides). Nucleosides and nucleotides are also sometimes referred to as “units” or “monomers” for the same reason.

[0025] Modified nucleoside In this specification, the terms “modified nucleoside” or “nucleoside modification” refer to a nucleoside modified compared to an equivalent DNA or RNA nucleoside by introducing one or more modifications to the sugar moiety or (nucleo)base moiety. In a preferred embodiment, the modified nucleoside includes a modified sugar moiety. The term “modified nucleoside” may also be used herein to mean synonymously with the terms “nucleoside analog” or modified “unit” or modified “monomer.”

[0026] Inter-modified nucleoside bonding The term “modified nucleoside bond” is widely understood by those skilled in the art as a bond other than a phosphodiester (PO) bond, and is defined as a bond that covalently bonds two nucleosides together. Nucleotides having modified nucleoside bonds are also called “modified nucleotides.” In some embodiments, modified nucleoside bonds increase the nuclease resistance of oligonucleotides compared to phosphodiester bonds. In naturally occurring oligonucleotides, nucleoside bonds contain phosphate groups that form phosphodiester bonds between adjacent nucleosides. Modified nucleoside bonds are particularly useful for stabilizing oligonucleotides for in vivo use and can protect the DNA or RNA nucleoside regions in the oligonucleotides of the present invention from nuclease cleavage, for example, within the gap regions of gapmer oligonucleotides and within the regions of modified nucleosides.

[0027] In some embodiments, the oligonucleotide comprises one or more nucleoside bonds modified from a natural phosphate diester to, for example, a bond that enhances resistance to nuclease attack. Methods for quantifying nuclease resistance 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. Nucleoside bonds that can enhance the nuclease resistance of an oligonucleotide are called nuclease-resistant nucleoside bonds. In some embodiments, at least 50% of the nucleoside bonds in the oligonucleotide or its sequence of nucleotides (e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, or e.g., at least 90%) are modified. In some embodiments, all nucleoside bonds in the oligonucleotide or its sequence of nucleotides are modified. In some embodiments, it will be recognized that the nucleoside (e.g., the bond) linking the oligonucleotide of the present invention to a non-nucleotide functional group may be a phosphodiester. In some embodiments, all internucleoside bonds in the oligonucleotide or its continuous nucleotide sequence are nuclease-resistant internucleoside bonds.

[0028] The modified nucleoside linkage can be selected from the group including phosphorothioates, diphosphorothioates, and boranophosphates. In some embodiments, the modified nucleoside linkage is compatible with supplementation of RNase H with oligonucleotides of the present invention, such as phosphorothioates, diphosphorothioates, or boranophosphates.

[0029] In some embodiments, the nucleoside bond includes sulfur (S) (e.g., a phosphorothioate nucleoside bond).

[0030] Phosphothioate nucleoside binding is particularly useful for nuclease resistance, beneficial pharmacokinetics, and ease of manufacture. In some embodiments, at least 50% of the nucleoside binding in an oligonucleotide or its sequential nucleotide sequence is phosphorothioate (e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, or e.g., at least 90% of the nucleoside binding in an oligonucleotide or its sequential nucleotide sequence is phosphorothioate). In some embodiments, all of the nucleoside binding in an oligonucleotide or its sequential nucleotide sequence is phosphorothioate.

[0031] In some embodiments, the oligonucleotide comprises one or more neutral nucleoside bonds, particularly those selected from phosphotriesters, methylphosphonates, MMIs, amide-3s, formacetals, or thioformacetals.

[0032] Further nucleoside linkages are disclosed in International Publication No. 2009 / 124238 (incorporated herein by reference). In some embodiments, the nucleoside linkage is selected from the linkers disclosed in International Publication No. 2007 / 031091 (incorporated herein by reference). In particular, the nucleoside linkages are -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 H- is selectable from, and / or the internucleoside linker is -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- (where R H is selected from hydrogen and C1-4-alkyl).

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

[0034] However, each of the design regions may contain internucleoside linkages other than phosphorothioates, such as nucleoside linkages such as phosphodiester linkages, in regions where modified nucleosides such as locked nucleic acids (LNAs) protect the linkage from nuclease degradation. In particular, by including one or two linkages such as phosphodiester linkages between modified nucleoside units (or, usually in non-nuclease-recruiting regions), the bioavailability and / or biodistribution of the oligonucleotide can be modified. For this, reference is made to WO 2008 / 113832 pamphlet, which is incorporated herein by reference.

[0035] In one embodiment, all nucleoside bonds within the oligonucleotide are phosphorothioate bonds and / or boranophosphate bonds. It is preferable that all nucleoside bonds within the oligonucleotide are phosphorothioate bonds.

[0036] Nucleic acid bases The term "nucleic acid base" encompasses nucleosides and nucleotides that form hydrogen bonds in nucleic acid hybridization, as well as purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present within nucleotides. In the context of this invention, the term "nucleic acid base" also encompasses modified nucleic acid bases that are different from naturally occurring nucleic acid bases but function during nucleic acid hybridization. In this context, "nucleic acid base" refers to both naturally occurring nucleic acid bases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as variants that do not exist in nature. 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 nucleic acid base moiety is modified by changing a purine or pyrimidine to a modified purine or pyrimidine (e.g., substituted purines or substituted pyrimidines such as isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolocytosine, 5-propynylcytosine, 5-propynyluracil, 5-bromouracil, 5-thiazolouracil, 2-thiouracil, 2'-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine).

[0038] The nucleic acid base portion may be represented by the corresponding nucleic acid base letter code (e.g., A, T, G, C, or U), and each letter may optionally contain a modified base having an equivalent function. For example, in the exemplary oligonucleotide, the nucleic acid base portion is selected from A, T, G, C, and 5-methylcytosine. Optionally, 5-methylcytosine LNA nucleoside can be used as the LNA gapmer.

[0039] Modified oligonucleotides The term "modified oligonucleotide" refers to an oligonucleotide containing one or more sugar-modified nucleosides and / or inter-modified nucleoside bonds. 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 can contain nucleosides with modified nucleic acid bases. For example, 5-methylcytosine is often used as a substitute for cytosine, so the term complementarity is understood to encompass Watson-Crick base pairing between unmodified and modified nucleic acid bases (see, e.g., Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0041] In this specification, the term "% complementary" means that the proportion (expressed as a percentage) of nucleotides in a sequence of nucleotides within a nucleic acid molecule (e.g., oligonucleotide) is complementary at a given position to a sequence of nucleotides in a separate nucleic acid molecule (e.g., target nucleic acid) at a given position (i.e., forms a Watson-Crick base pair with this sequence of nucleotides). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with oligonucleotide sequences from the target sequences 5'-3' and 3'-5'), dividing this by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleic acid bases / nucleotides that do not align (form a base pair) are called mismatches.

[0042] The term "perfectly 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' (Sequence ID: 772) 3'cgtcatctcggttaat5' (Sequence ID: 5)

[0044] identity In this specification, the term "identity" refers to the percentage (expressed as a percentage) of nucleotides in a sequence of nucleotides within a nucleic acid molecule (e.g., oligonucleotide) that is identical at a given position to a sequence of nucleotides at a given position in a separate nucleic acid molecule (e.g., target nucleic acid) (i.e., the ability to form Watson-Crick base pairs with complementary nucleosides). The percentage is calculated by counting the number of identical aligned bases between two sequences, including a gap, dividing this by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Identity % = (Identical × 100) / Length of Aligned Region (with gap)

[0045] Hybridization In this specification, the terms “hybridize” or “to hybridize” are understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) that form a double helix by forming hydrogen bonds between base pairs on opposite strands. The affinity of the bond between the two nucleic acid strands is the strength of hybridization and is often defined as the melting temperature (T) at which half of the oligonucleotide doubles with the target nucleic acid. m This is explained in relation to physiological conditions T. m It 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, where ΔG° = -RTln(K d The dissociation constant of the reaction (K) (wherein R is the gas constant and T is the absolute temperature) d) is related to this. Therefore, a very low reactivity 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 where the water concentration is 1 M, the pH is 7, and the temperature is 37°C. Hybridization of oligonucleotides to target nucleic acids is a spontaneous reaction, and G° is set to less than zero so that a spontaneous reaction Δ is induced. ΔG° can be experimentally measured by isothermal titration calorimetry (ITC) as described, for example, 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 the nearest neighbor points described in SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, and the appropriately derived thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34: 11211-11216 and McTigue et al., 2004, Biochemistry 43: 5388-5405. To have the potential to modulate their intended nucleic acid targets through hybridization, the oligonucleotides of the present invention hybridize to target nucleic acids with estimated ΔG° values ​​of less than -10 kcal for oligonucleotides of 10–30 nucleotide lengths. In some embodiments, the degree or intensity of hybridization is measured by the standard-condition Gibbs free energy ΔG°. Oligonucleotides, in the case of oligonucleotides with a length of 8 to 30 nucleotides, 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). In some embodiments, oligonucleotides hybridize to target nucleic acids having 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 mammalian PD-L1, and may be, for example, a gene, RNA, mRNA, and premRNA, mature mRNA, or cDNA sequence. Therefore, the target may be referred to as a PD-L1 target nucleic acid. The oligonucleotide of the present invention may be, for example, a target exon region of mammalian PD-L1, or, for example, a target intron region of PD-L1 premRNA (see Table 1).

[0047] [Table 1]

[0048] Preferably, the target nucleic acid encodes a PD-L1 protein, particularly mammalian PD-L1, such as human PD-L1 (see, for example, Tables 2 and 3 which describe the mRNA and premRNA sequences of human, monkey, and mouse PD-L1). In the context of the present invention, premRNA is also considered a nucleic acid that encodes 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 their naturally occurring variants (e.g., sequences encoding the mammalian PD-L1 protein).

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

[0051] In vivo or in vitro applications, the oligonucleotides of the present invention can typically inhibit the expression of PD-L1 target nucleic acids in cells expressing PD-L1 target nucleic acids. The sequence of nucleic acid bases of the oligonucleotides of the present invention is typically complementary to the PD-L1 target nucleic acid when measured over the length of the oligonucleotide, excluding optionally one or two mismatches and optionally a nucleotide-based linker region that can ligate the oligonucleotide to any functional group (e.g., a conjugate) or other non-complementary terminal nucleotides (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 premRNA). In some embodiments, the target nucleic acid is RNA or DNA encoding a mammalian PD-L1 protein, such as human PD-L1, where the mammalian PD-L1 protein is a human PD-L1 premRNA sequence, e.g., disclosed as SEQ ID NO: 1, or a human mRNA sequence having NCBI reference number NM_014143. Further details regarding exemplary target nucleic acids are shown in Tables 2 and 3.

[0052] [Table 2]

[0053] [Table 3-1]

[0054] target sequence In this specification, the term “target sequence” refers to a sequence of nucleotides present in a target nucleic acid, comprising a nucleic acid base sequence complementary to the oligonucleotide of the present invention. In some embodiments, the target sequence consists of a region on the target nucleic acid that is complementary to the continuous nucleotide sequence of the oligonucleotide of the present invention. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide and may represent, for example, a preferred region of the 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 sequence of nucleotides (e.g., a subsequence of the target nucleic acid, such as the target sequence described herein) that is complementary to or hybridizes with the target nucleic acid.

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

[0060] target cell In this specification, the term “target cell” refers to a cell expressing the target nucleic acid. In some embodiments, the target cell may be in vivo or in vitro. In some embodiments, the target cell may be a mammalian cell such as a rodent cell (e.g., mouse cell or rat cell) or a primate cell (e.g., monkey cell or human cell).

[0061] In a preferred embodiment, target cells express PD-L1 mRNA, such as PD-L1 pre-mRNA or PD-L1 mature mRNA. The poly(A) 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, which may differ due to the presence of premRNA resulting from genetic coding degeneracy or alternative splicing that causes multiple codons encoding the same amino acid, or due to polymorphism (e.g., single-nucleotide polymorphism and allelic variants). Therefore, the oligonucleotides of the present invention can target the target nucleic acid and its naturally occurring variants based on the presence of sufficiently complementary sequences to the oligonucleotide.

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

[0064] [Table 3-2]

[0065] [Table 3-3]

[0066] Regulation of expression In this specification, the term “modulation of expression” should be understood as a comprehensive 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, the modulation of expression can be quantified by reference to a control experiment. The control is generally understood to be a saline composition treated with a non-target oligonucleotide (mock), an individual, or an individual or target cells treated with a non-target oligonucleotide (mock), but may also be an individual treated according to care standards.

[0067] One type of regulation is the ability of oligonucleotides to inhibit, downregulate, reduce, suppress, remove, interrupt, block, prevent, mitigate, decrease, evade, or stop PD-L1 expression, for example, by degrading mRNA or interfering with transcription. Another type of regulation is the ability of oligonucleotides to restore, increase, or enhance PD-L1 expression. This can be achieved by preventing splicing, removal, or blockage of inhibitory mechanisms such as splice site repair or microRNA repression.

[0068] High affinity modified nucleoside When a high-affinity modified nucleoside is incorporated into an oligonucleotide, it affects the oligonucleotide's complementary target, for example, its melting temperature (T mThese are nucleotides modified to enhance affinity as measured by ). The high affinity modified nucleosides of the present invention result in an increase in melting temperature between preferably +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art, including, for example, numerous 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 The oligomers of the present invention may contain one or more nucleosides having modified sugar moieties, compared to the modified sugar moieties, i.e., the ribose sugar moieties found in DNA and RNA.

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

[0071] Such modifications include, for example, modifications to the ribose ring structure 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 by substitution of an unconnected 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 (International Publication No. 2011 / 017521) or tricyclic nucleic acids (International Publication No. 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is substituted with a non-sugar moiety, for example, peptide nucleic acids (PNA) or morpholino nucleic acids.

[0072] Sugar modifications also include modifications made by changing substituents on the ribose ring to groups other than hydrogen, or to 2'-OH groups naturally present in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions. Nucleosides with modified sugar moieties also include 2'-modified nucleosides, such as 2'-substituted nucleosides. In fact, considerable focus has been placed on the development of 2'-substituted nucleosides, and many of them have been found to exhibit beneficial properties such as enhanced nucleoside tolerance and increased affinity when incorporated into oligonucleotides.

[0073] 2'-modified nucleoside.

[0074] 2'-sugar-modified nucleosides include those having substituents other than H or -OH at the 2' position (2'-substituted nucleosides) or 2'-bonded biradides, and include 2'-substituted nucleosides and LNA (2'~4' biradical-bridged) nucleosides. For example, 2'-modified sugars can result in increased binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted nucleosides include 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, for instance, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, as well as Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are some diagrams of 2'-substituted nucleosides.

[0075] [ka]

[0076] [ka]

[0077] Locked nucleic acid nucleosides (LNAs). LNA nucleosides are modified nucleosides that contain a linker group (called a biradical or bridge) between the C2' and C4' ends. These nucleosides are also referred to as cross-linked nucleic acids or bicyclic nucleic acids (BNAs) in the literature. In some embodiments, the oligomer-modified nucleoside or LNA nucleoside of the present invention has 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 )-, for example, selected from -O- in some embodiments, where B represents a nucleic acid base or modified nucleic acid base moiety, Z represents an internucleoside bond to an adjacent nucleoside or 5' terminal group, Z * represents an internucleoside bond to an adjacent nucleoside or 3' terminal group, and X 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 Represents a base selected from a list consisting of )- 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 Selected from the group consisting of )-. 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 This represents a group selected from the group consisting of )- and >C=Z. In some embodiments, Y is -CH2-, -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 It is selected from the group consisting of )=N-. In some embodiments, Y is -CH2-, -CHR a -, -CHCH3-, CR a R b - Alternatively, selected from the group consisting of -XY-, both representing a divalent linker group (also called a substrate), and -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 This represents a divalent linker group consisting of 1, 2, 3, or 4 groups / atoms selected from the group consisting of )- and >C=Z. In some embodiments, -XY- is -X-CH2-, -X-CR a R b -, -X-CHR a-、 -XC(HCH3) -、-O-Y-, -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-, N-O-CH2, -S-CR a R b -and -S-CHR a represents a radical selected from the group consisting of

[0080] In some embodiments, -X-Y- represents -O-CH2- or -O-CH(CHs)-, wherein Z is selected from -O-, -S-, and -N(R a ), and when R a represents R b , each is independently 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, sulphono, C 1-6-alkyl sulfonoxy, nitro, azide, sulfanyl, C 1-6 -Selected from alkylthio and halogen, aryl and heteroaryl may be optionally substituted, and two geminal substituents R a and R b Together, they can represent a methylene group (=CH2) that may be substituted, where the chiral group can be found in either the R or S orientation for all chiral centers. R 1 , R 2 , R 3 , R 5 and R 5* These 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, sulfon, C 1-6 -Alkyl sulfonyloxy, nitro, azide, sulfanyl, C 1-6-Selected from the group consisting of alkylthio and halogen, aryl and heteroaryl are optionally substituted, and the 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 Selected from alkyl groups (e.g., methyl and hydrogen).

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

[0083] In some embodiments, R 1 , R 2 , R 3 Both are hydrogen, and similarly R 5 and R 5* Either of them is hydrogen, and R 5 and R 5* The other of these is something other than hydrogen (for example, C such as methyl 1-6 It is alkyl.

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

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

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

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

[0088] In some embodiments, R a and R b Both are 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* All of these are hydrogen. Such LNA nucleosides are disclosed in International Publication No. 99 / 014226, International Publication No. 00 / 66604, International Publication No. 98 / 039352, and International Publication No. 2004 / 046160, all of which are referred to herein by reference, and include those commonly known 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* All of these are hydrogen atoms. Such thio-LNA nucleosides are disclosed in International Publication No. 99 / 014226 and International Publication No. 2004 / 046160. These 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*All of these are hydrogen atoms. Such amino-LNA nucleosides are disclosed in International Publication No. 99 / 014226 and International Publication No. 2004 / 046160. These applications are incorporated herein by reference.

[0092] In some embodiments, the biradical -XY- is -O-CH2-CH2- or -O-CH2-CH2-CH2-, where W is O and R 1 , R 2 , R 3 , R 5 and R 5* All of these are hydrogen. Such LNA nucleosides are disclosed in International Publication No. 00 / 047599, which is incorporated herein by reference, and in Morita et al, Bioorganic & Med. Chem. Lett. 12 73-76, and include those commonly known as 2'-O-4'C-ethylene-bridged nucleic acids (ENAs).

[0093] In some embodiments, the biradical -XY- is -O-CH2-, W is O, and R 1 , R 2 and R 3 All of and R 5 and R 5* One of them is hydrogen, and these R 5 and R 5* The other of these is a C other than hydrogen, for example, methyl. 1-6 Such 5'-substituted LNA nucleosides are disclosed in International Publication No. 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 One or both of them are not hydrogen (e.g., methyl), W is O, and R 1 , R 2 and R 3All of and R 5 and R 5* One of them is hydrogen, and these R 5 and R 5* The other of these is something other than hydrogen (for example, C such as methyl). 1-6 Such bis-modified LNA nucleosides are disclosed in International Publication No. 2010 / 077578, which is incorporated herein by reference.

[0095] In some embodiments, the biradical -XY- represents the divalent linker group -O-CH(CH2OCH3)-(2'O-methoxyethyl bicyclic nucleic acid (Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). In some embodiments, the biradical -XY- represents the divalent linker group -O-CH(CH2CH3)-(2'O-ethyl bicyclic nucleic acid (Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). In some embodiments, the biradical -XY- represents -O-CHR a - and W is O and R 1 , R 2 , R 3 , R 5 and R 5* All of these are hydrogen atoms. Such 6'-substituted LNA nucleosides are disclosed in International Publication No. 10036698 and International Publication No. 07090071. Both of these applications are incorporated herein by reference.

[0096] In some embodiments, the biradical -XY- is -O-CH(CH2OCH3)-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* All of these are hydrogen atoms. Such LNA nucleosides are also known in the art as cyclic MOEs (cMOEs) and are disclosed in International Publication No. 07090071.

[0097] In some embodiments, within either the -R- or S- configuration, the biradical -XY- represents the divalent linker group -O-CH(CH3)-. In some embodiments, both biradicals -XY- represent the divalent linker group -O-CH2-O-CH2 (Seth at al., 2010, J. Org. Chem). In some embodiments, the biradical -XY- is -O-CH(CH3)-, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* All of these are hydrogen atoms. Such 6'-methyl LNA nucleosides are also known in the art as cET nucleosides and may be either (S)cET or (R)cET stereoisomers disclosed in International Publication No. 07090071 (β-D) and International Publication No. 2010 / 036698 (α-L). Both of these applications are incorporated herein by reference.

[0098] In some embodiments, the biradical -XY- is -O-CR a R b - and R a or R b None of them are hydrogen, W is O, and R 1 , R 2 , R 3 , R 5 and R 5* All of them are hydrogen. In some embodiments, R a and R b Both are methyl. Such 6'-disubstituted LNA nucleosides are disclosed in International Publication No. 2009006478, which is incorporated herein by reference.

[0099] In some embodiments, the biradical -XY- is -S-CHR a - and W is O and R 1 , R 2 , R 3 , R5 and R 5* All of these are hydrogen. Such 6'-substituted thioLNA nucleosides are disclosed in International Publication No. 11156202, which is incorporated herein by reference. In some embodiments of 6'-substituted thioLNA, R a It is methyl.

[0100] In some embodiments, the biradical -XY- is -C(=CH2)-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* All of these are hydrogen. Such vinyl carbonate LNA nucleosides are disclosed in International Publication No. 08154401 and International Publication No. 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* All of them are hydrogen. In some embodiments, R a C is a compound such as methyl 1-6 It is alkyl. Such LNA nucleosides are also known as N-substituted LNAs and are disclosed in International Publication No. 2008 / 150729, which is incorporated herein by reference. In some embodiments, the biradical -XY- is a divalent linker group -O-NR a -CH3 is specified together (Seth at al., 2010, J. Org. Chem). In some embodiments, the biradical -XY- is -N(R a )-, W is O, and R 1 , R2 , R 3 , R 5 and R 5* All of them are hydrogen. In some embodiments, R a C 1-6 Alkyl, for example, methyl.

[0102] In some embodiments, R 5 and R 5* One or both of them are hydrogen, and when substituted, R 5 and R 5* The other of these is C 1-6 Alkyl, for example, methyl. In such embodiments, R 1 , R 2 , R 3 Both can be hydrogen, and the biradical -XY- can be -O-CH2- or -OC(HCR a )-, for example, -OC(HCH3)- can be selected.

[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* All of them are hydrogen. In some embodiments, R a C 1-6ア Lukyl, for example, methyl. Such LNA nucleosides are also known as stereoconstricted nucleotides (CRNs) and are disclosed in International Publication No. 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* All of them are hydrogen. In some embodiments, R a is C 1-6 Alkyl, for example, methyl. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, which is incorporated herein by reference.

[0105] Unless otherwise specified, it will be recognized that LNA nucleosides can be β-D or α-L stereoisomers.

[0106] An example of an LNA nucleoside is illustrated in Scheme 1.

[0107] [ka]

[0108] As illustrated in the examples, in some embodiments of the present invention, the LNA nucleoside in the oligonucleotide is a β-D-oxy-LNA nucleoside.

[0109] Nuclease-mediated degradation Nuclease-mediated degradation refers to oligonucleotides that can mediate the degradation of complementary nucleotide sequences when they form a double helix with such sequences.

[0110] In some embodiments, the oligonucleotides of the present invention can function via nuclease-mediated degradation of a target nucleic acid having the ability to replenish a nuclease, particularly an endonuclease, preferably an endonuclease (RNase), such as RNase H. The nuclease-mediated mechanism is an oligonucleotide that typically comprises at least five or six DNA nucleoside regions and is flanked on one or both sides by affinity-enhancing nucleosides, such as gapmers, headers, and tailmers.

[0111] RNase H activity and supplementation The RNase H activity of an antisense oligonucleotide refers to its ability to replenish RNase H when double-stranded with a complementary RNA molecule. International Publication No. 01 / 23613 provides an in vitro method for measuring RNase H activity that can be used to determine the ability to replenish RNase H. Oligonucleotides are typically considered to have the ability to replenish RNase H and, when a complementary target nucleic acid sequence is provided, have an initial velocity of at least 5%, e.g., at least 10% or more than 20% of the quantified initial velocity when the initial velocity is measured in pmol / l / min (when using an oligonucleotide having the same nucleotide sequence as the modified oligonucleotide under test, provided that it contains only DNA monomers with phosphorothioate bonds between all monomers in the oligonucleotide, and using the methodology described in Examples 91-95 of International Publication No. 01 / 23613, which is incorporated herein by reference).

[0112] Gapmar In this specification, the term “gapmer” refers to an antisense oligonucleotide that contains a region of RNase H-replenishing oligonucleotide (gap) adjacent to the 5' and 3' ends by a region containing one or more affinity-enhancing modified nucleosides (sides or wings). Various gapmer designs are described herein and are characterized by their ability to replenish RNase H. Headers and tailmers are oligonucleotides having the ability to replenish RNase H, with one of their sides missing, i.e., only one of the oligonucleotide ends containing an affinity-enhancing modified nucleoside. In the case of a head, the 3' side is missing (i.e., the 5' side contains an affinity-modified nucleoside), and in a tailmer, the 5' side is missing (i.e., the 3' side contains an affinity-modified nucleoside).

[0113] LNA gapmer 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 gap mar The term mixed wing gapmer or mixed lateral gapmer refers to an LNA gapmer in which at least one of the lateral 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 an LNA nucleoside (e.g., 5' or 3') on one lateral side and a 2'-substituted modified nucleoside and optionally an LNA nucleoside on the other lateral side (3' or 5', respectively).

[0115] Gap Breaker The term "gap-breaker oligonucleotide" is used for gapmers that have the ability to maintain RNase H replenishment even when the number of consecutive DNA nucleosides within the gap region falls below 5 as a result of the gap region being disrupted by a non-RNase H replenishment nucleoside (gap-breaker nucleoside, E). Non-RNase H replenishment nucleosides are, for example, nucleosides with a 3' terminal conformation such as LNA, where the crosslink between the C2' and C4' of the ribose sugar ring of the nucleoside is in the β conformation, such as β-D-oxy LNA or ScET nucleosides. The ability of gap-breaker oligonucleotides to replenish RNase H is typically sequence-specific, or even compound-specific (see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487). This document discloses "gap-breaker" oligonucleotides. These gap-breaker oligonucleotides supplement RNase H, which in some cases provides more specific cleavage of target RNA.

[0116] In some embodiments, the oligonucleotides of the present invention are gap-breaker oligonucleotides. In some embodiments, the gap-breaker oligonucleotide comprises a 5'-side (F), a gap (G), and a 3'-side (F'), and the gap is broken by a non-RNase H-supplemented nucleoside (gap-blocker nucleoside, E) so that the gap contains at least 3 or 4 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, is located within the gap region, the gap breaker LNA nucleoside is at least 3'(5') and 3(3') DNA nucleosides, or at least 3(5') and 4(3') DNA nucleosides, or 5' and 3' adjacent to at least 4(5') and 3(3') DNA nucleosides, and the oligonucleotide has the ability to replenish RNase H.

[0117] A gap-breaker oligonucleotide can be represented by the following equation. 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] Areas D' and D'' are described in the section on "Gapmer Design".

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

[0120] Conjugate In this specification, the term "conjugate" refers to an oligonucleotide covalently bonded to a non-nucleotide portion (a binding portion or region C or a third region), and is also called an oligonucleotide conjugate.

[0121] When the oligonucleotide of the present invention is conjugated to one or more non-nucleotide moieties, the pharmacology of the oligonucleotide can be improved. This is achieved by influencing the activity, cell distribution, cell uptake, or stability of the oligonucleotide. In some embodiments, the conjugation moieties target 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), thereby blocking the targeted activity or activity in untarget cell types, tissues, or organs. In one embodiment of the present invention, the oligonucleotide conjugate of the present invention shows improved inhibition of PD-L1 in target cells compared to the unconjugated oligonucleotide. In another embodiment, the oligonucleotide conjugate of the present invention shows improved cell distribution between the liver and other organs, such as the spleen or kidneys, compared to the unconjugated oligonucleotide. That is, the conjugated oligonucleotide tends to migrate to the liver rather than the spleen or kidneys. In another embodiment, it is revealed that the oligonucleotide conjugate of the present invention improves the cell uptake of the conjugated oligonucleotide into the liver compared to the conjugated oligonucleotide.

[0122] International Publication No. 93 / 07883 and International Publication No. 2013 / 033230 provide suitable binding sites, and these applications are incorporated herein by reference. The binding site is more preferably one that has the ability to bind to asialocytic protein receptors (ASGPr). In particular, trivalent N-acetylgalactosamine binding sites are suitable for binding to ASGr. See, for example, International Publication Nos. 2014 / 076196, 2014 / 207232, and 2014 / 179620, which are incorporated herein by reference. This binding site is essentially part of an antisense oligonucleotide conjugate that is not composed of nucleic acids.

[0123] Oligonucleotide conjugates and their synthesis are also reported in 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, and these documents are incorporated herein by reference in their entirety.

[0124] In one embodiment, the non-nucleotide portion (binding portion) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations 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 via one or more covalent bonds. The linking portion can be attached directly to an oligonucleotide or via a linking portion (e.g., a linker or tether). A linker functions to covalently bond a third region, for example, the linking portion (region C), to a first region, for example, an oligonucleotide or sequence of nucleotides complementary to the target nucleic acid (region A).

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

[0127] Region B refers to a biocleavable linker that contains or consists of a physiologically variable bond that can be cleaved under conditions normally encountered or similar to those encountered in the mammalian body. Conditions under which a physiologically variable linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or drugs, and salt concentrations found in or similar to those encountered in mammalian cells. The intracellular conditions of mammals also include the presence of enzyme activity normally present in mammalian cells, such as proteases, hydrolases or nucleases. In one embodiment, the biocleavable linker is sensitive to cleavage by an 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 2 consecutive phosphodiester links, e.g., at least 3, 4, or 5 consecutive phosphodiester links, or 2 to 4 linked nucleosides. The nucleosides are preferably DNA or RNA. A phosphodiester-containing biocleavable linker is described in detail in International Publication No. 2014 / 076195, which is incorporated herein by reference.

[0128] Region Y is a linker that, while not necessarily biocleavable, primarily functions to covalently bond the binding portion (region C or third region) to an oligonucleotide or continuous nucleotide sequence (region A or first region) complementary to the target nucleic acid. The linker in region Y may include a chain structure or oligomer of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugate of the present invention can be configured as ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl group (e.g., a C2-C36 aminoalkyl group such as a C6-C12 aminoalkyl group). In preferred embodiments, 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 a disease, i.e., prophylaxis. Thus, it will be appreciated that in some embodiments the treatment referred to herein can be prophylactic.

[0130] Restoration of the immune response to a pathogen The immune response is classified into innate and adaptive immune responses. The innate immune system provides an immediate but non-specific response. The adaptive immune response is activated by the innate immune response and is highly specific for a particular pathogen. When a pathogen-derived antigen is presented on the surface of an antigen-presenting cell, immune cells of the adaptive immune response (i.e., T lymphocytes and B lymphocytes) are activated via antigen-specific receptors, leading to the generation of a pathogen-specific immune response and immune memory. Chronic viral infections such as HBV and HCV are associated with T cell exhaustion characterized by non-responsiveness of virus-specific T cells. T cell exhaustion has been well studied; for reviews, see, e.g., Yi et al 2010 Immunology 129, 474-481. Chronic viral infection is also associated with reduced function of natural killer (NK) cells, which are innate immune cells. Enhancement of the viral immune response is important for clearance of chronic infection. Restoration of the immune response to a pathogen mediated by T cells and NK cells can be evaluated by measurement of proliferation, cytokine secretion and cytolytic function (Dolina et al. 2013 Molecular Therapy-Nucleic Acids, 2 e72, and Example 6 herein).

Mode for Carrying Out the Invention

[0131] The present invention relates to the use of antisense oligonucleotides and conjugates thereof, as well as pharmaceutical compositions containing them, for restoring the immune response against pathogens that infect animals, particularly humans. The antisense oligonucleotide conjugates of the present invention are particularly useful against pathogens infecting the liver, particularly chronic liver infections such as HBV. The conjugate enables targeting of the distribution of the oligonucleotide and prevents systemic knockdown of the target nucleic acid.

[0132] The oligonucleotide of the present invention The present invention relates to an oligonucleotide capable of regulating the expression of PD-L1. The regulation can be achieved by hybridization to a target nucleic acid encoding PD-L1 or a target nucleic acid involved in the regulation 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 may be pre-mRNA, mRNA, or any RNA sequence expressed from mammalian cells that supports the expression or regulation of PD-L1.

[0133] The oligonucleotide of the present invention is an antisense oligonucleotide that targets PD-L1.

[0134] In one aspect of the present invention, the oligonucleotide of the present invention is bound to a binding moiety, particularly an asialoglycoprotein receptor-targeting binding moiety.

[0135] In some embodiments, the expression of the target can be modulated by inhibiting or downregulating the antisense oligonucleotides of the present invention. Such modification preferably results in a normal expression level of the target of at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the normal expression level of the target. When cells or organisms are administered an antigen with an infectious agent, or treated with an agent that simulates antigen administration with an infectious agent (e.g., poly(I:C) or LPS), such modification preferably results in inhibition of expression of at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression level. In some embodiments, the oligonucleotides of the present invention may be able to inhibit the expression level of PD-L1 mRNA by at least 60% or 70% in vivo using KARPAS-299 or THP1 cells. In some embodiments, the compounds of the present invention may be able to inhibit the expression level of PD-L1 protein by at least 50% in vivo using KARPAS-299 or THP1 cells. Preferably, the examples provide assays that can be used to measure PD-L1 RNA (e.g., Example 1). The modulation of the target is induced by hybridization between the sequential nucleotide sequence of the oligonucleotide and the target nucleic acid. In some embodiments, the oligonucleotides of the present invention include a mismatch 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 the binding affinity is reduced as a result of the mismatch, it may be advantageous to compensate by increasing the number of nucleotides in the oligonucleotide and / or by enhancing the binding affinity to the target (e.g., a 2' modified nucleoside such as LNA present in the oligonucleotide sequence).

[0136] In some embodiments, the antisense oligonucleotides of the present invention have the function of restoring pathogen-specific T cells. In some embodiments, the oligonucleotides of the present invention have the function of increasing pathogen-specific T cells by at least 40%, 50%, 60%, or 70% compared to an untreated control or a control treated with standard care. In one embodiment, the antisense oligonucleotide or conjugate of the present invention has the function of increasing HBV-specific T cells compared to an untreated control or a control 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 oligonucleotide or conjugate of the present invention has the function of increasing HCV-specific T cells compared to an untreated control or a control treated with standard care. In another embodiment, the antisense oligonucleotide or conjugate of the present invention can increase HDV-specific T cells compared to an untreated control or a control treated with standard care.

[0137] In some embodiments, the antisense oligonucleotides of the present invention can reduce HBs antigen levels in animals or humans. In some embodiments, the oligonucleotides of the present 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. It is most preferable that the oligonucleotides of the present invention can achieve serological conversion of HBs antigen in HBV-infected animals or humans.

[0138] Aspects of the present invention relate to antisense oligonucleotides comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, having at least 90% complementarity to PD-L1 target nucleic acids.

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

[0140] In preferred embodiments, the oligonucleotides of the present invention, or sequences thereof, are 100% complementary to the region of the target nucleic acid, or in some embodiments, there may be one or two mismatches between the oligonucleotide and the target nucleic acid.

[0141] In some embodiments, the oligonucleotide comprises a continuous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary, for example, perfectly (i.e., 100%) complementary, to the 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 sequence of 10 to 30 nucleotides in length having at least 90% complementarity, e.g., 100% complementarity, to a corresponding target nucleic acid region, wherein the sequence of nucleotides is complementary to a subsequence of the target nucleic acid selected from the group consisting of positions 371-3068, 5467-12107, and 15317-19511 on 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 on 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 sequence of 10 to 30 nucleotides in length that is at least 90% complementary, for example 100%, to the corresponding target nucleic acid region present in SEQ ID NO:1, and the target nucleic acid region is selected from the group consisting of regions a1 to a449 in 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, 269, a274, a350, a360, a364, a365, a370, a372, a381, a383, a386, a389, a400, a427, a435, and a438.

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

[0150] In some embodiments, the oligonucleotides of the present 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 oligonucleotide comprises or consists of 16 to 20 nucleotides in length. It should be understood that any range indicated herein includes the endpoints of the range. Thus, when it can be said that an oligonucleotide contains 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, or 20 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 sequential nucleotide sequence includes or consists of a sequence selected from the group consisting of the sequences listed in Table 5.

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

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

[0155] In some embodiments, the antisense oligonucleotide or sequential nucleotide sequence is 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 A sequence selected from the group consisting of 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 contains or consists of 10 to 30 nucleotide lengths that are at least 90% identical, preferably 100% identical.

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

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

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

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

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

[0161] In embodiments where the oligonucleotide is longer than a continuous nucleotide sequence (complementary to the target nucleic acid), the motif sequences in Table 5 form the continuous nucleotide sequence portion of the antisense oligonucleotide of the present invention. In some embodiments, the oligonucleotide sequence is equivalent to a continuous nucleotide sequence (for example, when no biocleavable linker is added).

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

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

[0164] In one embodiment, the oligonucleotide comprises at least 1 modified nucleoside, for example, 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 another embodiment, the oligonucleotide comprises 1 to 10 modified nucleosides, for example, 2 to 8 modified nucleosides, for example, 3 to 7 modified nucleosides, for example, 4 to 6 modified nucleosides, for example, 3, 4, 5, 6, or 7 modified nucleosides.

[0165] In embodiments, 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, arabino nucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides. More preferably, the one or more modified nucleosides are locked nucleic acid (LNA).

[0166] In further embodiments, the oligonucleotide comprises at least one modified nucleoside bond. In preferred embodiments, all nucleoside bonds in a sequence of oligonucleotides are phosphorothioate or boranophosphate nucleoside bonds. In some embodiments, all nucleoside bonds in a sequence of oligonucleotides are phosphorothioate bonds.

[0167] In some embodiments, the oligonucleotide of the present invention comprises at least one LNA nucleoside, for example, LNA nucleoside 1, 2, 3, 4, 5, 6, 7, or 8, for example, LNA nucleosides 2-6, for example, LNA nucleosides 3-7, LNA nucleosides 4-6, or LNA nucleosides 3, 4, 5, 6, or 7. In some embodiments, at least 75% of the modified nucleosides in the oligonucleotide are LNA nucleosides, and for example, 80%, 85%, or 90% of the modified nucleosides are LNA nucleosides. In further embodiments, all modified nucleosides in the oligonucleotide are LNA nucleosides. In further embodiments, the oligonucleotide may contain 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 a β-D or α-L configuration or a combination thereof. In further embodiments, all LNA cytosine units are 5-methylcytosine. In preferred embodiments, the oligonucleotide or continuous 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, the oligonucleotide of the present invention comprises at least one modified nucleoside, which 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 oligonucleotide of the present invention comprises at least one LNA nucleoside and at least one 2'-substituted modified nucleoside.

[0170] In some embodiments of the present invention, the oligonucleotide comprises both 2'-sugar modified nucleosides and DNA units. Preferably, the oligonucleotide comprises both LNA and DNA nucleosides (units). Preferably, the total number of LNA and DNA units is 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., a continuous nucleotide sequence) of the oligonucleotide consists of at least one or two LNA nucleosides, and the remaining nucleosides are DNA units. In some embodiments, the oligonucleotide comprises only LNA nucleosides and naturally occurring nucleosides (RNA or DNA, most preferably DNA nucleosides), and optionally those having modified nucleoside bonds (e.g., phosphorothioates).

[0171] In embodiments of the present invention, the oligonucleotide of the present invention has the ability to replenish RNase H.

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

[0173] Gapmar Design In a preferred embodiment, the oligonucleotide of the present invention has a gapmer design or structure, also referred to herein simply as “gapmer”. In the gapmer structure, the oligonucleotide comprises at least three distinct structural regions, namely “5->3'” oriented FG-F', on the 5' side, the gap, and the 3'- side. In this design, the flanking F and F' regions (also called wing regions) contain a contiguous stretch of modified nucleotides complementary to the PD-L1 target nucleic acid, and the gap region G has the ability to replenish a nuclease, preferably an endonuclease such as an RNase, e.g., RNase H, when the oligonucleotide containing the contiguous stretch of nucleotides is double-stranded with the target nucleic acid. The nucleoside having the ability to replenish a nuclease, particularly an RNase H, can be selected from the group consisting of DNA, α-L-oxy-LNA, 2'-fluoro(Flouro)-ANA, and UNA. Regions F and F' adjacent to the 5' and 3' ends of region G preferably contain non-nuclease-supplemented nucleosides (nucleosides having a 3'-terminal structure), more preferably one or more affinity-enhancing modified nucleosides. In some embodiments, the 3' side contains at least one LNA nucleoside, preferably at least two LNA nucleosides. In some embodiments, the 5' side contains at least one LNA nucleoside. In some embodiments, both the 5' and 3' side regions contain LNA nucleosides. In some embodiments, all nucleosides in the side region are LNA nucleosides. In other embodiments, the adjacent region may contain both LNA nucleosides and other nucleosides (mixed side), such as DNA nucleosides and / or non-LNA-modified nucleosides (e.g., 2'-substituted nucleosides). In this case, the gap is defined as a sequence of at least five RNase H-refilled nucleosides (nucleosides having a 2' end structure, preferably DNA) adjacent to the 5' and 3' ends via affinity-enhancing modified nucleosides, preferably LNA (e.g., β-D-oxy-LNA).As a result, the nucleosides in the 5' and 3' adjacent regions to the gap region are modified nucleosides, preferably non-nuclease-supplemented nucleosides.

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

[0175] In some embodiments, the modified nucleoside within region F has a 3' terminal structure.

[0176] In one embodiment, one or more modified nucleosides within region F are 2'-modified nucleosides. In another embodiment, all nucleosides within region F are 2'-modified nucleosides.

[0177] In another embodiment, region F includes DNA and / or RNA in addition to 2'-modified nucleosides. The side containing DNA and / or RNA is characterized by having 2'-modified nucleosides within the 5' and 3' ends (adjacent to region G) of region F. In one embodiment, region F includes DNA nucleosides (e.g., 1-3 consecutive DNA nucleosides, such as 1-3 or 1-2 consecutive DNA nucleosides). The DNA nucleosides located on the side are preferably capable of naturally replenishing RNase H. In some embodiments, the 2'-modified nucleosides of region F, as well as the DNA and / or RNA nucleosides, are alternating with 1-3 2'-modified nucleosides and 1-3 DNA and / or RNA nucleosides. Such sides are sometimes referred to as alternating sides. The length of the 5' side (region F) in an oligonucleotide having alternating sides 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 5' sides of the oligonucleotide are alternating. A specific example of a region F having alternating nucleosides is: 2' 1-3 -N' 1-4 -2' 1-3 2' 1-2 -N' 1-2 -2' 1-2 -N' 1-2 -2' 1-2 That is the case.

[0178] In the formula, 2' represents a modified nucleoside and N' represents RNA or DNA. In some embodiments, all modified nucleosides within the alternating surface are LNA and N' is DNA. In further embodiments, one or more of the 2' 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, arabino nucleic acid (ANA) units, and 2'-fluoro-ANA units.

[0179] In some embodiments, the F region contains both LNA and a 2'-substituted nucleoside. These are often referred to as mixed wings or mixed side oligonucleotides.

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

[0181] Region G (gap region) preferably contains or consists of at least 4, for example, 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 having the ability to replenish the aforementioned nuclease, particularly RNase H. In a further embodiment, region G contains or consists of 5-12, or 6-10, or 7-9 (for example, 8 consecutive nucleotide units having the ability to replenish the aforementioned nuclease).

[0182] In one embodiment, the nucleoside units of region G having the ability to replenish nucleases are selected from the group consisting of DNA, α-L-LNA, C4' alkylated DNA (International Patent Application PCT / EP2009 / 050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300; both of these documents are incorporated herein by reference), as well as 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; these documents are incorporated herein by reference). UNAs are typically unlocked nucleic acids in which the bond between C2 and C3 of ribose has been removed, resulting in an unlocked "sugar" moiety.

[0183] In further embodiments, at least one nucleoside unit within region G is a DNA nucleoside unit (e.g., 1-18 DNA units (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 DNA units), preferably 2-17 DNA units (e.g., 3-16 DNA units (e.g., 4-15 DNA units), preferably 5-14 DNA units (e.g., 6-13 DNA units (e.g., 7-12 DNA units (e.g., 8-11 DNA units), more preferably 8-17 DNA units, or 9-16 DNA units, 10-15 DNA units, or 11-13 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 a further embodiment, region G may consist of a mixture of DNA capable of mediating RNase H cleavage and other nucleosides. 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 further embodiments, at least one nucleoside unit in region G is an α-L-LNA nucleoside unit (for example, at least one α-L-LNA (for example, 2, 3, 4, 5, 6, 7, 8, or 9α-L-LNA). In further embodiments, region G includes at least one α-L-LNA which is an α-L-oxy-LNA. In further embodiments, region G includes a combination of DNA and an α-L-LNA nucleoside unit.

[0186] In some embodiments, the nucleoside within region G has a 2' terminal structure.

[0187] In some embodiments, region G may contain a gap-breaker nucleoside that leads to a gap-breaker oligonucleotide having the ability to replenish RNase H.

[0188] Area F' Region F' (3' side or 3' wing) attached to the 3' end of region G contains, comprises, 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 one embodiment, region F' contains or consists of 1-7 modified nucleosides (e.g., 2-6 modified nucleosides (e.g., 2-4 modified nucleosides (e.g., 1-3 modified nucleosides (e.g., 1, 2, 3, or 4 modified nucleosides)). Region F' is defined by having at least modified nucleosides at its 5' and 3' ends.

[0189] In some embodiments, the modified nucleoside within region F' has a 3' terminal structure.

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

[0191] In a embodiments, one or more of the modified nucleosides within region F' are 2'-modified nucleosides.

[0192] In one embodiment, all nucleosides within region F' are 2'-modified nucleosides. In another embodiment, region F' includes DNA or RNA in addition to 2'-modified nucleosides. The side containing DNA and / or RNA is characterized by having 2'-modified nucleosides within the 5' end (adjacent to region G) and 3' end of region F'. In one embodiment, region F' includes DNA nucleosides (e.g., 1-4 consecutive DNA nucleosides, such as 1-3 or 1-2 consecutive DNA nucleosides). The DNA nucleosides located on the side are preferably capable of naturally replenishing RNase H. In some embodiments, the 2'-modified nucleosides in region F', as well as the DNA and / or RNA nucleosides, are alternating 1-3 2'-modified nucleosides and 1-3 DNA and / or RNA nucleosides, and such sides may be called alternating sides. The length of the 3' side (region F') in an oligonucleotide with alternating sides can be 4-10 nucleosides (e.g., 4-8 nucleosides, e.g., 4-6 nucleosides, e.g., 4, 5, 6, or 7 modified nucleosides). In some embodiments, only the 3' sides of the oligonucleotide are alternating. A specific example of a region F' having alternating nucleosides is: 2' 1-2 -N' 1-4 -2' 1-4 2' 1-2 -N' 1-2 -2' 1-2 -N' 1-2 -2' 1-2 That is the case.

[0193] In the formula, 2' represents a modified nucleoside and N' represents RNA or DNA. In some embodiments, all modified nucleosides within the alternating surface are LNA and N' is DNA. In further embodiments, the modified nucleoside within region F' is 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, arabino nucleic acid (ANA) units, and 2'-fluoro-ANA units.

[0194] In some embodiments, the F' region contains both an LNA and a 2'-substituted nucleoside. These are often referred to as mixed wings or mixed lateral oligonucleotides.

[0195] In one embodiment, all modified nucleosides within region F' are LNA nucleosides. In a further embodiment, all modified nucleosides within region F' are LNA nucleosides. In a further embodiment, the LNA nucleosides within region F' are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and / or ENA, either in a 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 a continuous sequence.

[0196] Regions D' and D'' Regions D' and D'' can be joined to the 5' end of region F or the 3' end of region F', respectively. The choice between region D' and D'' is arbitrary.

[0197] Region D' or D'' may contain 0 to 5 additional nucleotides, e.g., 1 to 5, e.g., 2 to 4, e.g., 0, 1, 2, 3, 4, or 5, which may be complementary or non-complementary to the target nucleic acid. In this regard, the oligonucleotides of the present invention may, in some embodiments, include a sequence of nucleotides that can modulate the target adjacent to the 5' and / or 3' ends with further nucleotides. Such additional creotides may function as nuclease-sensitive biocleavable linkers (see definition of linker). In some embodiments, the further 5' and / or 3' terminal nucleosides may be bound to a phosphodiester bond and may be DNA or RNA. In another embodiment, the additional 5' and / or 3' terminal nucleosides may be modified nucleosides, for example, to enhance nuclease stability or for ease of synthesis. In one embodiment, the oligonucleotides of the present invention include region D' and / or D'' at the 5' or 3' end of a sequence of nucleotides. In a further embodiment, the D' and / or D'' regions consist of 1-5 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] The preferred number and types of nucleosides within regions F, G and F', D' and D'' are as described above. The oligonucleotide conjugate of the present invention has region C, in particular the gapmer oligonucleotide shown above, covalently bonded to either the 5' or 3' end of the oligonucleotide.

[0200] In one embodiment, the oligonucleotide conjugate of the present invention comprises an oligonucleotide of formula 5'-D'-FG-F'-3' or 5'-FG-F'-D”-3', wherein regions F and F' independently comprise 1-7 modified nucleosides, G is a region between 6-16 nucleosides having the ability to replenish RNase H, and region D' or D” comprises 1-5 phosphodiester linked nucleosides. Region D' or D” is preferably located at the terminal end of the oligonucleotide intended to bind to the binding site.

[0201] An example of an oligonucleotide with alternating sides can be represented by the following equation. 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 denoted as F or F' and contain only 2'-modified nucleosides such as LNA nucleosides. The number and types of preferred 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, and 22 nucleotides in length, where each F and F' region independently consists of 1, 2, 3, or 4 modified nucleoside units complementary to the PD-L1 target nucleic acid, and region G consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17 nucleoside units that can supplement nucleases when double-stranded with the PD-L1 target nucleic acid, and region D' consists of two phosphodiester-linked DNAs.

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

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

[0206] In a more specific embodiment, the oligonucleotide is a gapmer in which each F region and F' region consists of three LNA units independently, and region G consists of 8, 9, 10, 11, 12, 13, or 14 nucleoside units, preferably DNA units. Specific gapmer designs with this property 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 in which each F and F' region consists of four LNA units, and region G consists of 8, 9, 10, 11, or 12 nucleoside units, preferably DNA units. Specific gapmer designs with this property include 4-8-4, 4-9-4, 4-10-4, 4-11-4, and 4-12-4.

[0208] Specific gapmer designs for this property include FG-F' designs selected from the group consisting of a gap containing 6 nucleosides and independently 1-4 modified nucleosides within the wings, such as 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] Examples of specific gapmer designs for this property include FG-F' designs, which are selected from the group consisting of a gap containing 7 nucleosides and independently 1-4 modified nucleosides within the wings, such as 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] Examples of specific gapmer designs for this property include FG-F' designs, which are selected from the group consisting of gapmers having 8 nucleosides and independently 1 to 4 modified nucleosides within the wings, such as 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] Examples of specific gapmer designs for this property include FG-F' designs, which are selected from the group consisting of a gap containing 9 nucleosides and independently 1-4 modified nucleosides within the wings, such as 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 for this property include FG-F' designs selected from the group consisting of gapmers containing 10 nucleosides, such as 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 for this property include FG-F' designs selected from the group consisting of gapmers containing 11 nucleosides, such as 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.

[0214] Specific gapmer designs for this property include FG-F' designs selected from the group consisting of gapmers containing 12 nucleosides, such as 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.

[0215] Specific gapmer designs for this property include FG-F' designs selected from the group consisting of gapmers containing 13 nucleosides, such as 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.

[0216] Specific gapmer designs for this property include FG-F' designs selected from the group consisting of gapmers containing 14 nucleosides, such as 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.

[0217] A specific gapmer design for this property is the FG-F' design, which is selected from the group consisting of gapmers containing 15 nucleosides, such as 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.

[0218] A specific gapmer design for this property is the FG-F' design, which is selected from the group consisting of gapmers containing 16 nucleosides, such as 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] A specific gapmer design for this property is the FG-F' design, which is selected from the group consisting of gapmers containing 17 nucleosides, such as 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.

[0220] In all examples, 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 in regions F and F' are preferably modified nucleosides, and the nucleotides in region G are preferably unmodified nucleosides.

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

[0222] In another embodiment, all nucleoside bonds in the gap within the gapmer are phosphorothioate and / or boranophosphate bonds. In another embodiment, all nucleoside bonds in the sides (F and F' regions) within the gapmer are phosphorothioate and / or boranophosphate bonds. In another preferred embodiment, all nucleoside bonds in the D' and D'' regions of the gapmer are phosphodiester bonds.

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

[0224] In certain embodiments, the gapmer is a so-called shortmer as described in International Publication No. 2008 / 113832, which is incorporated herein by reference.

[0225] Further Gapmer designs are disclosed in International Publication No. 2004 / 046160 and International Publication No. 2007 / 146511, which are incorporated by reference.

[0226] In one embodiment of the present invention, the oligonucleotide is selected from the group consisting of oligonucleotide compounds with CMP-ID numbers 5_1 to 743_1 and 771_1.

[0227] In one embodiment of the present invention, 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, 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 The oligonucleotide compounds are selected from the group consisting of 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 a preferred embodiment of the present invention, the oligonucleotide is CMP-ID number: 287_1.

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

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

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

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

[0233] In further embodiments of the present invention, the oligonucleotide motifs and oligonucleotide compound sequences of the present invention include a sequence of nucleotides (e.g., region D') at the 5' end of a linked nucleoside of 2-4 additional phosphodiesters. In one embodiment, the nucleoside acts as a biocleavable linker (see the section “Biocleavable Linker”). In a preferred embodiment, a ca(cytidine-adenosine) dinucleotide is linked to the 5' end of the sequence of nucleotides (i.e., any one of the motif sequences or oligonucleotide compounds listed in Table 5) via a phosphodiester bond. In a preferred embodiment, the double-stranded nucleotide is not complementary to the target sequence at a position where the reminder of the sequence of nucleotides is complementary.

[0234] In some embodiments of the present invention, the oligonucleotide or continuous nucleotide sequence is selected from the group consisting of motif sequences of sequence numbers 766, 767, 768, 769 and 770.

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

[0236] Carbohydrate binding portion The carbohydrate binding sites 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, European Patent No. 1495769, International Publication No. 99 / 65925, Yang et al., Bioconjug Chem (2009) 20(2): 213-21, and Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10): 1401-17.

[0237] In some embodiments, the carbohydrate-binding moiety is polyvalent, and for example, two, three, or four identical or non-identical carbohydrate moieties can optionally be covalently bonded to the oligonucleotide via a linker or linker. In some embodiments, the present invention provides a conjugate comprising the oligonucleotide and the carbohydrate-binding moiety of the present invention.

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

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

[0240] One aspect of the present invention is an antisense oligonucleotide conjugate comprising: a) an oligonucleotide (region A) having a continuous nucleotide sequence of 10 to 30 nucleotides in length having at least 90% complementarity to a PD-L1 target nucleic acid; and b) at least one asialocrypoprotein receptor targeting binding moiety (region C) covalently bonded to the oligonucleotide in a). The oligonucleotide or continuous nucleotide sequence may be as described in any of the sections “Oligonimbus of the Present Invention”, “Oligonimbus 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., it comprises one, two, three, or four terminal carbohydrate moieties capable of binding to the asialoglycoprotein receptor). The asialoglycoprotein receptor targeting binding moiety is divalent, and more preferably trivalent. In preferred embodiments, the asialoglycoprotein receptor targeting binding moiety comprises one to three N-acetylgalactosamine (GalNAc) moieties (also called GalNAc conjugates). In some embodiments, the oligonucleotide conjugate includes an asialocrycoprotein receptor targeting binding moiety, which is a trivalent N-acetylgalactosamine (GalNAc) moiety. The GalNAc conjugate is a phosphodiester, methylphosphonate, and PNA antisense oligonucleotide (e.g., U.S. Patent No. 5,994517, 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., International Publication No. 2009 / 126933, International Publication No. 2012 / 089352, and International Publication No. 2012 / 083046), and have been used in combination with LNA and 2'-MOE modified nucleosides (International Publication No. 2014 / 076196, International Publication No. 2014 / 207232, and International Publication No. 2014 / 179620). These documents are incorporated herein by reference.

[0242] To generate an asialoglycoprotein receptor targeting binding moiety, the ASPGr-binding carbohydrate moiety (preferably GalNAc) is bound 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 5885968; Biessen et al. J. Med. Chern. 1995 Vol. 39 p. 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 binding of two or three terminal ASPGr-binding carbohydrate moieties and further allows binding to oligonucleotides at the branching point. A typical branched molecule is a dyridine. The dyridine molecule contains three amine groups to which three ASPGr-binding carbohydrate moieties can be bound, and a carboxyl reactive group to which the dyridine can bind to an oligonucleotide. Alternative branched molecules may be doublers or treblers, such as those supplied by Glen Research. In some embodiments, the blanchers are 1,3-bis-[5-(4,4'-pentylamide]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 The following can be selected from the group consisting of Research catalog number: 10-1922-xx), tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 1-[5-(4,4'-dimethoxy-trityloxy)pentylamide]-3-[5-fluormethoxycarbonyloxy-pentylamide]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research catalog number: 10-1925-xx).International Publication No. 2014 / 179620 and International Application PCT / EP2015 / 073331 describe the formation of various GalNAc binding moieties, 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 the linkers described in “Linkers” and its subsections.

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

[0244] Pharmacokinetic modifiers related to siRNA delivery are described in International Publication No. 2012 / 083046 (incorporated herein by reference). In some embodiments, the carbohydrate-binding moiety comprises a hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16 to 20 carbon atoms, a pharmacokinetic modifier selected from the group consisting of palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl and cholesterol. In preferred embodiments, the pharmacokinetic modifier containing the carbohydrate-binding moiety is a GalNAc conjugate.

[0245] The carbohydrate binding moiety preferably contains 1 to 3 terminal ASPGr-binding carbohydrate moieties, preferably N-acetylgalactosamine moieties. In some embodiments, the carbohydrate binding moiety contains 3 ASPGr-binding carbohydrate moieties, preferably N-acetylgalactosamine moieties, linked to the branched molecule via a spacer. The spacer molecule may be 8 to 30 atoms long. A preferred carbohydrate binding moiety contains 3 terminal GalNAc moieties linked to the dilysine branched molecule via a PEG spacer. A preferred PEG spacer is a 3PEG spacer. A preferred asialoglycoprotein receptor targeting binding moiety is shown in Figure 1. A preferred asialoglycoprotein receptor targeting binding moiety is shown in Figure 3.

[0246] Other GalNAc binding sites include, for example, small peptides with a bound GalNAc moiety such as Tyr-Glu-Glu-(aminohexylGalNAc)3 (YEE(ahGalNAc)3); glycotripeptides that bind to asialoglycoprotein receptors on hepatocytes (see, e.g., Duff, et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose colonies (L3G4; Biessen, et al., Cardovasc. Med., 1999, 214); and coran-based galactose colonies (e.g., sugar recognition motifs of asialoglycoprotein receptors).

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

[0248] In a preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound shown in Figure 4.

[0249] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound illustrated in Figure 5.

[0250] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound illustrated in Figure 6.

[0251] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound illustrated in Figure 7.

[0252] In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound illustrated in Figure 8.

[0253] Linker Biologically cleavable linker (region B) The use of conjugates is often associated with enhancing pharmacokinetic or pharmacodynamic properties. However, the presence of a binding site can impede the activity of the intended target oligonucleotide, for example, through steric hindrance that prevents hybridization or nuclease replacement (e.g., RNase H). By using a physiologically readily variable linker (biodegradable linker) between the oligonucleotide (region A or 1st region) and the binding site (region C or 3rd region), it is possible to improve the properties caused by the presence of the binding site, while ensuring that the binding site does not impede the effective activity of the oligonucleotide once it is in the target tissue.

[0254] When molecules containing mutable bonds reach a suitable intracellular or extracellular environment, physiologically mutable bonds spontaneously arise. For example, when a molecule enters an acidified endosome, pH-mutable bonds may be cleaved. Therefore, pH-mutable bonds can be considered endosomal cleavable bonds. Enzyme-cleavable bonds may be cleaved when exposed to enzymes, such as enzymes present in endosomes, lysosomes, or the cytoplasm. When a molecule enters the highly reducing environment of the cytoplasm, disulfide bonds may be cleaved. Therefore, disulfides can be considered cytoplasmic cleavable bonds. In this specification, pH-mutable bonds are mutable bonds that are selectively destroyed under acidic conditions (pH < 7). Since cellular endosomes and lysosomes have a pH of less than 7, such bonds may also be called endosomal mutable bonds.

[0255] For biocleavable linkers associated with binding sites for targeted delivery, it is preferable that the cleavage rate observed in target tissue (e.g., muscle, liver, kidney, or tumor) is greater than that observed in serum. A preferred method for quantifying the level of cleavage (%) in target tissue relative to serum or S1 nuclease cleavage is described in the "Materials and Methods" section. In some embodiments, biocleavable linkers (also called physiologically mutable linkers, nuclease-sensitive linkers, or region B) are cleaved by at least about 20%, e.g., at least about 30%, e.g., at least about 40%, e.g., at least about 50%, e.g., at least about 60%, e.g., at least about 70%, e.g., at least about 75%) compared to the standard.

[0256] In some embodiments, the oligonucleotide conjugate of the present invention comprises three regions: i) a first region (region A) containing 10 to 25 consecutive nucleotides complementary to the target nucleic acid; ii) a second region (region B) containing a biocleavable linker; and iii) a third region (region C) containing a binding site such as an asialoclyoglycoprotein receptor targeting binding site, wherein the third region is covalently bonded to the second region, which is covalently bonded to the first region.

[0257] In one embodiment of the present invention, the oligonucleotide conjugate includes a biocleavable linker (region B) between a continuous nucleotide sequence (region A) and an asialoclyoglycoprotein receptor targeting binding region (region C).

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

[0259] In some embodiments, the cleavable linker is sensitive to a nuclease that may be expressed in the target cell, for example. In some embodiments, the biocleavable linker consists of 2 to 5 consecutive phosphodiester bonds. The linker may be a short region of phosphodiester-linked nucleosides (e.g., 1 to 10 as detailed in the definition of the linker). In some embodiments, the nucleosides in biocleavable linker region B are selected (optionally and independently) from the group consisting of DNA and RNA or their modifications that do not interfere with nuclease cleavage. Modifications of DNA and RNA nucleosides that do not interfere with nuclease cleavage may be nucleic acid bases that do not exist in nature. Certain sugar-modified nucleosides may also enable nuclease cleavage, such as α-L-oxy-LNA. In some embodiments, all nucleosides in region B contain (optionally and independently) either 2'-OH ribose sugar (RNA) or 2'-H sugar (i.e., RNA or DNA). In a preferred embodiment, at least two consecutive nucleosides in region B are DNA or RNA nucleosides (such as at least 3, 4, or 5 consecutive DNA or RNA nucleotides). In a more preferred embodiment, it is preferable that the nucleosides in region B are DNA nucleosides consisting of 1 to 5 or 1 to 4 (e.g., 2, 3, 4 consecutive phosphodiester-linked DNA nucleosides). In a preferred embodiment, region B is short enough that RNase H is not added. 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] If region B consists of phosphodiester-linked nucleosides, regions A and B may collectively form an oligonucleotide linked to region C. In this context, region A can be distinguished from region B, and region A contains at least one and two modified nucleosides with enhanced binding affinity to the target nucleic acid (e.g., LNA or nucleoside having a 2'-substituted sugar moiety) and region A, which can themselves regulate the expression of the target nucleic acid in the relevant cell lineage. Furthermore, if region A contains DNA or RNA nucleosides, these nucleosides are linked to nuclease-resistant nucleoside bonds, such as phosphorothioates or boranophosphates. On the other hand, region B contains phosphate ester bonds 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 is not complementary to the sequence of nucleotides complementary to the target nucleic acid within region A.

[0262] In some embodiments, region B is complementary to the target nucleic acid sequence. In this respect, regions A and B together can form a single continuous sequence complementary to the target sequence.

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

[0264] In some embodiments, the nucleotide sequence of region B is selected to provide an optimal endonuclease cleavage site based on the major endonuclease cleavage enzyme present in the compartment of the target tissue or cell or subcell. In this regard, by isolating cell extracts from target and non-target tissues, the endonuclease cleavage sequence for use in region B can be selected based on its preferential cleavage activity in the desired target cell (e.g., liver / hepatocyte) compared to non-target cells (e.g., kidney). In this regard, the potency of the down-control compound can be optimized for the desired tissue / cell.

[0265] In some embodiments, region B comprises a dinucleotide of sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, where C may be 5-methylcytosine and / or T may be substituted with U, and the nucleoside bond is preferably a phosphodiester bond. In some embodiments, region B preferably comprises the sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, and GGG trinucleotides, where C may be 5-methylcytosine and / or T may be substituted with U, and the nucleoside bond is preferably a phosphodiester bond. 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 The trinucleotides include 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, where X can be selected from the group consisting of A, T, U, G, C and their analogs, C may be 5-methylcytosine, and / or T can be substituted with U. The nucleoside bond is preferably a phosphodiester bond.When referring to (naturally occurring) nucleic acid bases A, T, U, G, and C, it is recognized that these are substituted with nucleic acid base analogs that function as equivalent natural nucleic acid bases (e.g., base pairs with complementary nucleosides).

[0266] Other linkers (region Y) A linker may have at least two functional groups (one bonded to an oligonucleotide and the other to the binding site). Exemplary linker functional groups may be electrophilic to react with nucleophilic groups on the oligonucleotide or binding site, or nucleophilic to react with electrophilic groups. In some embodiments, linker functional groups include, but are not limited to, phosphorothioates, phosphates, phosphites, and unsaturated groups (e.g., double or triple bonds). 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-amide-caproic acid) (LCSMCC), succinimidyl m-maleimido-benzoylate (MBS), succinimidyl Examples include Ne-maleimido-caproic acid (EMCS), succinimidyl 6-(beta-maleimido-propionamide) 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), allylenediol, polyethylene glycol, and amino acids. In some embodiments, the linker (region Y) includes an aminoalkyl group, such as a C2-C36 aminoalkyl group or a C6-C12 aminoalkyl group. In preferred embodiments, 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 aminoalkylphosphoramidites.The linking group between the aminoalkyl and oligonucleotide may be, for example, a phosphorothioate or phosphodiester, or one of the other nucleoside linking groups referred to herein. The aminoalkyl group is covalently bonded to the 5' or 3' end of the oligonucleotide. Commercially available aminoalkyl linkers include, for example, 5'-aminomodifying enzyme C6, a 3'-aminomodifying reagent for linking at the 3' end of oligonucleotides and for linking at the 5' end of oligonucleotides. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar compounds were used by Krieg, et al, Antisense Research and Development 1991, 1, 161, for the purpose of linking fluorescein to the 5' end of oligonucleotides. A wide variety of further linker groups are known in the art and may be useful for linking the binding moiety 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 polymethylene bonds (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 further linkers (region Y-Y' or region YB or BY).

[0267] The use of linkers and linkers during the preparation of oligonucleotide conjugates is described in International Publication No. 96 / 11205, International Publication No. 98 / 52614, U.S. Patent 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. These specifications are provided through the technical fields in International Publication No. 2012 / 083046, and the entirety of each of these applications is incorporated by reference.

[0268] Manufacturing method In a further embodiment, the present invention provides a method for producing oligonucleotides of the present invention, which comprises reacting nucleotide units to form a covalently linked sequence of nucleotide units contained within the oligonucleotide. This method preferably utilizes phosphoramidite chemistry (see, for example, Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment, the method further comprises reacting the sequence of nucleotides with a binding site (ligand). Further embodiments provide a method for producing a composition of the present invention, comprising mixing the oligonucleotide or bound oligonucleotide of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or auxiliary agent.

[0269] Pharmaceutical composition In a further embodiment, the present invention provides a pharmaceutical composition comprising the aforementioned oligonucleotides and / or oligonucleotide conjugates, and any of a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or auxiliary agent. Examples of pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). Examples of pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate-buffered saline. In some embodiments, the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50 to 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 overview 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 International Publication No. 2007 / 031091 (incorporated herein by reference). Suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also described in International Publication No. 2007 / 031091.

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

[0272] These compositions can be sterilized using conventional sterilization techniques or by sterile filtration. The resulting aqueous solutions may be packaged for immediate use, or they may be freeze-dried, and the freeze-dried formulation may be combined with a sterile aqueous carrier before administration. The pH of the preparations 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). The compositions obtained in solid form can be packaged in multiple single-dose units, each containing a fixed amount of the drug, such as in a sealed tablet or capsule package. The solid compositions can also be packaged in flexible containers, such as a squeeze tube designed for topically applied creams or ointments.

[0273] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is a prodrug. In particular, with respect to oligonucleotide conjugates, when the prodrug is delivered to the site of action (e.g., target cells), the binding site is cleaved from the oligonucleotide.

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

[0275] In this research, such oligonucleotides or oligonucleotide conjugates are used to specifically regulate the synthesis of PD-L1 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating the functional analysis of the target or evaluation of its usefulness as a target for therapeutic intervention. Target regulation is typically achieved by degrading or inhibiting the protein-producing mRNA, thereby preventing protein synthesis, or by degrading or inhibiting the protein-producing gene or mRNA regulators.

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

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

[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 preferred embodiments, the target cells are located in the liver. Hepatocyte target cells can be selected from parenchymal cells (e.g., hepatocytes) as well as non-parenchymal cells such as Kupffer cells, LSECs, astrocytes (or Ito cells), cholangiocarcinomas, 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 that are complexed with major histocompatibility complex (MHC) class I or class II. In some embodiments, the antigen-presenting cells express MHC class II (i.e., professional antigen-presenting cells such as dendritic cells, macrophages, and B cells).

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

[0280] The oligonucleotides or oligonucleotide conjugates of the present invention, or pharmaceutical compositions thereof, can be administered to animals or humans suspected of having a disease or disorder, and the disease or disorder 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 a therapeutically or preventively effective amount of the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention to a subject who is suffering from or prone to the disease.

[0282] Furthermore, the present invention relates to oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions for use as pharmaceuticals.

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

[0284] The present invention also provides the use of oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the present invention, as described herein in relation to the manufacture of pharmaceuticals for treating diseases or disorders mentioned herein. In one embodiment, the diseases are 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 metastasis in the liver.

[0285] In one embodiment, the present invention relates to oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions for use in the treatment of diseases or disorders selected from viral or parasitic infections. In further embodiments, the diseases are 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 metastasis in the liver.

[0286] The diseases or disorders referred to herein are related to immune exhaustion. In particular, the diseases or disorders are associated with exhaustion of the virus-specific T cell response. In some embodiments, the diseases or disorders can be mitigated 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 related to immune exhaustion.

[0288] In one embodiment of the present invention, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention is used to restore the immune response to 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 may be found in the liver. The pathogen may be a virus or a parasite, in particular those described herein. In a preferred embodiment, the pathogen is HBV.

[0290] The present invention further relates to the use of oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions as defined herein for the purpose of producing drugs for restoring immunity against viral or parasitic infections as described herein.

[0291] The oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions 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). Viral hepatitis infections can be selected from the group consisting of hepatitis viruses, particularly HBV, HCV, and HDV, and especially chronic forms of these infections. In one embodiment, the oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention are used for the treatment of HBV, particularly chronic HBV. Indicators of chronic HBV infection are high levels of viral load (HBV DNA) and higher levels of empty HBs antigen particles in the circulatory system (more than 100 times the number of viral particles).

[0292] The oligonucleotides or oligonucleotide conjugates of the present invention can also be used to treat viral hepatitis infections that develop as co-infections with HIV. Other viral infections that can be treated with the oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention include lcmv (lymphochoroidal meningitis virus), as well as HIV, HSV-1, HSV-2, and other herpesviruses as monoinfections. These viruses are not hepatic trophoblastic but may be susceptible to PDL1 downregulation.

[0293] In some embodiments, the restoration of immunity or immune response includes improvement of T cell and / or NK cell responses, and / or mitigation of T cell depletion, in particular, the restoration of HBV-specific T cell responses, HCV-specific T cell responses, and / or HDV-specific T cell responses. Improvement of the T cell response can be assessed, for example, as an increase in T cells in the liver (particularly an increase in CD8+ and / or CD4+ T cells) 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, in particular, 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, CD8+ T cells specific to HBV s antigen (HBs antigen), and / or CD8+ T cells specific to HBV e antigen (HBe antigen), and / or CD8+ T cells specific to HBV core antigen (HBcAg) are administered an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of interferon-γ (IFN-γ) or tumor necrosis factor α (TNF-α). It is preferable that the HBV antigen-specific CD8+ T cells produce one or more cytokines, such as interferon-γ (IFN-γ) or tumor necrosis factor α (TNF-α). The increase in CD8+ T cells is observed particularly in the liver. The increase described herein should be statistically significant compared to a control. This increase is preferably 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 oligonucleotides or oligonucleotide conjugates, or pharmaceutical compositions of the present invention, can be used to treat parasitic infections, particularly parasitic infections affecting the PD-1 pathway (e.g., 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). See pp 58-64; Wykes et al. 2014 Front Microbiol Vol 5 pp 249). Parasitic infections can be selected from the group consisting of malaria, toxoplasmosis, leishmaniasis, and trypanosomiasis. Malaria is caused by protozoa of the Plasmodium genus, particularly Plasmodium vivax, Plasmodium malariae, and Plasmodium 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. Chaga disease in tropical regions is caused by the species Trypanosoma cruzi, while sleeping sickness is caused by the species Trypanosoma brucei.

[0295] In some embodiments, the restoration of immunity involves the restoration of parasite-specific T cell and NK cell responses, particularly Plasmodium falciparum-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 present invention can be administered topically (skin, inhalation, eyes, or ears), enterally (oral or gastrointestinal tract, etc.), or parenterally (intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly, or subarachnoidally).

[0297] In preferred embodiments, the oligonucleotide or pharmaceutical composition of the present invention is administered by intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or by parenteral routes including intra-arachnoid or intracranial routes, such as intrathecal or intracranial routes. These include intracerebral or intracavitary and intravitreous 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 oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the present invention are administered in doses 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 oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the present invention are used in combination therapy with other therapeutic agents. The therapeutic agent may, for example, be 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 modifiers is recommended. Effective antiviral drugs against HBV include, for example, nucleoside analogs. There are five nucleoside analogs approved for treatment.

[0301] HBV, specifically lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread), terbivudine (Tyzeka), and entecavir (Baraclude), are effective in suppressing viral replication but have no effect on HBs antigen levels. Other antiviral drugs include ribavirin and HBV antibody therapy (monoclonal or polyclonal). Immune system modifiers can be, for example, interferon α-2a and PEGylated interferon α-2a (Pegasys), or TLR7 agonists (e.g., GS-9620), or therapeutic vaccines. While the effect of IFN-α treatment on reducing viral load has been shown to be very low, this treatment results in a somewhat (less than 10%) reduction in HBs antigen, albeit very inefficiently.

[0302] The oligonucleotides or oligonucleotide conjugates of the present invention can be used in combination with other antiviral agents effective against HBV, such as the antisense oligonucleotides described in International Publication No. 2012 / 145697 and International Publication No. 2014 / 179629, or the siRNA molecules described in International Publication No. 2005 / 014806, International Publication No. 2012 / 024170, International Publication No. 2012 / 2055362, International Publication No. 2013 / 003520 and International Publication No. 2013 / 159109.

[0303] When the oligonucleotides or oligonucleotide conjugates of the present invention are administered in combination therapy with other agents, they can be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical compositions of the present invention may consist of combinations of oligonucleotides or oligonucleotide conjugates of the present invention in conjunction with pharmaceutically acceptable excipients described herein and other therapeutic or prophylactic agents known in the art.

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

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

[0306] 2. The oligonucleotide according to Embodiment 1, wherein the continuous nucleotide sequence is at least 90% complementary to the PD-L1 target nucleic acid.

[0307] 3. The oligonucleotide according to Embodiment 1 or 2, wherein the continuous 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. Oligonucleotides according to Embodiments 1 to 3, wherein the continuous nucleotide sequence is complementary to the regions at positions 1 and 15720 on SEQ ID NO: 1.

[0309] 5. The oligonucleotide according to 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. Oligonucleotides according to Embodiments 1 to 5, wherein the continuous 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 according to 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. Oligonucleotides according to embodiments 2 to 7, wherein the target nucleic acid is RNA.

[0313] 9. The oligonucleotide according to Embodiment 8, wherein the RNA is mRNA.

[0314] 10. The oligonucleotide according to Embodiment 9, wherein mRNA is precursor mRNA or mature mRNA.

[0315] 11. Oligonucleotides according to Embodiments 1 to 10, wherein the continuous nucleotide sequence comprises or consists of at least 14 consecutive nucleotides, particularly 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides.

[0316] 12. Oligonucleotides according to Embodiments 1 to 10, wherein the continuous nucleotide sequence comprises or consists of 16 to 20 nucleotides.

[0317] 13. The oligonucleotide according to Embodiments 1 to 10, wherein the oligonucleotide contains or consists of 14 to 35 nucleotides in length.

[0318] 14. The oligonucleotide according to Embodiment 13, wherein the oligonucleotide contains or consists of 18 to 22 nucleotides in length.

[0319] 15. The oligonucleotide according to Embodiments 1 to 14, wherein the oligonucleotide or continuous nucleotide sequence is single-stranded.

[0320] 16. The oligonucleotide according to Embodiments 1 to 15, wherein the continuous nucleotide sequence is complementary to the subsequence of the 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, 269, A274, A350, A360, A364, A365, A370, A372, A381, A383, A386, A389, A400, A427, A435 and A438.

[0321] 17. Oligonucleotide according to Embodiment 16, wherein the subsequence is selected from the group consisting of A221, A360, A180, A160, and A269.

[0322] 18. Oligonucleotides according to Embodiments 1 to 17, wherein the oligonucleotide is not an siRNA and is not self-complementary.

[0323] 19. Oligonucleotides according to Embodiments 1 to 18, wherein the continuous nucleotide sequence comprises or consists of a sequence selected from SEQ ID NOs: 5 to 743 or 771.

[0324] 20. The sequence of consecutive nucleotides is: 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, 466, 47 Oligonucleotides according to embodiments 1 to 19, comprising or consisting of sequences 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. Oligonucleotides according to Embodiments 1 to 20, wherein the continuous nucleotide sequence comprises or consists of a sequence selected from SEQ ID NOs: 466, 640, 342, 287, and 566.

[0326] 22. Oligonucleotides according to Embodiments 1 to 21, wherein the sequence of nucleotides has 0 to 3 mismatches compared to the target nucleic acid, and the sequence of nucleotides is complementary to the target nucleic acid.

[0327] 23. The oligonucleotide according to Embodiment 22, wherein the continuous nucleotide sequence has one mismatch compared to the target nucleic acid.

[0328] 24. The oligonucleotide according to Embodiment 22, wherein the continuous nucleotide sequence has two mismatches compared to the target nucleic acid.

[0329] 25. The oligonucleotide according to Embodiment 22, wherein the continuous nucleotide sequence is fully complementary to the target nucleic acid sequence.

[0330] 26. Oligonucleotides according to Embodiments 1 to 25, comprising one or more modified nucleosides.

[0331] 27. The oligonucleotide according to Embodiment 26, wherein one or more modified nucleosides are high-affinity modified nucleosides.

[0332] 28. The oligonucleotide according to Embodiment 26 or 27, wherein one or more modified nucleosides are 2'-saccharide modified nucleosides.

[0333] 29. The oligonucleotide according to Embodiment 28, wherein one or more 2'-saccharide-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, arabino nucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides.

[0334] 30. The oligonucleotide according to Embodiment 28, wherein one or more modified nucleosides are LNA nucleosides.

[0335] 31. The oligonucleotide according to Embodiment 30, wherein the modified LNA nucleoside is oxy-LNA.

[0336] 32. The oligonucleotide according to Embodiment 31, wherein the modified nucleoside is β-D-oxy-LNA.

[0337] 33. The oligonucleotide according to Embodiment 30, wherein the modified nucleoside is thioLNA.

[0338] 34. The oligonucleotide according to Embodiment 30, wherein the modified nucleoside is amino-LNA.

[0339] 35. The oligonucleotide according to Embodiment 30, wherein the modified nucleoside is cET.

[0340] 36. The oligonucleotide according to Embodiment 30, wherein the modified nucleoside is ENA.

[0341] 37. The oligonucleotide according to 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. Oligonucleotides according to embodiments 30 to 37, wherein at least one 2'-substituted modified nucleoside is present in addition to the modified LNA nucleoside.

[0343] 39. The oligonucleotide according to Embodiment 38, wherein the 2'-substituted 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 according to any one of Embodiments 1 to 39, wherein the oligonucleotide comprises at least one modified nucleoside bond.

[0345] 41. The oligonucleotide according to Embodiment 40, wherein the modified nucleoside bond is nuclease-resistant.

[0346] 42. The oligonucleotide according to Embodiment 40 or 41, wherein at least 50% of the nucleoside-nucleoside bonds in the continuous nucleotide sequence are phosphorothioate nucleoside bonds or boranophosphate nucleoside bonds.

[0347] 43. The oligonucleotide according to Embodiment 40 or 41, wherein all nucleoside-to-nucleoside bonds within the continuous nucleotide sequence are phosphorothioate nucleoside-to-nucleoside bonds.

[0348] 44. Oligonucleotides according to Embodiments 1 to 43, wherein the oligonucleotide has the ability to replenish RNase H.

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

[0350] 46. ​​The oligonucleotide according to Embodiment 44 or 45, wherein the oligonucleotide is a gapmer of formula 5'-FG-F'-3', regions F and F' independently contain or consist of 1 to 7 modified nucleosides, and G is a region between 6 to 16 nucleosides having the ability to replenish RNase H.

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

[0352] 48. The oligonucleotide according to Embodiment 47, wherein D' or D'' is optional.

[0353] 49. The oligonucleotide according to Embodiment 47, wherein region D' consists of a linked nucleoside of two phosphodiesters.

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

[0355] 51. The oligonucleotide according to Embodiment 46 or 47, wherein the modified nucleoside is a 2'-saccharide 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, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleoside.

[0356] 52. Oligonucleotides according to embodiments 46 to 51, wherein one or more of the modified nucleosides in the F region and F' region are LNA nucleosides.

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

[0358] 54. The oligonucleotide according to Embodiment 53, wherein the F region and F' region consist of an LNA nucleoside.

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

[0360] 56. The oligonucleotide according to Embodiment 52, wherein at least one of region F or F' further comprises at least one 2'-substituted 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. Oligonucleotides according to Embodiments 46 to 56, wherein the RNase H supplementation nucleoside in region G is independently selected from DNA, α-L-LNA, C4' alkylated DNA, ANA, and 2'F-ANA and UNA.

[0362] 58. The oligonucleotide according to Embodiment 57, wherein the nucleoside in region G is DNA and / or an α-L-LNA nucleoside.

[0363] 59. The oligonucleotide according to Embodiment 57 or 58, wherein region G consists of at least 75% DNA nucleoside.

[0364] 60. Oligonucleotides according to Embodiments 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 with 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_ Oligonucleotides according to Embodiments 1 to 60, 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 according to 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 (region A) according to any one of claims 1 to 62; b. At least one binding portion (region C) covalently bonded to the oligonucleotide; Antisense oligonucleotide conjugates, including those mentioned above.

[0368] 64. The oligonucleotide conjugate according to Embodiment 63, wherein the binding site is selected from carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins, vitamins, viral proteins, or combinations thereof.

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

[0370] 66. The oligonucleotide conjugate according to Embodiment 65, wherein the carbohydrate-binding portion comprises at least one asialoclycoprotein receptor-targeting portion and is covalently bonded to the oligonucleotide according to any one of claims 1 to 62.

[0371] 67. The oligonucleotide conjugate according to Embodiment 66, wherein the asialocrycoprotein 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 according to Embodiment 66 or 67, wherein the asialocryprotein receptor targeting binding moiety is monovalent, divalent, trivalent, or tetravalent.

[0373] 69. The oligomer conjugate according to Embodiment 68, wherein the asialocrypoprotein receptor targeting binding portion consists of 2 to 4 terminal GalNAc portions and PEG spacers that link each GalNAc portion to a branched molecule.

[0374] 70. Oligonucleotide conjugates according to embodiments 66 to 69, wherein the asialocrypoprotein receptor targeting binding moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety.

[0375] 71. Oligonucleotide conjugates according to embodiments 66 to 70, wherein the binding site is selected from one of the trivalent GalNAc sites in Figure 1.

[0376] 72. The oligonucleotide conjugate according to Embodiment 71, wherein the binding portion is the trivalent GalNAc portion shown in Figure 3.

[0377] 73. Oligonucleotide conjugates according to embodiments 63 to 72, wherein a linker is present between the oligonucleotide or a sequence of oligonucleotides and the binding portion.

[0378] 74. The oligonucleotide conjugate according to Embodiment 73, wherein the linker is a physiologically mutable linker (region B).

[0379] 75. The oligonucleotide conjugate according to Embodiment 74, wherein the physiologically mutable linker is a nuclease-sensitive linker.

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

[0381] 77. The oligonucleotide conjugate according to Embodiment 76, wherein the physiologically mutable linker is equal to region D' or D'' present in Embodiments 47 to 50.

[0382] 78. An oligonucleotide conjugate according to 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 according to Embodiment 78, wherein the oligonucleotide conjugate is selected from the oligonucleotide conjugates shown in Figures 4, 5, 6, 7, and 8.

[0384] 80. Oligonucleotide conjugates according to embodiments 63 to 76, which exhibit improved inhibition of PD-L1 in target cells, improved cell distribution between the liver and spleen, or improved uptake of the conjugated oligonucleotide into liver cells, compared to unbound oligonucleotides.

[0385] 81. A pharmaceutical composition comprising an oligonucleotide according to Embodiments 1 to 62, or a conjugate according to Embodiments 63 to 80, and a pharmaceutically acceptable diluent, carrier, salt, and / or auxiliary agent.

[0386] 82. A method for producing an oligonucleotide according to Embodiments 1 to 62, comprising reacting nucleotide units to form a series of covalently bonded nucleotide units contained within the oligonucleotide.

[0387] 83. The method according to Embodiment 82, further comprising reacting a continuous nucleotide sequence with a non-nucleotide binding site.

[0388] 84. A method for producing the composition according to Embodiment 81, comprising mixing an oligonucleotide with a pharmaceutically acceptable diluent, carrier, salt, and / or auxiliary agent.

[0389] 85. An in vivo or in vitro method for regulating the expression of PD-L1 in target cells expressing PD-L1, the method comprising administering to the cells an effective amount of 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.

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

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

[0392] 88. The method according to Embodiment 87, wherein immune recovery is an increase in CD8+ T cells specific to one or more HBV antigens in the liver compared to a control.

[0393] 89. Oligonucleotides according to Embodiments 1 to 62, conjugates according to Embodiments 63 to 80, or pharmaceutical compositions according to Embodiment 81, for use as pharmaceuticals for the treatment or prevention of disease in a subject.

[0394] 90. Use of oligonucleotides according to Embodiments 1 to 62, or use of conjugates according to Embodiments 63 to 80, for preparing a drug for the treatment or prevention of a disease in a subject.

[0395] 91. Oligonucleotides according to Embodiments 1 to 62, conjugates according to Embodiments 63 to 80, or pharmaceutical compositions according to Embodiment 81, for use in restoring immunity against viruses or parasites.

[0396] 92. The use according to Embodiment 91, wherein immune recovery is an increase in CD8+ T cells specific to 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 the HBs antigen.

[0398] 94. The methods, oligonucleotides, or uses according to Embodiments 86 to 93, wherein the disease is related to the in vivo activity of PD-L1.

[0399] 95. The methods, oligonucleotides, or uses according to Embodiments 86 to 94, wherein the disease is associated with increased expression of PD-L1 in antigen-presenting cells.

[0400] 96. The method, oligonucleotide, or use according to Embodiment 95, wherein PD-L1 is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the expression of PD-L1 when not treated with the oligonucleotide described in Embodiments 1 to 62, or the conjugate described in Embodiments 63 to 80, or the pharmaceutical composition described in Embodiment 81, or the expression of PD-L1 before treatment.

[0401] 97. The methods, oligonucleotides, or uses according to Embodiments 86 to 95, wherein the disease is selected from viral hepatitis infection or 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 methods, oligonucleotides, or uses according to embodiments 86 to 95, wherein the disease is chronic HBV.

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

[0405] 101. The methods, oligonucleotides, or uses according to Embodiments 86 to 100, wherein the subject is a mammal.

[0406] 102. The method, oligonucleotide, or use according to Embodiment 101, wherein the mammal is a human. [Examples]

[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] A motif sequence represents a sequence of nucleic acid bases present within an oligonucleotide.

[0428] The design refers to the gapmer design FG-F'. Each number represents the number of consecutive modified nucleosides. A 2' modified nucleoside (first number = 5' side), followed by the number of DNA nucleosides (second number = gap region), followed by the number of modified nucleosides (e.g., including a 2' modified nucleoside (third number = 3' side) preceded or followed by further repeating regions of DNA and LNA that are not part of a contiguous sequence complementary to the target nucleic acid).

[0429] Oligonucleotide compounds represent a specific design of motif sequences. Uppercase letters represent β-D-oxy LNA nucleosides, and lowercase letters represent DNA nucleosides. All LNA Cs are 5-methylcytosine, and all nucleoside bonds are phosphorothioate nucleoside bonds.

[0430] [Table 6]

[0431] A motif sequence represents a sequence of nucleic acid bases present within an oligonucleotide.

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

[0433] Oligonucleotide compounds represent a specific design of motif sequences. Uppercase letters represent β-D-oxy LNA nucleosides, and lowercase letters represent DNA nucleosides. All LNA Cs are 5-methylcytosine, and all nucleoside bonds are phosphorothioate nucleoside bonds.

[0434] [Table 7]

[0435] Uppercase letters represent β-D-oxy LNA nucleosides, and lowercase letters represent DNA nucleosides. All LNA Cs are 5-methylcytosine, the subscript o represents a phosphodiester nucleoside bond, and unless otherwise specified, other nucleoside bonds are phosphorothioate nucleoside bonds.

[0436] [Table 8]

[0437] GN2 represents the trivalent GalNAc colony shown in Figure 3, C6 represents an aminoalkyl group with six carbon atoms, uppercase letters represent β-D-oxyLNA nucleosides, and lowercase letters represent DNA nucleosides. All LNA Cs are 5-methylcytosine, the subscript o represents a phosphodiester nucleoside bond, and unless otherwise specified, nucleoside bonds are phosphorothioate nucleoside bonds. Chemical diagrams representing some molecules are shown in Figures 4 to 8.

[0438] AAV / HBV Mouse Model Pasteur model: At the Pasteur Institute, HLA-A2.1- / HLA-DR1-transgenic H-2 class I- / class II-knockout mice (referred to herein as HLA-A2 / DR1 mice) were created and bred. These mice are an in vivo experimental model for human immunoassay that does not interfere with the mouse MHC response (Pajot et al 2004 Eur J Immunol.34(11):3060-9).

[0439] These studies used adeno-associated virus (AAV) vectors and AAV serotype 2 / 8 containing a replicable HBV DNA genome. The AAV-HBV vector (batch GVPN#6163) was divided into 5 × 10⁻¹⁵ units. 11 The solution was diluted with sterile phosphate-buffered saline (PBS) to reach a titer of vg / mL. 100 μL of this diluted solution (dosage / mouse: 5 × 10⁴) 10 (vg) was intravenously (iv) injected into the tail vein of mice. Complete viral particles containing HBV DNA were detected in the blood of HBV-carrier mice. HBc antigen was detected in the liver for up to one year, along with circulating HBV proteins HBe antigen and HBs antigen. In all AAV2 / 8-HBV transduced mice, serum HBs antigen, HBe antigen, 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 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] Male C57BL / 6 mice (4-6 weeks old) free of specific pathogens were purchased from SLAC (Shanghai Laboratory Animal Center of Chinese Academy of Sciences) and housed in individual ventilated cages in the animal housing facility. Guidelines for animal care and use, as instructed by WuXi IACUC (WUXI IACUC Protocol No. R20131126-Mouse), were followed. The mice were allowed to acclimate to the new environment for three days and then classified according to the experimental design.

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

[0443] On day 0, 200 μL of AAV-HBV was injected into the tail vein of all mice. On days 6, 13, and 20 after AAV injection, blood was collected from the submandibular region of all mice (0.1 ml blood / mouse), and serum was collected. On day 22 after injection, preparations for oligonucleotide treatment were completed in mice with stable viremia. Oligonucleotides can be in unbound or GalNAc-bound form.

[0444] DNA vaccine The plasmid DNA was endotoxin-free and manufactured at Plasmid-Factory (Germany). pCMV-S2.S encodes the pre-S2 domain and S domain of the HBs antigen (genotype D), and its expression is regulated 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 containing the hepatitis core (HBc) antigen (Dion et al 2013 J Virol 87: 5554-5563).

[0445] Treatment with a DNA vaccine was performed as described here. Five days before vaccination, cardiotoxin (CaTx, Latoxan refL81-02, 50 μl / muscle) was injected into the muscles of the mice. CaTx depolarizes muscle fibers and induces cytopathology, resulting in the appearance of new muscle fibers five days after injection, improving the transfection effect with DNA vaccine administration. pCMV-S2.S ayw and pCMVCore were mixed in equal volumes of 1 mg / ml each, and a total of 100 μg was administered to each mouse by bilateral intramuscular injection into the cardiotoxin-treated tibialis anterior muscle under anesthesia (100 μL of 12.5 mg / mL ketamine, 1.25 mg / mL xylazine), as previously described in Michel et al 1995 Proc Natl Acad Sci USA 92:5307-5311.

[0446] Anti-PD-L1 antibody This is clone 6E11, a mouse anti-mouse PD-L1 IgG1 antibody internally produced at Genetech, which blocks atezolizumab and is a surrogate antibody with similar in vitro blocking activity to atezolizumab internally produced atezolizumab 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. The following is an applicable protocol. The oligonucleotides of the present invention may be produced by methods that vary slightly in terms of the equipment used, the support, and the concentration.

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

[0449] Oligonucleotide extension: 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) were coupled using acetonitrile solution (0.1 M) of 5'-O-DMT-protected amidite and acetonitrile solution (0.25 M) of DCI (4,5-dicyanoimidazole) as activators. For the final cycle, phosphoramidites with the desired modifications can be used. These are C6 linkers or binding groups for attaching the binding group. Thiolation for the introduction of phosphorothioate bonds is carried out using xanthan hydride (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] To prepare for conjugation after solid-phase synthesis, a commercially available C6 aminolinker phosphoramidite can be used in the final cycle of solid-phase synthesis to isolate the amino-bonded deprotected oligonucleotide after deprotection and cleavage from the solid support. Using standard synthetic methods, the conjugate is introduced via activation of the functional group.

[0451] Alternatively, as described in International Patent Application PCT / EP2015 / 073331 or European Patent Application Publication No. 15194811.4, it is possible to add the oligonucleotide while the binding portion remains on a solid support by using phosphoramidites of GalNAc- or GalNAc colonies.

[0452] Purification by RP-HPLC: The crude compound is purified by preparative RP-HPLC using a 10 μm 150 × 10 mm column of Phenomenex Jupiter C18. 0.1 M ammonium acetate pH 8 and acetonitrile are used as buffer solutions at a flow rate of 5 mL / min. The collected fractions are lyophilized to obtain 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: Reverse-phase high-performance liquid chromatography

[0454] T m Assay Oligonucleotides and RNA target (phosphate-bound, PO) double strands were diluted to 3 mM in 500 ml of RNase-free water, and 2×T was added to 500 ml. m Mix with buffer solution (200 mM NaCl, 0.2 mM EDTA, 20 mM sodium phosphate, pH 7.0). Heat the solution at 95°C for 3 minutes, then anneal at room temperature for 30 minutes. Record the double-strand melting temperature (T) using a Lambda 40 UV / VIS spectrophotometer equipped with a Peltier temperature programmer PTP6 with PE Templab software (Perkin Elmer). m) is measured. The temperature is increased from 20°C to 95°C, then decreased to 25°C, and the absorption is recorded at 260 nm. The first derivative, as well as the maximum values ​​for both melting and annealing, are used to measure the double-stranded T. m Evaluate.

[0455] Tissue-specific in vivo linker cleavage assay FAM-labeled oligonucleotides having a biocleavable linker (e.g., a DNA phosphodiester linker (PO linker)) to be tested are cleaved in vitro using homogenates of the relevant tissue (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 a buffer solution (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, and then extracted with phenol-chloroform. AIE HPLC analysis of the 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 relative to the standard was quantified using both a fluorescence detector (615 nm) and a UV detector (260 nm).

[0457] S1 nuclease sensitivity assay FAM-labeled oligonucleotides are cleaved in vitro in an S1 nuclease extract or serum using an S1 nuclease-sensitive linker (e.g., a DNA phosphate diester linker (PO linker)).

[0458] In vitro cleavage of 100 μM oligonucleotides with S1 nuclease in nuclease buffer (60 U / 100 μL) was performed for 20 minutes and 120 minutes. Enzyme activity was interrupted by adding EDTA to the buffer. AIE HPLC analysis of the 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 relative to the standard substance was quantified using both a fluorescence detector (615 nm) and a UV detector (260 nm).

[0459] Preparation of liver mononuclear cells Hepatocytes derived from AAV / HBV mice were prepared using the method described by Tupin et al. 2006 Methods Enzymol 417: 185-201, with some modifications. After euthanasia, 10 ml of sterile PBS was perfused into the liver of the mice via the portal vein using a syringe with a G25 needle. When the organ was pale, it was collected with Hanks equilibrium salt solution (HBSS) (GIBCO® HBSS, 24020) + 5% fetal bovine fetal serum (FCS). The collected liver was passed through a 100 μm cell strainer (BD Falcon, 352360) and slowly pressurized 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 15 ml units in 35% isotonic Percoll solution (GE Healthcare Percoll #17-0891-01, diluted in RPMI 1640 (GIBCO, 31870)) at room temperature and transferred to 15 ml tubes. The cells were further centrifuged at 1360 g at room temperature for 25 minutes. The supernatant was discarded by aspirate, and the pellet containing mononuclear cells was washed twice with HBSS + 5% FCS.

[0460] Cells were cultured in complete medium consisting of α 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 in a U-bottom 96-well plate and washed with PBS FACS (PBS containing 1% bovine serum albumin and 0.01% sodium azide). Cells were incubated for 10 minutes at 4°C in the dark with 5 μL of PBS FACS containing rat anti-mouse CD16 / CD32 antibody and the survival marker LD fixable yellow (Thermofisher, L34959). Next, cells were stained with 25 μL of PBS FACS containing monoclonal antibodies (Mab) 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) in the dark at 4°C for 20 minutes. Two supplemental surface markers, PDL1 (rat Mab anti-mouse PD1 PE, BD Biosciences, 551892) and PDL1 (rat Mab anti-mouse PDL1 BV711, Biolegend, 124319), were added.

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

[0463] Cells cultured overnight were washed with PBS FACS and incubated with 5 μL of PBS FACS containing rat anti-mouse CD16 / CD32 antibody and survival marker LD-fixed yellow (Thermofisher, L34959) for 10 minutes in the dark at 4°C. The cells were then stained with 25 μL of PBS FACS containing Mab for 20 minutes in the dark at 4°C. This 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). The cells were fixed after several washes, permeabilized with Cytofix / Cytoperm for 20 minutes in the dark at room temperature, and then washed with Perm / Wash solution (BD Biosciences, 554714) at 4°C.

[0464] Intracellular cytokines were stained for 30 minutes 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 α (TNFα) (rat Mab anti-mouse TNFα-FITC, clone MP6-XT22;1 / 250 (BD Biosciences 554418)). Prior to analysis by flow cytometry using a MACSQuant Analyzer, cells were washed with Perm / Wash and resuspended in PBS FACS containing 1% formaldehyde.

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

[0466] The positive threshold was defined according to the experimental background (i.e., the mean percentage of stained cells obtained for each group in the medium alone and at two standard deviations). Only the percentage of cytokines representing at least five events was considered positive.

[0467] [Table 9]

[0468] Example 1: In vitro efficacy test Gene walks were performed on the entire human PD-L1 transcript, primarily using 16-20mer gapmers. Efficacy studies were conducted in vitro using the human leukemia monocyte cell lineage THP1 and the human non-Hodgkin lymphoma cell lineage (KARPAS-299).

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

[0470] Effects of oligonucleotides THP-1 cells (3.104 in RPMI-GLutamax, 10% FBS, 1% Pen-Strep (Thermo Fisher Scientific)) were added to oligonucleotides (4-5 μl), placed in 96-well round-bottom plates, and cultured for 6 days at a final volume of 100 μl / well. Oligonucleotides were screened at a single concentration (20 μM) and in a dose range of 25 μM to 0.004 μM (1:3 dilution aqueous solution). 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, pre-designed 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 Scientific (CSI) RT-qPCR. Relative PD-L1 mRNA expression levels were calculated using the 2ΔΔC(T)(2(-Delta Delta C(T))) method and as the inhibition percentage compared to the control sample (untreated cells).

[0471] Karpas-299 cells were cultured in RPMI 1640, 2 mM glutamine, and 20% FBS (Sigma). These cells were seeded at 10,000 cells / well in 96-well plates and incubated for 24 hours, after which oligonucleotides lysed in PBS were added. The final oligonucleotide concentrations were either a single dose of 5 μM at a final culture volume of 100 μL / well, or a range dose response 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. Cells were harvested on day 3 after the addition of the oligonucleotide compounds, 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, an 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). The relative mRNA expression levels of PD-L1 are shown in Table 10 as a percentage of the control sample (cells treated in PBS).

[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 test in dose-response curve Oligonucleotides selected from Table 10 were tested in KARPAS-299 cells using half-log serial dilutions of PBS (oligonucleotides in concentrations of 50 μM, 15.8 μM, 5.0 μM, 1.58 μM, 0.5 μM, 0.158 μM, μM, 0.05 μM, and 0.0158 μM), and evaluated using the in vitro efficacy assay described in Example 1. IC50 and the oligonucleotide with the greatest inhibition (residual %PD-L1 expression level) were evaluated.

[0492] EC50 was calculated using GraphPad Prism6. Table 11 shows the IC50 and maximum PD-L1 knockdown level as a percentage of control (PBS) treated cells.

[0493] [Table 11-1]

[0494] [Table 11-2]

[0495] [Table 11-3]

[0496] Oligonucleotides selected from Table 6 were tested in THP-1 cells using the in vitro efficacy assay described in Example 1, employing 1:3 serial dilutions with 25 μM to 0.004 μM water. IC50 and maximum inhibition (residual PD-L1 expression rate) were evaluated for each oligonucleotide.

[0497] EC50 was calculated using GraphPad Prism6. IC50 and the maximum PD-L1 knockdown level are shown in Table 12 as percentages of treated cells in the control (PBS).

[0498] [Table 12]

[0499] The results in Tables 7 and 8 are also shown in Figure 2, along with their targeting locations for the PD-L1 premRNA of Sequence ID No. 1.

[0500] This indicates that for almost all compounds, the EC50 value was less than 1 μM, and the targeted knockdown was below 25% of the PD-L1 expression level in control cells (treated with physiological saline).

[0501] Example 3 - In vitro efficacy and effectiveness, and in vivo PD-L1 reduction, in poly(I:C)-induced mice using naked and GalNAc-bound PD-L1 antisense oligonucleotides. Efficacy and efficacy studies were conducted in vitro in dose-response experiments in MCP-11 cells using the oligonucleotides listed in Table 6. The same oligonucleotides, as well as GalNAc-bound versions (CMP ID numbers 755_2~765_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 assay MCP-11 cells (originally purchased from ATCC), suspended in DMEM (Sigma catalog number 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. These cells were then cultured for 3 days in a humidified incubator at 37°C with 5% CO2 at a final volume of 200 μl / well. Oligonucleotides were screened at dose-range 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. cDNA was synthesized using M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion), and a 100mM 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 the percentage of residual PD-L1 expression in the PBS control sample (PBS-treated cells). EC50 was calculated using GraphPad Prism6. Table 13 shows the EC50 and maximum PD-L1 knockdown levels as percentages of control (PBS) cells.

[0504] In vivo assay Female C57BL / 6J mice (20-23g; 5 mice per group) were subcutaneously injected with either 5 mg / kg of unbound oligonucleotide to mouse PD-L1 or 2.8 mg / kg of GalNAc-bound oligonucleotide to mouse PD-L1. Three days later, 10 mg / kg of poly(I:C) (LWM, Invivogen) was intravenously injected into the mice. Five hours after poly(I:C) injection, the mice were sacrificed, and liver samples were placed in RNAlater (Thermo Fisher Scientific) for RNA extraction or frozen with dry ice in preparation 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, an 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 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 percentages of control samples from mice injected with physiological saline and poly(I:C).

[0506] Liver homogenates were prepared by homogenizing 2 ml of liver sample per 100 mg of tissue T-PER® tissue protein extract reagent (Thermo Fisher Scientific) mixed with a 1× stop protease inhibitor cocktail, EDTA-free (Thermo Fisher Scientific). The protein concentration in the liver homogenates was measured using the Coomassy 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 1× MOPS electrophoresis 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 parts at the 64 kDa band. The membranes were blocked in TBS containing 5% skim milk and 0.05% Tween20, and then incubated overnight at 4°C with either rabbit monoclonal anti-vinculin (Abcam catalog no. ab129002) diluted 1:10000 (upper membrane) or goat polyclonal anti-mPD-L1 (R&D Systems catalog no. AF1019) diluted 1:1000 in TBS containing 5% skim milk and 0.05% Tween20 (lower membrane). The membranes were washed in TBS containing 0.05% Tween20, and then exposed to HRP-conjugated porcine anti-rabbit IgG (DAKO) diluted 1:3000, or HRP-conjugated rabbit anti-goat IgG (DAKO) diluted 1:2000 in TBS containing 5% skim milk and 0.05% Tween20, for 1 hour at room temperature. After washing the membranes, reactivity was detected using ECL Select (Amersham GE Healthcare). For each group treated with oligonucleotides, the intensity of the PD-L1 band relative to the vinculin band was evaluated by comparing it to the PD-L1 / vinculin band intensity of mice injected with physiological saline and poly(I:C) (control).The results are shown in Table 13, and Western blots using pairs of naked oligonucleotides and bound oligonucleotides are shown in Figures 9A to 9E.

[0507] [Table 13]

[0508] As can be seen from the data in Table 13, it is clear that the binding of oligonucleotides to GalNAc improves the reduction of in vivo PD-L1. The reduction in mRNA generally correlates with the reduction of PD-L1 protein. With the exception of CMP ID number: 754_1, the low in vitro EC50 values ​​generally reflect the good reduction of in vivo PD-L1 mRNA after the oligonucleotides have bound to GalNAc.

[0509] Example 4 - In vivo PK / PD in selected hepatocytes and non-parenchymal cells derived from poly(I:C)-induced mice The distribution of naked oligonucleotides and GalNAc-binding oligonucleotides, as well as the reduction of PD-L1 mRNA, were investigated in hepatocytes and non-parenchymal cells isolated from poly(I:C)-inducible mice.

[0510] Female C57BL / 6J mice were subcutaneously injected with either 5 mg / kg of an unbound oligonucleotide (748_1) or 7 mg / kg of a GalNAc-bound oligonucleotide (759_2) that targets mouse PD-L1 mRNA (n=3 per group). Two days later, 15 mg / kg of poly(I:C) (LWM, InvivoGen) was intraperitoneally injected into the mice. The mice were anesthetized for 18-20 hours after poly(I:C) injection, and their livers were perfused for 5 minutes at a flow rate of 7 ml / min with a Hanks equilibrium salt solution containing 15 mM Hepes and 0.38 mM EGTA. Next, the cells were washed for 12 minutes with a collagenase solution (Hanks equilibrium 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 excised, the hepatic sac was opened, and the liver suspension was filtered using Williams E medium through a 70 μm cell filter. Aliquots of the cell suspension (= mixed hepatocytes) were extracted for subsequent 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 E medium (Sigma catalog number W1878: 1 × Pen / Strep, supplemented with 2 mM L-glutamine and 10% FBS (ATCC#30-2030)), mixed with 25 ml of William E medium containing 90% Percoll and hepatocytes, and centrifuged at 50 × g for 10 minutes to precipitate the hepatocytes. After washing twice with William E medium, the precipitated hepatocytes were resuspended in William E medium. The supernatant containing non-physical cells was centrifuged at 500 × g for 7 minutes, the cells were resuspended in 4 ml of RPMI medium, and the cells were passed through two Percoll layers (25% and 50% Percoll) and centrifuged at 1800 × g for 30 minutes. After the non-physical 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 total 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 transcriptionase, 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 TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1). Table 10 shows the relative expression levels of PD-L1 mRNA as percentages of control samples from mice injected with physiological saline and poly(I:C).

[0512] Oligonucleotide contents were analyzed using ELISA with a biotinylation capture probe having the sequence 5'-TACCGT-s-Bio-3' and a digoxigenin-binding detection probe having the sequence 5'-DIG-C12-S1-CCTGTG-3'. The probes consisted solely of LNA with a phosphodiester skeleton. Liver samples (approximately 50 mg) were homogenized in 1.4 mL of pure MagNa lysis buffer (Roche catalog number 03604721001) in a 2 mL Eppendorf tube containing one 5 mm diameter stainless steel bead. The samples were homogenized using a Retsch MM400 homogenizer (Merck Eurolab) until a uniform lysate was obtained. The samples were incubated at room temperature for 30 minutes. Standards were prepared by spiking untreated liver samples with a specified concentration of unbound antisense oligonucleotide compound (CMP ID number 748_1) and treating these samples. Select the spike-in concentration to match the expected sample oligo content (within approximately 10 times).

[0513] Homogenized samples were diluted at least 10-fold in 5×SSCT buffer (containing 750 mM NaCl and 75 mM sodium citrate, 0.05% (v / v) Tween-20 (pH 7.0)), and then serially diluted 6×2-fold using a capture-detection solution (35 nM capture probe and 35 nM detection probe in 5×SSCT buffer). The samples were incubated at room temperature for 30 minutes. The samples were transferred to a 96-well streptavidin-coated plate (Nunc catalog no. 436014) at a rate of 100 μL per well. The plates were incubated at room temperature for 1 hour with gentle agitation. Each well was washed three times with 2×SSCT buffer, and 100 μL of anti-DIG-AP Fab fragment (Roche Applied Science, catalog no. 11093274910), diluted 1:4000 in freshly prepared 0.05% (v / v) Tween-20 (pH 7.2) PBST (phosphate-buffered saline), was added. The wells were incubated at room temperature for 1 hour with gentle stirring. After washing three times with 2×SSCT buffer, 100 μL of alkaline phosphatase (AP) substrate solution (Blue Phos substrate, KPL product code 50-88-00, freshly prepared) was added. After incubation for 30 minutes with gentle stirring, the color intensity was measured spectrophotometrically at 615 nm. The raw data was 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] As is evident from the results, both the naked (CMP ID: 748_1) and conjugated (CMP ID: 759_2) oligonucleotides similarly and effectively reduce PD-L1 mRNA in total hepatocytes. In isolated hepatocytes, the conjugated oligonucleotide was approximately five times more potent than the naked oligonucleotide, and the naked oligonucleotide was twice as potent as the GalNAc-conjugated oligonucleotide in non-parenchymal cells. In hepatocytes and non-parenchymal cells, the reduction in PD-L1 mRNA expression 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 PD-L1 antisense oligonucleotides and GalNAc-bound PD-L1 antisense oligonucleotides. In this study, AAV / HBV mice were treated with either naked or GalNAc-bound PD-L1 antisense oligonucleotides, and PD-L1 mRNA expression levels and HBV gene expression in the liver were evaluated.

[0517] In the first week, 5-8 week old female HLA-A2 / DR1 mice (5 animals per group) were pre-treated with a vehicle (physiological saline), naked PD-L1 antisense oligonucleotide (CMP ID 752_1 (5 mg / kg sc)), and GalNAc (5 mg / kg sc) PD-L1 antisense oligonucleotide (7 mg / kg subcutaneously, CMP ID 763_2). These doses correspond to equimolar concentrations of the oligonucleotides. 5 × 10⁶ mice were treated at week 0. 10 The mice were transduced using vg AAV-HBV. For details, please refer to the description of the AAV / HBV mouse model in the "Materials and Methods" section. From week 1 to week 4 after AAV-HBV transduction, the mice were further administered PD-L1 oligonucleotide or vehicle (physiological saline) by subcutaneous injection four more times at one-week intervals.

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

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

[0520] To measure HBV gene expression, DNA was extracted from serum using the QIAamp One-For-All Nucleic Acid kit (catalog number 965672) with a Qiagen Biorobot. The serum was then diluted 1:20 in PBS, and a total of 100 μl was eluted in 200 μl of AL buffer. Finally, DNA was eluted in 100 μl units from the kit.

[0521] For real-time qPCR, TaqMan Gene Expression Master Mix (catalog number 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) in a 1:1:0.5 ratio. Forward (F3_core): CTG TGC CTT GGG TGG CTT T (Sequence ID: 784) Reverse (R3_core): AAG GAA AGA AGT CAG AAG GCA AAA (Sequence ID: 785) Probe (P3_core): 56-FAM-AGC TCC AAA / ZEN / TTC TTT ATA AGG GTC GAT GTC CAT G-3IABkFQ (Sequence ID: 786)

[0522] A calibration curve using HBV plasmid (genotype D, GTD) was created for the first 1 × 10⁻⁶ 9 The solution was prepared using 10-fold dilutions ranging from copies / μl to 1 copy / μl, and 5 μl was used per reaction.

[0523] For each reaction, 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 were added, and qPCR was performed.

[0524] For analysis, the copy number / ml / well was calculated using a calibration curve. The results are shown in Table 15.

[0525] The mRNA expression level of PD-L1 was measured by qPCR.

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

[0527] Liver tissue samples 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 RNA containing the clear phase (approximately 500 μl) 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, and Qiagen (including an RNA digestion RNase-free DNase set, catalog number 79254). The final eluted RNA concentration in 50 μl of H2O was measured, and all samples were adjusted to 100 ng / μl.

[0528] qPCP was performed on 7.5 μl of RNA using the Taqman RNA-to-ct 1-step Kit (catalog number 4392938, Thermo Fisher) according to the manufacturer's instructions. The fprimers used in the mixture contained PD-L1_1-3 (primer numbers Mm00452054_m1, Mm03048247_m1, and Mm03048248_m1), as well as endogenous controls (ATCB Mm00607939_s1, CANX Mm00500330_m1, YWHAZ Mm03950126_s1, and GUSB Mm01197698_m1).

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

[0530] [Table 15]

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

[0532] Example 6 - In vivo effect on T cell response in AAV / HBV mice In this study, AAV / HBV mice obtained from Pasteur were treated with PD-L1-targeting antibodies or antisense oligonucleotides. The antisense oligonucleotides were either naked or GalNAc-conjugated. During treatment, the animals were immunized with DNA vaccines against HBs and HBc antigens to ensure efficient T-cell priming by antigen-presenting cells (see the "Materials and Methods" section). We evaluated how this treatment affected the cell populations in the liver and spleen, as well as PD-L1 expression in these populations, and whether it could identify HBV-specific T-cell responses.

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

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

[0535] [Table 16]

[0536] [Table 17]

[0537] Mononuclear cells were collected from the spleen and liver of each mouse group at the time of slaughter, and erythrocytes were depleted (Lysing Buffer, BD biosciences, 555899). As described in the "Materials and Methods" section, specific preparations were required for liver mononuclear cells.

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

[0539] Compared to the control groups (i.e., the vehicle and DNA immunization groups), no significant changes were observed in the frequency of NK cells in the spleen and liver of treated mice. As shown in Table 18, in the liver, the groups treated with naked PD-L1 oligonucleotide (CMP ID: 748_1) and GalNAc-bound PD-L1 oligonucleotide (CMP ID: 759_2) showed a significant increase in T cell count compared to either control group (i.e., the vehicle and DNA immunization groups). This is similarly shown in Figure 10A. This increase was attributed to an increase in both the CD4+ and CD8+ T cell populations (Table 18, Figure 10B, and Figure 10C, respectively).

[0540] [Table 18]

[0541] PD-L1 expression level : The expression of PD-L1 protein in macrophages, B cells, and T cells derived from the spleen and liver at the time of slaughter was evaluated. By presenting PD-L1 antibody in a surface-labeled antibody mixture (see "Materials and Methods"), it became possible to quantify PD-L1-expressing cells using cytometry.

[0542] No significant differences were observed in PD-L1 expression rates (%) within macrophages, B cells, and CD4+ T cells in the spleen between treatment groups. 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-bound PD-L1 oligonucleotide (CMP ID 759_2) compared to other treatments (data not publicly available).

[0543] In the liver, PD-L1 was primarily expressed on CD8+ T cells. In the control group (Figure 11A, a combination of the vehicle-vaccinated group and the DNA-vaccinated group), the mean frequencies were 32% and 41%, respectively. Treatment with naked PD-L1 oligonucleotides or GalNAc PD-L1 oligonucleotides reduced the frequency of PD-L1-expressing CD8+ T cells (see Table 19 and Figure 11A). These cell types had significantly lower PD-L1 expression rates than CD8+ T cells, but significant differences in the percentage of PD-L1-expressing cells were also observed in B cells and CD4+ T cells after ASO treatment (see Table 19, Figures 11B and 11C). Furthermore, a reduction in PD-L1 expression was evident in all cell types with treatment with anti-PD-L1 antibodies. However, this reduction is due to the partial blockade of the PD-L1 epitope by the anti-PD-L1 antibody used in treatment, which in turn prevents the PD-L1 detection antibody in the surface-labeled antibody mixture from binding to PD-L1. Therefore, what appears to be PD-L1 downregulation via the anti-PD-L1 antibody used in treatment may be a result of epitope competition between the treatment antibody and the detection antibody.

[0544] [Table 19]

[0545] HBV-specific T cell response: Intracellular cytokine staining assays that detect IFNγ and TNFα production (see the "Materials and Methods" section) 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%). In mice treated with either naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide, or DNA vaccine alone (data not publicly available), IFNγ-producing CD8+ T cells targeting two HBV antigens were detected.

[0547] At sacrifice, IFNγ-producing NK cells were not detected in the livers of DNA-immunized HBV carrier mice, whereas IFNγ-secreting CD4+ T cells were specific to the core or S2+S antigen in the livers of some DNA-immunized mice (<0.4%, data not publicly available). IFNγ-producing HBV S2+S-specific CD8+ T cells were detected in most DNA-immunized mice. Mice treated with a combination of DNA vaccine and naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide showed an increased frequency of IFNγ-secreting CD8+ T cells, whereas treatment with anti-PD-L1 antibody did not show a clear additional effect compared to DNA vaccination (Figure 12). IFNγ-producing CD8+ T cells targeting envelope and core antigens were detected in most DNA-immunized groups (excluding those with anti-PD-L1 antibody) (Figure 12B). Most S2-S-specific T cells produced both IFNγ and TNFα (Figure 12C). The results are shown in Table 20.

[0548] [Table 20]

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

[0550] The effects of the treatment on serum HBe and HBs antigens and HBV DNA levels were evaluated in comparison to vehicle-treated animals.

[0551] Treatment protocol: As described in the "Materials and Methods" section for the Shanghai model, this study used male C57BL / 6 mice infected with recombinant adeno-associated virus (AAV) (AAV / HBV) carrying the HBV genome. Both 5 mg / kg of antisense oligonucleotide CMP ID number: 759_2 in a medium (saline) were injected into the mice (6 mice per group) once a week for 8 weeks. The injections were administered subcutaneously (sc). Blood samples were collected weekly during treatment and again 6 weeks post-treatment. HBV DNA, HBs antigen, and HBe antigen levels were measured by serological tests as described below. Results for the first 10 weeks are shown in Table 21 and Figure 13. Data for the remaining 4 weeks are unavailable as the investigation was still ongoing at the time of filing.

[0552] Detection of HBs antigen and HBe antigen: Serum HBs antigen and HBe antigen levels in infected AAV-HBV mouse serum were measured using the HBs antigen-chemiluminescent immunoassay (CLIA) and HBe antigen CLIA kits (Autobio diagnostics Co. Ltd., Zhengzhou, China, catalog numbers CL0310-2 and CL0312-2, respectively), following the manufacturer's protocol. Briefly, 50 μl of serum was transferred to each antibody-coated microtiter plate, and 50 μl of enzyme-binding reagent was added. After incubating the plates at room temperature on a shaker for 60 minutes, all wells were washed six times with washing 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 plates at room temperature for 10 minutes, luminescence was measured using an Envision luminescence reader. HBs antigen levels are given in units IU / ml, assuming 1 ng of HBs antigen = 1.14 IU. HBe antigen levels are given in units 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 no. 06374913001) in a treatment cartridge and incubated for 10 minutes. Then, DNA was extracted using the "MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit" (Roche, catalog no. 06543588001) and the "Viral NA Plasma SV external lysis 2.0" protocol. The DNA elution volume was 50 μl.

[0554] Quantification of extracted HBV DNA was performed using a Taqman qPCR machine (ViiA7, Life Technologies). Each DNA sample was tested in duplicate by PCR. PCR was performed by adding 5 μl of DNA sample to 15 μl of PCR master mix containing 10 μl of TaqMan Gene Expression Master Mix (Applied Biosystems, catalog no. 4369016), 0.5 μl of PrimeTime XL qPCR Primer / Probe (IDT), and 4.5 μl of distilled water in a 384-well plate. The settings used were as follows: UDG incubation (2 min, 50°C), enzyme activation (10 min, 95°C), and 40 PCR cycles (denaturation at 95°C for 15 seconds; and annealing and extension at 60°C for 1 minute). DNA copy number was determined based on the HBV plasmid DNA assay line using ViiA7 software. t It was calculated from the values.

[0555] TaqMan primer sequence and probe (IDT): Forward core primer (F3_core): CTG TGC CTT GGG TGG CTT T (Sequence ID: 784) Reverse primer (R3_core): AAG GAA AGA AGT CAG AAG GCA AAA (Sequence ID: 785) Taqman probe (P3_core): 56-FAM / AGC TCC AAA / ZEN / TTC TTT ATA AGG GTC GAT GTC CAT G / 3IABkFQ (Sequence ID: 786).

[0556] [Table 21]

[0557] As this study demonstrates, GalNAc-bound PD-L1 antisense oligonucleotide CMP number 759_2 has a significant effect on reducing HBV-DNA, HBs antigen, and HBe antigen levels after 6 weeks of treatment, and this effect persists 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 We investigated the ability of GalNAc-bound PD-L1 antisense oligonucleotide compounds to reduce PD-L1 transcripts in primary human hepatocytes using genomics.

[0559] cell culture Frozen human liver cells were incubated in WME (Whole Membrane Extract) supplemented with 10% fetal bovine serum, penicillin (100 U / ml), streptomycin (0.1 mg / ml), and L-glutamine (0.292 mg / ml) for 5 × 10⁶ times. 6 Dilute to cells / ml and 2 × 10 5Cells were seeded at a cell / well density in collagen-coated 24-well plates (Becton Dickinson AG, Allschwil, Switzerland). Cells were pre-cultured for 4 hours and attached to the cell culture plate before treatment with oligonucleotides at a final concentration of 100 μM was initiated. The oligonucleotides used are shown in Tables 21 and 8, and PBS was used as the vehicle. 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). 35 μl of oligonucleotide stock solution in 1 mM PBS was added to the cell culture, and the cells were left on the surface for 24 or 66 hours.

[0560] Library preparation Transcript expression profiling was performed using Stranded mRNA chemistry with a sequencing strategy of 2×51bp paired terminal reads and 30M / sample (Q squared EA). Cells were lysed by adding 350 μl of Qiagen RLT buffer to the wells and submitted under a randomization scheme.

[0561] mRNA was purified using the QiagenRNeasy Mini Kit. mRNA was quantified and its integrity was assessed using an Agilent Bioanalyzer. Upon initial quality assessment of the isolated RNA, all samples were observed to have a RIN score > 7.0, meeting the 100 ng input quality metric.

[0562] Starting with 100 ng of total RNA, sequencing libraries were prepared for all samples using Illumina TruSeq Stranded mRNA Library Preparation. The final cDNA libraries were analyzed for size distribution and using Agilent Bioanalyzer (DNA1000 kit), quantified by qPCR (KAPA Library Quant Kit), and standardized to 2 nM for sequencing preparation. Using the Standard Cluster Generation Kit v5, the cDNA libraries were bound to the flow cell surface, and cBot was isothermally attached to amplify the attached cDNA constructs to approximately 1000 clonal colonies. DNA sequencing was performed using synthetic sequencing techniques with the TruSeq SBS kit.

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

[0564] Table 22 shows the decrease in PD-L1 transcripts after incubation with GalNAc-bound PD-L1 antisense oligonucleotide compounds.

[0565] [Table 22-1]

[0566] Compared to samples treated with the vehicle, all five GalNAc-conjugated antisense compounds showed a significant decrease in PD-L1 transcripts after 24-hour and 66-hour incubation.

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

[0568] cell lineage HepaRG cells (Biopredic International, Saint-Gregoire, France) were cultured in William E medium (supplemented with 10% HepaRG proliferation supplement (Biopredic)). Using lentiviral technology, HepaRG cell lines stably overexpressing human ASGPR1 and ASGPR2 were generated from this cell line. Lentiviruses encoding human ASGPR1 and 2, produced as needed by Sirion biotech (CLV-CMV-ASGPR1-T2a_ASGPR2-IRES-Puro) under the control of the CMV promoter and puromycin resistance gene, were transduced into proliferation HepaRG cells at a 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 expression of the transgenes. ASGPR1 / 2 overexpression was confirmed at both the mRNA level by RT-qPCR (ASGPR1: 8560x compared to non-transduced, ASGPR2: 2389x compared to 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 isolated from the HepG2.2.15 cell culture supernatant and concentrated using PEG precipitation. To evaluate the activity of the test compound against HBV, differentiated ASGPR-HepaRG cells in a 96-well plate were infected with HBV for 20 hours at an infection multiplicity (MOI) of 20-30, and the cells were then washed four times with PBS to remove the HBV inoculum.

[0570] Effectiveness of oligonucleotides The following oligonucleotides

[0571] [Table 22-2]

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

[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] The data were analyzed using the 2^-ddct method. DCT values ​​were calculated using actin B as the endogenous control. PD-L1 expression levels are associated with both the endogenous control and the saline vehicle.

[0575] The EC50 calculation was performed using GraphPad Prism6. The results are shown in Table 23.

[0576] [Table 23]

[0577] As these data clearly show, GalNAc-bound PD-L1 antisense oligonucleotides significantly improve EC50 values.

[0578] Example 10 - Stimulated T cell function in PBMCs derived 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 stimulation of peripheral blood mononuclear cells (PBMCs) with HBV antigen.

[0579] Frozen PBMCs collected from three chronic HBV-infected patients were thawed and seeded at a density of 200,000 cells / well in 100 μl of medium (RPMI1640 + GlutaMax + 8% human serum + 25 mM Hepes + 1% PenStrep). The following day, cells were stimulated with 1 μM PepMixHBV large envelope protein or 1 μM PepMixHBV core protein, with or without 5 μM CMP ID number: 466_1 or CMP ID number: 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, the cells were re-stimulated for 24 hours with PepMix or 5 μg / ml concanavalin A + PD-L1 antisense oligonucleotide. 0.1 μl of Brefeldin A, 0.1 μl of Monensin, and 3 μl of anti-human CD-107 (APC) were added to ensure stimulation over the following 5 hours.

[0580] After 24 hours, the 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 viability and death staining (BV510) (BD Biosciences)] was applied at 4°C for 30 minutes. The cells were fixed in BD fixation buffer at 4°C for 15 minutes. The following morning, the cells were permeabilized with BD Perm / Wash Buffer at 4°C for 15 minutes, and intracellular staining [anti-human INF (PE)] was performed at 4°C for 30 minutes. After washing with Perm / Wash Buffer, the cells were dissolved in 250 μl of staining buffer.

[0581] FACS measurements were performed using BD Fortessa (BD Biosciences). The entire cell population for analysis was first gated with live cells (viable and dead cells, BV510), and then with CD3+ (BV605) cells. Subsequently, CD3+ cells were graphed as CD107a+ (APC) versus IFN+ (PE).

[0582] The results are shown in Table 24.

[0583] [Table 24]

[0584] As these data show, antigen stimulation alone can induce T cell activation (an increase in the percentage of CD3+ cells expressing INF and / or CD107a) in PBMCs of CHB patients (n=3). Addition of PD-L1 antisense oligonucleotides CMP 466_1 or 640_1 further increased the CD3+ T cell response. This increase was primarily observed in the HBV envelope-stimulated group. [1] a. Oligonucleotides (region A) comprising a sequence of 10-30 nucleotides in length having at least 90% complementarity to the PD-L1 target nucleic acid; b. At least one asialocryprotein receptor targeting binding site (region C) is covalently bonded to the oligonucleotide of (a); An antisense oligonucleotide conjugate comprising [the specified element]. [2] The oligonucleotide conjugate according to item 1, wherein the sequence of nucleotides 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 continuous 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 items 1 to 3, wherein the continuous 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 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 described in items 1 to 5, comprising one or more modified nucleosides in the continuous nucleotide sequence (for example, one or more 2'-sugar modified nucleosides). [7] The oligonucleotide conjugate described in item 6, wherein one or more 2'-saccharide 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, arabino nucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides. [8] The oligonucleotide conjugate according to either item 6 or 7, wherein all of the modified nucleosides are LNA nucleosides. [9] The oligonucleotide conjugate according to any one of items 1 to 8, wherein the continuous nucleotide sequence comprises at least one modified nucleoside bond (e.g., at least one phosphorothioate nucleoside bond).

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

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

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

[13] The oligonucleotide conjugate according to any one of items 1 to 12, wherein the asialoclycoprotein receptor targeting binding site is monovalent, divalent, trivalent, or tetravalent.

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

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

[16] The oligonucleotide conjugate according to 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] Antisense oligonucleotides comprising an oligonucleotide or a sequence of nucleotides as described in any one of items 1 to 11.

[18] A pharmaceutical composition comprising an oligonucleotide conjugate as described in items 1 to 16, or an oligonucleotide as described in item 17, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or auxiliary agent.

[19] An in vivo or in vitro method for regulating the expression of PD-L1 in target cells expressing PD-L1, the method comprising administering an effective amount of an oligonucleotide conjugate according to item 1 to 16 or an oligonucleotide according to item 17 to the cells.

[20] Oligonucleotide conjugates as described in items 1 to 16, or oligonucleotides as described in item 17, or pharmaceutical compositions as described in item 18, for use in restoring an immune response to a virus or parasite.

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

[22] Oligonucleotide conjugates as described in items 1 to 16, or oligonucleotides as described in item 17, or pharmaceutical compositions as described in item 18, for use as pharmaceuticals.

[23] Oligonucleotide conjugates according to items 1 to 16, or oligonucleotides according to item 17, or pharmaceutical compositions 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 oligonucleotide conjugates described in items 1 to 16, or oligonucleotides described in item 17, or pharmaceutical compositions described in item 18, for the preparation of drugs 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. An antisense oligonucleotide comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, capable of reducing PD-L1 expression, wherein the continuous nucleotide sequence is at least 90% complementary to a PD-L1 target nucleic acid, and the continuous nucleotide sequence is complementary to a partial sequence of the target nucleic acid, wherein the partial sequence is selected from the group consisting of positions 5467 to 12107, 15317 to 15720, 15317 to 18083, 15317 to 19511, 9812 to 9859, 15690 to 15735, 371 to 3068, 7300 to 7333, 8028 to 8072, 11787 to 11873 and 18881 to 19494 of SEQ ID NO:

1.

2. It has at least 90% complementarity to the PD-L1 target nucleic acid, and the sequence numbers are 466, 640, 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, 4 An antisense oligonucleotide comprising a sequence of 10 to 30 nucleotides in length, having at least 90% identity with a sequence selected from the sequences 74, 490, 493, 512, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636, 637, 638, 645, 650, 651, 652, 653, 658, 659, 660, 665, 678, 679, 680, 682, 683, 684, 687, 694, 706, 716, 728, 733, 734, and 735.

3. The aforementioned continuous nucleotide sequence is sequence numbers 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, 474 The antisense oligonucleotide according to claim 2, having 100% identity with a sequence selected from the group consisting of 490, 493, 512, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636, 637, 638, 645, 650, 651, 652, 653, 658, 659, 660, 665, 678, 679, 680, 682, 683, 684, 687, 694, 706, 716, 728, 733, 734, and 735.

4. An antisense oligonucleotide conjugate comprising an oligonucleotide according to any one of claims 1 to 3 and a conjugate portion covalently bonded to the oligonucleotide.

5. The antisense oligonucleotide conjugate according to claim 4, wherein a linker is provided between the oligonucleotide and the conjugate portion.

6. The antisense oligonucleotide conjugate according to claim 4 or 5, wherein the conjugate portion is an asialoglycoprotein receptor targeting portion.

7. The antisense oligonucleotide conjugate according to claim 6, wherein the asialoclycoprotein receptor targeting portion is a trivalent N-acetylgalactosamine (GalNAc) portion.

8. The antisense oligonucleotide conjugate according to any one of claims 5 to 7, wherein the linker is a physiologically unstable linker.

9. The antisense oligonucleotide conjugate according to claim 8, wherein the physiologically unstable linker is a nuclease-sensitive linker.

10. The antisense oligonucleotide conjugate according to claim 8 or 9, wherein the physiologically unstable linker comprises a cytidine-adenosine dinucleotide.

11. The antisense oligonucleotide conjugate according to claim 4, wherein a linker is provided between the oligonucleotide and the conjugate portion, the conjugate portion comprises an asialocrypoprotein receptor targeting portion which is a trivalent N-acetylgalactosamine (GalNAc) portion, the linker is a physiologically unstable linker, and the physiologically unstable linker further comprises a cytidine-adenosine dinucleotide.

12. A pharmaceutical composition comprising an antisense oligonucleotide according to claims 1 to 3, or an antisense oligonucleotide conjugate according to any one of claims 4 to 11, and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutically acceptable diluent is sterile phosphate-buffered saline.

14. The pharmaceutical composition according to claim 12 or 13, wherein the pharmaceutically acceptable salt is a sodium salt.

15. The pharmaceutical composition according to claim 12 or 13, wherein the pharmaceutically acceptable salt is a potassium salt.

16. An in vivo or in vitro method for regulating PD-L1 expression in target cells expressing PD-L1, comprising administering an effective amount to the cells an antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15.

17. An antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15, used for restoring an immune response to a virus.

18. The antisense oligonucleotide, antisense oligonucleotide conjugate, or pharmaceutical composition according to claim 17, wherein the virus is hepatitis B virus (HBV).

19. An antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15, for use in restoring an immune response to a parasite.

20. The antisense oligonucleotide, antisense oligonucleotide conjugate, or pharmaceutical composition according to any one of claims 17 to 19, wherein the recovery of the immune response is an increase in CD8+ T cells specific to one or more HBV antigens in the liver compared to a control.

21. An antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15, for use as a pharmaceutical.

22. An antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15, for use in the treatment of hepatitis B virus (HBV) infection.

23. Use of an antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15 for the manufacture of a pharmaceutical for the treatment of hepatitis B virus (HBV) infection.

24. A method for treating or preventing a disease, comprising administering to a subject suffering from or at risk of suffering from the disease an effective amount therapeutically or prophylactically, an antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15.

25. A method for treating or preventing hepatitis B virus (HBV) infection, comprising administering to a subject who is infected with or at risk of being infected with HBV in a therapeutically or prophylactically effective amount an antisense oligonucleotide according to any one of claims 1 to 3, an antisense oligonucleotide conjugate according to any one of claims 4 to 11, or a pharmaceutical composition according to any one of claims 12 to 15.

26. A pharmaceutically acceptable salt of an antisense oligonucleotide according to any one of claims 1 to 3, or an antisense oligonucleotide conjugate according to any one of claims 4 to 11.

27. A pharmaceutically acceptable sodium salt of an antisense oligonucleotide according to any one of claims 1 to 3, or an antisense oligonucleotide conjugate according to any one of claims 4 to 11.

28. A pharmaceutically acceptable potassium salt of an antisense oligonucleotide according to any one of claims 1 to 3, or an antisense oligonucleotide conjugate according to any one of claims 4 to 11.

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