NZ793076A - Novel approach for treating cancer - Google Patents

Abstract

The present invention relates to a novel approach for treating cancer, which is based on targeting PD-L1 mRNA. The invention is directed to oligonucleotides comprising 10 to 20 modified or unmodified nucleotides complementary to specifically selected regions of the PD-L1.

Description

The present invention relates to a novel approach for treating cancer, which is based on targeting PD-L1 mRNA. The invention is directed to oligonucleotides comprising 10 to 20 modified or fied nucleotides complementary to specifically selected regions of the PD-L1.

NZ 793076 NOVEL APPROACH FOR TREATING CANCER This is a divisional application of New Zealand Patent Application 752539 the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to a novel approach for treating a tumor, which is based on targeting PD-L1 mRNA. The invention is directed to oligonucleotides comprising 10 to 20 modified or unmodified nucleotides complementary to ically selected regions of PDL1 mRNA.

BACKGROUND OF THE INVENTION During the last decades of cancer research it became obvious that the immune system is indispensable to initiate and release an ive anti-tumor response. Therefore it needs to be integrated in common cancer ies. However, cancer cells developed mechanisms to circumvent anti-tumor immune responses, e.g. by gulating HLA molecules leading to impaired antigen presentation, by the secretion of inhibitory soluble mediators such as IL-10 or adenosine, or by expressing T cell inhibitory s.

The most prominent inhibitory ligands expressed on the surface of antigen presenting cells and cancer cells are Programmed death-ligand 1 and 2 (PD-L1/PD-L2). Programmed death-ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that is encoded in humans by the CD274 gene. While PD-L2 (B7- DC or CD273) is expressed ily on professional antigen presenting cells (such as B cells and dendritic cells), PD-L1 is expressed on non-lymphoid cells, such as parenchymal cells, virus-infected cells and tumor cells, as well as on other immune cells.

The two ligands interact with their receptor Programmed death-1 (PD-1), expressed on several immune cells, such as activated T cells, B cells, natural killer cells and d cells in the periphery. The initial role of the interaction between the ve receptor PD- 1 and its ligands is thought to regulate the old of antigen responses of T cells and B cells in the ery. Activation of PD-1 by its ligands during infection or mation in normal tissue is critically important in maintaining homeostasis of immune response to prevent autoimmunity. Their interaction in tumor microenvironments, however, provides an immune escape for tumor cells by dephosphorylating key ns downstream of the T cell receptor after antigen encounter, mainly in the late phase of an immune response ing exhaustion or anergy of effector T cells. Engagement of PD-1 by its ligands during n recognition induces cross linkage of the antigen-receptor complex with PD- 1. This mediates phosphorylation of the tyrosine residue in the immunoreceptor tyrosinebased switch motif (ITSM) leading to the tment of tyrosine phosphatases that dephosphorylate and inactivate proximal effector molecules such as Zap70 in T cells.

These molecular mechanisms lead to decreased TCR signaling which in turn drives d proliferation and cytokine production of effector T cells.

Deficiency of PD-1 in mice renders them resistant to viral infection and induces the suppression of tumor growth and metastasis in ent tumor models. Thus, blocking PD- 1/PD-L1 interactions can result in therapeutic benefit in tumor-bearing mice, as it leads to improved tumor cell killing by cytotoxic T cells. In addition, deficient PD-1 expression in mice results in loss of peripheral tolerance and the subsequent development of autoimmune diseases such as lupus-like glomerulonephritis, arthritis, hepatitis or cardiomyopathy.

In humans, genetic alterations of the PD-1 encoding gene (PDCD1) is associated with increased susceptibility s several autoimmune diseases, such as systemic lupus matosus, type 1 diabetes, multiple sclerosis, toid arthritis, Grave´s disease and ankylosing spondylitis. However, distinct from other negative immune regulators, PD- 1 deficiency specifically and only affects antigen-specific autoimmune responses whereas deficiency of other negative regulators results in systemic, non-antigen-specific autoimmune phenotypes.

Until present, the blockade of PD-1/PD-L1 interactions by monoclonal antibodies or by genetic manipulation of PD-1 expression led to enhanced tumor ation. rmore, clinical data suggest that ed PD-L1 expression in tumors correlates with poorer survival prognosis of ent cancer patients. These results led to the development of several different fully humanized monoclonal dies targeting either PD-1 or PD-L1.

Application of those antibodies showed positive response rates in humans in al trials of e.g. non-small-cell lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma with drug-related adverse events in a subset of patients. Nonetheless, therapeutic blockade of the PD-1 y is the most powerful target for immunological anti-tumor therapies in the s at present.

However, a large proportion of cancer patients (>70%) do not d well to eutic blockade of PD-1 using monoclonal antibody therapies. These data suggest the importance of accessing combinatorial therapies using agents to block additional negative or to activate positive regulators that might have additive and/or synergistic s in order to improve antitumor immunotherapies. The application of antisense oligonucleotides targeting PD-L1 expression on mRNA level in combination with therapies that target other known negative (e.g. LAG-3; TIM-3; 2B4; CD160) or positive (e.g. CD137; CD40) immuneregulatory pathways could provide better therapeutic efficacy than targeting the PD-1 pathway alone.

Several studies indicate the presence of an immune inhibitory soluble form of PD-L1 (sPDL1 ) in sera of cancer patients, correlating with disease severity and a negative patient survival outcome. Thus, it is very likely that the soluble form of PD-L1 cannot be fully captured by conventional monoclonal antibodies directed against PD-L1 on a systemic level.

Furthermore, antibodies are huge in molecular size and therefore might not reach s sed on dense and packed tissues as it is the case for many different tumors.

Thus, while targeting PD-L1 appears to be a promising approach to develop and improve novel immunotherapies against different cancers, no satisfactory solution for achieving that has yet been found. Hence, there is still a high scientific and medical need for therapeutic agents, which reduce or inhibit PD-L1 expression and/or activity.

Based on experiences with similar signaling ctions, it appeared to be likely that tumor cells are more accessible to be targeted by inhibition of gene expression, e.g. by antisense oligonucleotides. Thus, the tion of target expression could be a more promising ch to develop and improve novel immunotherapies against different cancers than conventional antibody ies. Currently two competing logies are predominantly used for ic suppression of mRNA expression: Antisense oligonucleotides and siRNA.

Due to its double stranded nature, siRNA does not cross the cell ne by itself and delivery systems are required for its activity in vitro and in vivo. While delivery s for siRNA exist that efficiently r siRNA to liver cells in vivo, there is currently no system that can deliver siRNA in vivo to extra-hepatic tissues such as tumors with sufficient efficacy. Therefore siRNA approaches to target PD-L1 are tly limited to ex vivo approaches, for example for the generation of dendritic cell-based tumor es.

For antisense oligonucleotides efficacy in cell culture is lly determined after transfection using transfection reagents or oporation. Antisense approaches directed t PD-L1 are described, for example, in in Mazanet et al., J. l. 169 (2002) 3581-3588.

It was recently discovered that antisense oligonucleotides that are modified by so called 3rd generation chemistries, such as 2‘,4‘-LNA (see, for example, constrained ethyl bridged nucleic acids (c-ET), can enter cells in vitro and in vivo t a delivery system to achieve target gulation.

Additionally, double-stranded RNA molecules (see WO 27180) and so-called “3rd generation nse compounds”, which comprise two antisense constructs linked via their 5’ ends (see However, in approaches described in the prior art only moderate target suppression levels were achieved and relatively high concentrations of oligonucleotides were required for efficient target suppression. For example, in US 8,563,528 a concentration of 10 µM resulted in a target inhibition of just 70%. IC50 values for 3rd generation oligonucleotides without ection reagent typically range between 300 and 600 nM (Zhang et al. Gene Therapy (2011) 18, 326-333).

After systemic administration in vivo, only relatively low oligonucleotide concentrations can be achieved in relevant target tissues. Therefore antisense oligonucleotides that reach high maximal target suppression at low concentration would clearly result in an enhanced therapeutic effect.

Furthermore, in the case of gene silencing using LNA-modified antisense molecules, it has been observed that after removal of the antisense constructs, the target protein expression level rapidly raised again, and reached 50% of baseline expression in 24 h, and 100% in 72 h (see Stein et al., Nucleic Acids Res. 38 (2010) e3 [doi:10.1093/nar/gkp841]).

Thus, while targeting PD-L1 appears to be a ing approach to develop and improve novel immunotherapies against different s, no satisfactory solution for achieving that has yet been found. Hence, there is still a high scientific and medical need for therapeutic , which efficiently reduce or inhibit PD-L1 expression and/or activity, in particular for a prolonged period of time.

SUMMARY OF THE INVENTION Despite the suboptimal results obtained with the approaches described in the prior art that suppress disease causing targets with nse approaches, the present inventors surprisingly identified that certain specific antisense constructs were able to achieve the inhibition of the expression of PD-L1 relative to untreated control in HDLM-2 cells by at least 80%, with several candidates achieving inhibition of more than 85%, of more than 90%, or of more than 95%, and/or relative to untreated control in U-87MG cells by at least 50%, with several ates achieving inhibition of more than 75%, more ularly of more than 80%. IC50 values in HDLM-2 cells were below 100 nM for several candidates or even below 20 nM for ed candidates. Most importantly, the inhibition of PD-L1 expression continued after removal of the anti-PD-L1 ucts, in ular for at least 96 h.

Thus, in a first , the present invention relates to an oligonucleotide consisting of from 10 to 20 nucleotides, particularly from 13 to 18 nucleotides, wherein the sequence of said oligonucleotide corresponds to the antisense strand of the PD-L1 nucleic acid coding sequence of SEQ ID NO. 115, wherein one or more nucleotide(s) of the ucleotide is/are optionally modified, and wherein said oligonucleotide inhibits the expression of PDL1 relative to untreated control in HDLM-2 cells by at least 80%.

In a second aspect, the present invention relates to a pharmaceutical composition comprising the oligonucleotide according to the t invention.

In a third , the present invention relates to the oligonucleotides or the pharmaceutical composition according to the present invention for use in a method of preventing and/or treating a disease or disorder selected from the list of: a malignant tumor and a benign tumor.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of a first oligonucleotide screen for PD-L1 tion in HDLM- 2 cells, as described in Example 2: for each uct, a pair of bars is shown: left, brighter bar: PD-L1 expression; right, darker bar: HPRT1 expression, including bars for a positive control (third pair from the left), and for a negative control oligonucleotide and a no- oligonucleotide l (two pairs on the right).

Figure 2 shows the results of a first oligonucleotide screen for PD-L1 inhibition in U-87MG cells, as described in Example 2: for each construct, a pair of bars is shown: left, brighter bar: PD-L1 expression; right, darker bar: HPRT1 sion, including bars for a positive control (15th pair from the left), and for a negative control oligonucleotide (17th pair from the right, and a no-oligonucleotide control (last pair on the .

Figure 3 shows the results of the addition of different trations (10 µM; 1 µM; 0.5 µM; 0.1 µM) of selected oligonucleotides (A03021; A03053; A03043; A03037; A03014) to HDLM-2 cells as described in Example 3. PD-L1 protein levels were determined by flow cytometry at different time points (24 h = black bars; 48 h = light grey bars; 72 h = dark grey bars; 96 h = white bars) after oligo treatment. PD-L1 n expression per cell is depicted as Mean Fluorescence Intensity (MFI).

Figure 4 shows the results of the addition of different concentrations (10 µM; 5 µM; 1 µM; 0.5 µM; 0.1 µM; 0.05 µM) of oligonucleotide A03043H to primary human dendritic cells as described in Example 3. PD-L1 n levels were determined by flow try 72 h after oligo treatment. PD-L1 protein expression per cell is depicted as Median Fluorescence Intensity (MFI).

Figure 5 shows a number of different modified nucleotides that may be used in the context of the present invention.

Figure 6 shows the results of an oligonucleotide screen for murine PD-L1 inhibition in RENCA cells, as described in Example 5: for each construct, a pair of bars is shown: left, brighter bar: murine PD-L1 expression; right, darker bar: murine HPRT1 expression, both in relation to a no oligo control (=1.00), including bars for a negative control oligonucleotide (pair of bars on the right).

Figure 7 shows the results of a first oligonucleotide screen for murine PD-L1 inhibition in 4T1 cells, as described in Example 5: for each construct, a pair of bars is shown: right, er bar: murine PD-L1 sion; left, darker bar: murine HPRT1 expression, both in relation to a no oligo control ), including bars for a negative control oligonucleotide (second pair of bars from the right).

Figure 8 shows that the antigen-specific stimulation of T cells with PD-L1 oligonucleotide- treated dendritic cells increases the frequency of antigen-specific T cells.

Figure 9 shows the results of a knockdown of human PD-L1 in human myeloid d suppressor cells (MDSC).

Figure 10 shows the analysis of PD-L1 protein expression by flow cytometry in HDLM-2 cells 1, 2, 3 and 4 days after removal of selected oligonucleotides. PD-L1 protein expression is depicted as mean fluorescence intensity (MFI) and was calculated by subtracting the MFI of PD-L1 by the MFI of unspecific isotype control. Relative expression ed to untreated control cells (set as 1) is depicted. PD-L1 expression was analyzed in duplicates for each condition (specific staining and isotype control). Data were analyzed using Two-tailed student’s t-test, P ≤ 0.0001 (****).

Embodiments of the invention: 1. An oligonucleotide consisting of from 10 to 20 tides, particularly from 13 to 18 nucleotides, wherein the sequence of said ucleotide ponds to the antisense strand of the PD-L1 nucleic acid coding sequence of SEQ ID NO. 115, n one or more tide(s) of the oligonucleotide is/are optionally modified, and wherein said ucleotide inhibits the expression of PD-L1 relative to the expression of untreated control in HDLM-2 cells by at least 80%. 2. The oligonucleotide of embodiment 1, wherein said oligonucleotide inhibits the expression of PD-L1 in HDLM-2 cells relative to untreated cells by at least 85%, particularly by at least 90%, most particularly by at least 95%. 3. The oligonucleotide of embodiment 1 or 2, wherein one or more nucleotide(s) in said oligonucleotide are modified, wherein the modified nucleotide is an LNA, a c-ET, an ENA, a polyalkylene , a 2'-fluoro-, a 2'-O-methoxy-, a FANA and/or a 2'-O- -modified nucleotide. 4. The oligonucleotide of embodiment 3, wherein the modified nucleotide(s) is/are located within the stretch of 5 tides at the 5'- and/or 3'-end of the oligonucleotide, particularly at the 5'- and the 3'-end of the oligonucleotide.

. The ucleotide according to embodiment 4, wherein said oligonucleotide is a Gapmer comprising at least one LNA nucleotide within the stretch of 5 nucleotides at the 5’-end of said oligonucleotide, and at least one LNA nucleotide within the h of 5 nucleotides at the 3’-end of said oligonucleotide. 6. The oligonucleotide according to any one of embodiments 1 to 5, wherein the ucleotide comprises a sequence selected from the group ting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, SEQ ID NO. 16, SEQ ID NO. 76, SEQ ID NO. 72, SEQ ID NO. 54, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 112, and SEQ ID NO. 104, particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, and SEQ ID NO. 16, more particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, and SEQ ID NO. 84, more particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, and SEQ ID NO. 74, and more ularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, and SEQ ID NO. 28. 7. Pharmaceutical composition comprising the oligonucleotide according to any one of embodiments 1 to 6. 8. The oligonucleotide according to any one of embodiments 1 to 6 or the pharmaceutical composition according to embodiment 7 for use in a method of preventing and/or treating a disease or disorder selected from the list of: a malignant tumor, and a benign tumor. 9. The oligonucleotide or pharmaceutical composition for use according to embodiment 8, wherein the tumor is selected from the group consisting of solid tumors, blood born tumors, leukemias, tumor metastasis, hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas, psoriasis, astrocytoma, acoustic neuroma, blastoma, Ewing's tumor, craniopharyngioma, ependymoma, medulloblastoma, glioma, hemangioblastoma, Hodgkin’s lymphoma, medullablastoma, leukaemia, mesothelioma, neuroblastoma, neurofibroma, non- Hodgkin’s lymphoma, pinealoma, retinoblastoma, sarcoma, seminoma, mas, and Wilms’ tumor, or is selected from the group consisting of bile duct carcinoma, bladder carcinoma, brain tumor, breast , bronchogenic carcinoma, carcinoma of the kidney, al cancer, choriocarcinoma, choroid carcinoma, cystadenocarcinoma, embryonal carcinoma, epithelial carcinoma, esophageal cancer, cervical carcinoma, colon carcinoma, colorectal carcinoma, endometrial cancer, gallbladder cancer, c cancer, head cancer, liver carcinoma, lung oma, ary carcinoma, neck cancer, non-small-cell bronchogenic/lung carcinoma, ovarian cancer, pancreas carcinoma, papillary carcinoma, papillary adenocarcinoma, prostate cancer, small intestine carcinoma, prostate carcinoma, rectal cancer, renal cell carcinoma, retinoblastoma, skin cancer, small-cell bronchogenic/lung carcinoma, squamous cell carcinoma, ous gland carcinoma, testicular carcinoma, and uterine cancer.

DETAILED DESCRIPTION OF THE INVENTION Thus, in a first aspect, the present ion relates to an oligonucleotide consisting of from 10 to 20 nucleotides, particularly from 13 to 18 nucleotides, wherein the sequence of said oligonucleotide corresponds to the antisense strand of the PD-L1 nucleic acid coding ce of SEQ ID NO. 115 (Table 15), n one or more nucleotide(s) of the oligonucleotide is/are optionally modified, and n said oligonucleotide inhibits the expression of PD-L1 relative to untreated control in HDLM-2 cells by at least 80%.

In particular embodiments of the t invention, said oligonucleotide ts the expression of PD-L1 relative to untreated control in HDLM-2 cells by at least 85%, more particularly by at least 90%, and most ularly by at least 95%.

In particular such embodiments, said oligonucleotide inhibits the expression of PD-L1 relative to untreated control in HDLM-2 cells for at least 24 h after removal of said oligonucleotide, in particular for at least 48 h, for at least 72 h, or in particular for at least 96 h.

In particular embodiments of the present invention, one or more nucleotide(s) in said oligonucleotide are modified.

A tide forms the ng block of an oligonucleotide, and is for example composed of a nucleobase genous base, e.g., purine or pyrimidine), a five-carbon sugar (e.g., , 2-deoxyribose, arabinose, xylose, lyxose, allose, altrose, glucose, e, gulose, idose, galactose, talose or stabilized modifications of those sugars), and one or more phosphate groups. Examples of modified phosphate groups are orothioate or phosphonate. Each compound of the nucleotide is modifiable, and is naturally or non-naturally occurring. Examples of the latter are: locked nucleic acid (LNA), 2’, 4’ constrained ethyl nucleic acids (c-ET), 2'-0,4'-C-ethylene-bridged c acid (ENA), polyalkylene oxide- (such as triethylene glycol (TEG)), 2'-fluoro-, 2'-deoxy-2'-fluoro-beta- D-arabinonucleic acid , 2'methoxy- and 2'-O-methyl-modified nucleotides.

Figure 5 shows examples of a number of different modified nucleotides that may be used in the context of the t invention.

An “LNA” is a modified RNA nucleotide, wherein the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon (2'- 4 'ribonucleoside). The bridge locks the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleosides and nucleotides, respectively, comprise for example the forms of thio-LNA, oxy-LNA, or amino-LNA, in alpha-D- or beta-L-configuration, and can be mixed or combined, respectively, with DNA or RNA residues in the oligonucleotide.

A “bridged nucleic acid” is modified RNA nucleotide, sometimes also referred to as ained or inaccessible RNA molecule, which may contain a five-membered, sixmembered or even a seven-membered bridged structure with a “fixed” C3’-endo sugar puckering. The bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. Specific examples are “ENA” nucleotides, n the bridge is an ethylene bridge. Figure 5 shows a number of BNA tides that may be used in the context of the present invention.

In a particular embodiment, one or more nucleotide(s) in said oligonucleotide are modified, wherein the modified nucleotide contains a modified phosphate group, particularly selected from a phosphorothioate and a methylphosphonate, particularly a phosphorothioate. In particular ments, all phosphate groups of the oligonucleotide are modified ate groups, particularly independently selected from phosphorothioates and methylphosphonates, particularly wherein all phosphate groups are phosphorothioates.

In a ular embodiment, one or more tide(s) in said oligonucleotide are modified, wherein the modified nucleotide is an LNA, a c-ET, an ENA, a polyalkylene oxide-, a 2'- fluoro-, a 2'-O-methoxy-, a FANA and/or a 2'-O-methyl-modified nucleotide.

In particular embodiments, the modified nucleotide(s) is/are located within the stretch of 5 nucleotides at the 5'- and/or 3'- end of the oligonucleotide, particularly at the 5'- and the 3'- end of the oligonucleotide.

In particular ments, the oligonucleotides of the t ion comprise at least one modified nucleotide, particularly at least one LNA, c-ET and/or ENA, at the 5'- and/or 3'-end of the oligonucleotide. In a particular embodiment, the oligonucleotide comprises 1, 2, 3, or 4 LNAs or c-ETs or ENAs within the h of up to 5 nucleotides at the 5'-end, and 1, 2, 3, or 4 LNAs or c-ETs or ENAs within the stretch of up to 5 nucleotides at the 3 '-end. In another particular embodiment, the ucleotide comprises 1, 2, 3, or 4 LNAs, c-ETs, or ENAs at the within the stretch of 5 nucleotides 5'-end or 3'-end, and a polyalkylene oxide such as TEG within the stretch of 5 nucleotides at the 3'- or 5'-end.

In ular embodiments, said ucleotide is a Gapmer comprising at least one LNA nucleotide within the stretch of 5 nucleotides at the 5’-end of said oligonucleotide, and at least one LNA nucleotide within the stretch of 5 nucleotides at the 3’-end of said oligonucleotide. In particular ments, said Gapmer comprises 2 or 3 LNA nucleotides within the stretch of 5 nucleotides at the 5’-end of said oligonucleotide, and 2 or 3 LNA nucleotides within the stretch of 5 nucleotides at the 3’-end of said oligonucleotide.

In the context of the present invention, the term “Gapmer” refers to a chimeric nse oligonucleotide that contains a central block of deoxynucleotide rs sufficiently long to induce RNase H cleavage. The central block of a Gapmer is flanked by blocks of 2’-O modified cleotides or other artificially modified ribonucleotide monomers such as bridged nucleic acids (BNAs) that protect the internal block from nuclease degradation.

In many earlier studies modified DNA analogs were investigated for their stability in biological fluids. In the majority of these experiments phosphorothioate DNA analogs were used. More recently, several types of artificial nucleotide monomers including BNA monomers have been investigated for their usefulness in the design of Gapmers.

Gapmers have been used to obtain RNase-H mediated cleavage of target RNAs, while ng the number of phosphorothioate linkages. Phosphorothioates possess increased resistance to nucleases compared to unmodified DNA. However, they have several disadvantages. These include low g capacity to complementary nucleic acids and non-specific binding to proteins that cause toxic side-effects limiting their applications. The occurrence of toxic side-effects together with non-specific binding g off-target effects has stimulated the design of new artificial nucleic acids for the pment of modified oligonucleotides that provide efficient and specific antisense activity in vivo without exhibiting toxic ffects.

LNA Gapmers are powerful tools for loss of function studies of proteins, mRNA and lncRNAs. These single strand antisense oligonucleotides catalyze RNase H-dependent degradation of complementary RNA targets. LNA Gapmers are typically 12-20 nucleotides long enriched with LNA in the ng regions and DNA in a LNA free central gap - hence the name . The LNA-containing flanking regions confers nuclease resistance to the antisense oligo while at the same time increases target binding affinity less of the GC content. The central DNA “gap” activates RNase H cleavage of the target RNA upon binding.

In particular embodiments of the present in ion, the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, SEQ ID NO. 16, SEQ ID NO. 76, SEQ ID NO. 72, SEQ ID NO. 54, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 112, and SEQ ID NO. 104, particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, and SEQ ID NO. 16, more particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, and SEQ ID NO. 84, more ularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, and SEQ ID NO. 74, and more particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, and SEQ ID NO. 28.

In a particular embodiment, the oligonucleotide is a variant of a sequence selected from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, SEQ ID NO. 16, SEQ ID NO. 76, SEQ ID NO. 72, SEQ ID NO. 54, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 112, and SEQ ID NO. 104, particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, and SEQ ID NO. 16, more ularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, and SEQ ID NO. 84, more particularly from the group ting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, and SEQ ID NO. 74, and more particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, and SEQ ID NO. 28, wherein in such variant one or more of the phosphorothioates are independently replaced by an unmodified phosphate of a modified phosphate other than a phosphorothioate, particularly a methylphosphonate.

In a particular embodiment, the oligonucleotide is a variant of a sequence selected from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, SEQ ID NO. 16, SEQ ID NO. 76, SEQ ID NO. 72, SEQ ID NO. 54, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 112, and SEQ ID NO. 104, particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, SEQ ID NO. 84, SEQ ID NO. 82, SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 92, SEQ ID NO. 102, SEQ ID NO. 100, SEQ ID NO. 58, and SEQ ID NO. 16, more particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, SEQ ID NO. 74, SEQ ID NO. 94, SEQ ID NO. 108, SEQ ID NO. 88, SEQ ID NO. 56, SEQ ID NO. 46, SEQ ID NO. 96, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 62, SEQ ID NO. 114, SEQ ID NO. 34, SEQ ID NO. 98, and SEQ ID NO. 84, more particularly from the group consisting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, SEQ ID NO. 28, SEQ ID NO. 110, and SEQ ID NO. 74, and more particularly from the group ting of SEQ ID NO. 42, SEQ ID NO. 106, SEQ ID NO. 86, and SEQ ID NO. 28, wherein such variant comprises one or more nucleotide ches particularly one or two mismatches, more particularly one mismatch, provided that any such variant including the ch(es), when analyzed with the bioinformatic tools described in Example 1, contains at least one mismatch relative to human whole genome screening while maintaining gy to relevant species.

In a second aspect, the present invention relates to a pharmaceutical composition comprising an oligonucleotide according to the t invention.

In particular embodiments, the pharmaceutical composition further comprises a pharmaceutically able carrier.

In particular embodiments, the pharmaceutical ition further comprises at least one additional component selected from: a further anti-sense compound, an antibody, a chemotherapeutic nd, an anti-inflammatory compound, an ral compound, an immuno-modulating compound, a pharmaceutically acceptable binding agents and an adjuvant.

In one embodiment, the oligonucleotide and the pharmaceutical composition, respectively, is formulated as dosage unit in form of capsules, tablets and pills etc., respectively, which contain for example the following compounds: microcrystalline cellulose, gum or gelatin as binders; starch or lactose as excipients; stearates as lubricants, various sweetening or flavouring agents. For capsules the dosage unit may contain a liquid carrier like fatty oils.

Likewise coatings of sugar or enteric agents may be part of the dosage unit.

The oligonucleotide and/or the pharmaceutical composition is administrable via different routes. These routes of administration include, but are not limited to, electroporation, epidermal, impression into skin, intra-arterial, intra-articular, intracranial, intradermal, intra-lesional, intra-muscular, intranasal, intra-ocular, intrathecal, intracameral, intraperitoneal, intraprostatic, intrapulmonary, intraspinal, intratracheal, intratumoral, intravenous, intravesical, rectal, placement within cavities of the body, nasal inhalation, oral, pulmonary tion (e.g., by inhalation or insufflation of powders or aerosols, ing by nebulizer), subcutaneous, subdermal, topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), or transdermal stration.

For eral, subcutaneous, intradermal or topical administration the oligonucleotide and/or the pharmaceutical composition include for example a sterile diluent, buffers, regulators of ty and antibacterials. In a preferred embodiment, the oligonucleotide or pharmaceutical composition is ed with carriers that protect t degradation or immediate elimination from the body, including implants or microcapsules with lled release ties. For intravenous administration the preferred carriers are for e physiological saline or phosphate ed saline. An oligonucleotide and/or a pharmaceutical composition comprising such oligonucleotide for oral administration es for example powder or granule, microparticulate, nanoparticulate, sion or solution in water or non-aqueous media, capsule, gel capsule, sachet, tablet or minitablet.

An oligonucleotide and/or a pharmaceutical composition comprising for parenteral, intrathecal, intracameral or intraventricular administration includes for example sterile s solutions which optionally contain buffer, diluent and/or other suitable additive such as penetration enhancer, carrier compound and/or other pharmaceutically acceptable carrier or excipient.

A pharmaceutically acceptable carrier is for example liquid or solid, and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical ition. Typical pharmaceutically acceptable rs include, but are not limited to, a binding agent (e.g. pregelatinized maize starch, nylpyrrolidone or hydroxypropyl methylcellulose, etc.); filler (e.g. e and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricant (e.g., magnesium stearate, talcum, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrate (e.g., starch, sodium starch ate, etc.); or wetting agent (e.g., sodium lauryl sulfate, etc.). Sustained release oral delivery systems and/or enteric gs for orally administered dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404. An adjuvant is included under these phrases.

Besides being used in a method of human e prevention and/or treatment, the oligonucleotide and/or the pharmaceutical composition according to the present invention is also used in a method for prevention and/or treatment of other ts including veterinary animals, reptiles, birds, exotic animals and farm animals, including mammals, rodents, and the like. Mammals include for example horses, dogs, pigs, cats, or primates (for example, a , a chimpanzee, or a . Rodents include for example rats, rabbits, mice, squirrels, or guinea pigs.

In a third aspect, the present invention relates to a ceutical composition comprising the oligonucleotide according to the present invention for use in a method of preventing and/or treating a disease or disorder selected from the list of: a malignant tumor and a benign tumor.

In particular other embodiments of the t invention, the tumor is selected from the group consisting of solid tumors, blood born tumors, leukemias, tumor metastasis, hemangiomas, acoustic neuromas, ibromas, trachomas, pyogenic granulomas, sis, astrocytoma, acoustic neuroma, blastoma, Ewing's tumor, craniopharyngioma, ependymoma, medulloblastoma, glioma, hemangioblastoma, Hodgkin’s lymphoma, medullablastoma, leukaemia, mesothelioma, neuroblastoma, neurofibroma, non- Hodgkin’s lymphoma, pinealoma, retinoblastoma, sarcoma, seminoma, trachomas, and Wilms’ tumor, or is selected from the group of bile duct carcinoma, bladder carcinoma, brain tumor, breast cancer, bronchogenic carcinoma, carcinoma of the kidney, al cancer, choriocarcinoma, choroid carcinoma, cystadenocarcinoma, embryonal carcinoma, lial oma, geal cancer, cervical carcinoma, colon carcinoma, colorectal carcinoma, endometrial cancer, gallbladder , gastric cancer, head cancer, liver carcinoma, lung carcinoma, medullary carcinoma, neck cancer, ll-cell bronchogenic/lung carcinoma, ovarian , pancreas oma, papillary carcinoma, papillary arcinoma, prostate cancer, small intestine carcinoma, te carcinoma, rectal cancer, renal cell carcinoma, retinoblastoma, skin cancer, small-cell bronchogenic/lung carcinoma, squamous cell carcinoma, sebaceous gland oma, testicular oma, and uterine cancer.

For the purpose of y and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include ments having combinations of all or some of the features described.

The following es will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that the scope of the present invention refers to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention.

Examples e 1: Sequence selection and oligonucleotide modification process At first suitable sequences enting possible target sites were identified using proprietary bioinformatics tools. In a second step the effects of chemical modifications on these sequences were predicted in silico to optimize modification patterns.

Stepwise Sequence Selection Process.

Sequence lengths ranging from 13mer length to 17mer length were considered.

In the primary analysis the cross reactivity of potential target sites on the human PD-L1 mRNA (NM_014143.3) and cynomolgus monkey mRNA was investigated. Cross reactivity to non-human primates was considered as important for evaluation of rated pharmacology. From the 16,380 sequences initially analyzed, about 50% showed 100% gy to the lgus PD-L1 mRNA.

These ces were further analyzed for specificity to the spliced human and cynomolgus transcriptomes applying following filters:  No ce should tly match (100%) any off-target region in human or monkey.

 By allowing one ch, a higher number of off-target mRNA matches were allowed for shorter sequences (e.g. 13mers and 14mers) compared to 15mers and 16mers.

 No 17mer should show any predicted off-target binding in human and monkey ng one mismatch.

Sequences fulfilling these ia were further analyzed for specificity in the primary unspliced human and monkey transcriptome. Following filters were set:  No sequence should perfectly match (100%) any pre-mRNA off-target region.

 No 17mer should show any predicted off-target binding of pre-mRNA allowing one mismatch.

Analysis of the s of chemical modifications The effects of al modifications on physicochemical properties such as melting temperature and tendency to form hairpins or dimers were evaluated using available prediction tools. In total, 217 potential oligonucleotides based on the sequences with highest predicted specificity were analyzed using these tools Oligonucleotides with the most ble predicted physicochemical properties were selected for synthesis (see Table 3) and in vitro screening (see Examples 2 to 4).

Example 2: In vitro screening of antisense oligonucleotides Material: HDLM-2 cell line - DSMZ (Deutsche ng für Mikroorganismen und Zelllinien, Braunschweig, Germany / ACC-17) Table 1. ent used in the screening protocol Manufacturer Serial Number Centrifuge Heraeus Megafuge 16R