WO2018065627A1 - Immunosuppression-reverting oligonucleotides inhibiting the expression of cd73 - Google Patents

Abstract

The present invention refers to immunosuppression-reverting oligonucleotides comprising 12 to 18 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of an ectoenzyme (CD73) of SEQ ID NO.1 (human) and/or of a sequence of SEQ ID NO.2 (mouse), wherein the oligonucleotide inhibits at least 50 % of the CD73 expression. The invention is further directed to a pharmaceutical composition comprising such oligonucleotide.

Description

Immunosuppression-reverting oligonucleotides inhibiting the expression of

CD73 The present disclosure refers to an immunosuppression-reverting oligonucleotide hybridizing with a nucleic acid sequence of an ectoenzyme (NT5E or CD73) and to a pharmaceutical composition comprising such immunosuppression-reverting

oligonucleotide and a pharmaceutically acceptable carrier, excipient and/or dilutant. Technical background

In recent years the treatment of several different diseases such as malignant tumors was very successful by application of immune therapy, in particular by inhibitors of so called "immune checkpoints". These checkpoints are molecules in the immune system that either turn up (co-stimulatory molecules) or down a signal. The concept of the

therapeutic approach is based on the activation of endogenous anti-tumor immune reactions. Many cancers for example protect themselves from the immune system by inhibiting T cell and NK cell activity, respectively. Immune checkpoint modulators, i.e., stimulators or inhibitors are for example directed to one or more of CTLA-4, PD-1, PD- LI, LAG- 3, VISTA, A2AR, BTLA, IDO, CD39, CD73, STAT3, TD02, TIM-3, MICA, NKG2A, KIR, TIGIT, TGF-beta, Ox40, GITR, CD27, CD 160, 2B4 and 4- IBB.

CD73 needs to be considered as one novel and promising candidate to improve immunity towards different types of cancers. CD73 is an ectoenzyme (NTPdase) and catalyzes the conversion of AMP to immunosuppressive adenosine. CD73 acts in concert but downstream of CD39, known to convert ATP to ADP and ADP to AMP. Adenosine exerts its effects via adenosine receptor Al, adenosine receptor A2A, adenosine receptor AiB and adenosine receptor A3. The range of immune cells expressing adenosine receptors and therefore potentially affected by the immunomodulatory effects of adenosine include T lymphocytes, natural killer (NK) cells, NKT cells, macrophages, DCs, neutrophils, mast cells and B cells.

CD73 is found in most tissues and many cell types including subsets of lymphocytes, macrophages, dendritic cells, endothelial cells and epithelial cells. Hypoxia induces CD73 mRNA, protein expression and increases CD73 activity in mouse microvascular endothelial cells. Particularly, CD73 is highly expressed in many different human (solid and hematologic) tumors, and its elevated expression and activity are associated with tumor invasiveness and metastasis and with shorter patient survival. The RNA expression and enzyme activity of CD73 are variable in different breast cancer cell lines.

Dying cancer cells release ATP to the extracellular space in the tumor microenvironment. Living tumor cells express often high levels of CD39 and CD73 and convert ATP to the immunosuppressive adenosine. By this, tumor cells are competent to perform uncontrolled proliferation and expansion. By binding to A2A or A2B receptors on lymphocytes, adenosine mediates an immunosuppressive signal towards these cells. For example, T cells are inhibited in their proliferation, cytotoxic cytokine production and activation. NK cells show reduced cytotoxic potential. Adenosine induces alternative activation in macrophages (immune suppressive M2 phenotype) resulting in reduced proinflammatory cytokine production but increased generation of the immunosuppressive cytokine IL-10. The important role of CD73 as relevant therapeutic target in different tumors is underlined by the fact that tumor models using CD73 or A2A receptor knockout mice reveal improved disease outcome.

Anti-human CD73 monoclonal antibodies such as anti-CD73-antibodies of Innate Pharma (e.g., Innate Pharma Poster #iph_poster_aarc2016_cd73) are currently under pre-clinical investigation in immune-oncology cell-based assays. However, because of steric hindrance monoclonal antibodies against CD73 might fail to localize to the tumor microenvironment. Furthermore, non-hydrolysable small molecular inhibitors of CD73 such as AMPCP (adenosine 5'-(a,6-methylene)diphosphate; e.g., Structure20, 2161-2173, December 5, 2012) an ADP analog competitively inhibiting the CD73 activity, have been tested in vitro and in vivo in animal models but relatively high concentrations and repetitive dosing are needed to successfully block CD73 enzymatic activity.

Immune therapies have resulted in long-term remission, but only of small patient groups so far. The reason may be that numerous immune checkpoints and optionally further immunosuppressive mechanisms are involved in the interaction between for example the immune system and the tumor cells. The combination of immune checkpoints and potential other mechanisms may vary depending on the tumor and individual conditions of a subject to escape the body's defenses. For the inhibition of several immunosuppressive mechanisms common approaches using an antibody and/or a small molecule are not or hardly suitable as the molecular target is located intracellularly or does not have enzymatic activity. Accordingly, an agent which is safe and effective in inhibiting the function of an "immune checkpoint" such as CD73 would be an important addition for the treatment of patients suffering from diseases or conditions affected for example by the activity of this enzyme.

Oligonucleotides of the present invention are very successful in the inhibition of the expression and activity of CD73, respectively. The mode of action of an oligonucleotide differs from the mode of action of an antibody or small molecule, and oligonucleotides are highly advantageous regarding for example

(i) the penetration of tumor tissue in solid tumors,

(ii) the blocking of multiple functions and activities, respectively, of a target,

(iii) the combination of oligonucleotides with each other or an antibody or a small molecule, and

(iv) the inhibition of intracellular effects which are not accessible for an antibody or inhibitable via a small molecule.

Therefore, targeting CD73 expression on cancer and immune cells on mRNA-level by antisense-oligonucleotides is a promising state-of-the-art approach to develop and improve for example immunotherapies against different cancers and immune diseases, respectively.

Summary

The present invention refers to an oligonucleotide such as an immunosuppression- reverting oligonucleotide comprising about 10 to 20 nucleotides, wherein at least one of the nucleotides is modified. The oligonucleotide hybridizes for example with a nucleic acid sequence of an ectoenzyme CD73 of SEQ ID NO. 1 (human) and/or a sequence of SEQ ID NO.2 (mouse). The modified nucleotide is for example selected from the group consisting of a bridged nucleic acid (e.g., LNA, cET, ENA, 2'Fluoro modified nucleotide or 2O-Methyl modified nucleotide). In some embodiments, the oligonucleotide inhibits at least 50 % of the CD73 expression and in some embodiments the oligonucleotide inhibits the expression of CD73 at a nanomolar concentration. Antisense oligonucleotides have significant advantages in comparison to RNAi.

Antisense oligonucleotides can be transfected without transfecting reagent in vitro and thus, the transfection is closer to in vivo conditions than transfections using transfecting reagents which are obligatory for the transfection of RNAi. In vivo systemic

administration of antisense oligonucleotides is possible in different tissues whereas the administration of RNAi in vivo is dependent on delivery systems such as GalNAc for example in liver. Moreover, antisense oligonucleotides are shorter than RNAi and therefore, are less complex in synthesis and in the uptake into cells. RNAi regularly show off- target effects of passenger strands which likewise can initiate RNAi passenger strand RISC loading is a significant concern for RNAi drugs because the passenger strand could direct RNAi activity towards unintended targets, resulting in toxic side effects" (see Chackalamannil, Rotella, Ward, Comprehensive Modicinal Chemistry III Elsevier, 03.06.2017). Antisense oligonucleotides do not comprise a passenger strand. The present invention is further directed to a pharmaceutical composition comprising an immunosuppression-reverting oligonucleotide of the present invention and optionally a pharmaceutically acceptable carrier, excipient and/or dilutant. In some embodiments, this pharmaceutical composition additionally comprises a chemotherapeutic such as platinum or gemcitabine, another oligonucleotide, an antibody and/or a small molecule which is for example effective in tumor treatment.

In some embodiments, the oligonucleotide of the present invention is in combination with another oligonucleotide, an antibody and/or a small molecule, either each of these compounds is separate or combined in a pharmaceutical composition, wherein the oligonucleotide, the antibody and/or the small molecule inhibits or stimulates an immune suppressive factor such as IDOl, ID02, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TD02, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop, and/or Xbpl. In addition or alternatively, the oligonucleotide, the antibody and/or the small molecule inhibits or stimulates or an immune stimulatory factor such as 4- IBB, Ox40, KIR, GITR, CD27 and/or 2B4.

Furthermore, the present invention relates to the use of the oligonucleotide or the pharmaceutical composition of the present invention in a method of preventing and/or treating a disorder, where a CD73 imbalance is involved. In some embodiments, the disorder is for example an autoimmune disorder, for example autoimmune arthritis or gastrointestinal autoimmune diseases such as inflammatory bowel disease (IBD) or colitis, an immune disorder, for example an immune exhaustion due to chronic viral infections such as HIV infection, a cardiovascular disorder, an inflammatory disorder, for example a chronic airway inflammation, a bacterial, viral and/or fungal infection, for example sepsis or a Mycobacterium bovis infection, a liver disorder, a chronic kidney disorder, a psychiatric disorder and/or cancer. CD73 has many physiological roles, such as regulation of barrier function, adaptation to hypoxia, ischemic preconditioning, anti- inflammation, leukocyte extravasation. Expression and activity of CD73 on cancer cells is associated with poor prognosis and may promote metastasis. CD73 facilitates the adhesion, migration, invasion of human breast cancer cells and proliferation of glioma cells and these processes are dependent upon the enzyme's production of adenosine. In some embodiments, the oligonucleotide or the pharmaceutical composition of the present invention is for example administered locally or systemically. All documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Description of figures

Fig. 1 depicts the distribution of human (h)CD73 antisense oligonucleotide binding sites on the hCD73 mRNA of SEQ ID No. 1 (NM_002526.3) as well as their modification(s) and length. hCD73 antisense oligonucleotides were aligned to the hCD73 mRNA sequence of SEQ ID No. 1. The different grayscales indicate the different LNA

modifications and symbols indicate the different length of the antisense oligonucleotides.

Fig. 2A-2E depict hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotides in human cancer cell lines A- 172 (human glioblastoma) in a first and second screening round (Fig. 2A and 2B (parts 1 and 2)), and EFO-21 (human ovary cystadenocarcinoma;) in one screening round (Fig. 2C (parts 1 and 2)), in one murine cell line 4T1 (breast cancer) in one screening round (Fig. 2D (parts 1 and 2)) and human SKOV-3 (human ovarian adenocarcinoma cancer) cells in one screening round (Fig. 2E). A- 172, EFO-21, 4T1 and SKOV-3 cells were treated for 3 days with 10 μΜ of the respective antisense oligonucleotide. As control, cells were treated with negl, an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT (described in WO2014154843 Al). Residual human or mouse CD73 mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression values of the housekeeping gene GAPDH or HPRTl. Depicted is the mean of triplicate wells +/- SD.

Fig. 3A and 3B show a correlation analysis of hmCD73 antisense oligonucleotide efficacy in human EFO-21 cells compared to human A- 172 cells (Fig. 3A) and human A- 172 cells compared to mouse 4T1 cells (Fig. 3B). Fig. 4A and 4B shows concentration dependent hmCD73 mRNA knockdown by selected hmCD73 antisense oligonucleotides in SKOV-3 cells and EFO-21 (human ovarian cancer), which were A05008HM (SEQ ID No. 4), A05009HM (SEQ ID No. 6), A05018HM (SEQ ID No. 3), A05026HM (SEQ ID No. 8), and A05028HM (SEQ ID No. 5) (Fig. 4A) and A05018HM (SEQ ID No.3), A05027HM (SEQ ID No.30), A05028HM (SEQ ID No.5), and A05037HM (SEQ ID No.39) in EFO-21 cells (Fig. 4B). SKOV-3 and EFO-21 cells were treated for 3 days with the indicated concentration of the respective antisense oligonucleotide. Residual hCD73 expression is depicted compared to untreated control cells (set as 100). hCD73 mRNA expression values were normalized to expression of the housekeeping gene HPRTl. Depicted is the mean of triplicate wells +/- SD.

Concentration-dependent target knockdown was used for calculation of IC50 values shown in Table 7 for SKOV-3 cells and in Table 8 for EFO-21 cells.

Figure 5A to 5C depict hmCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotides in human and mouse cancer cell lines. EFO-21 (human ovary

cystadenocarcinoma (Fig. 5A)), SKOV-3 (human ovarian adenocarcinoma (Fig. 5B)), and 4T1 (mouse breast cancer (Fig. 5C)) cells were treated for 3 days with 10 μΜ of the respective antisense oligonucleotide. Residual hmCD73 mRNA expression relative to untreated cells (set as 1) is depicted. Expression values were normalized to expression of the housekeeping gene HPRTl. Depicted is the mean of triplicate wells +/- SD. Fig. 6 shows EFO-21 cells were treated for 3 days with the indicated concentrations of the respective antisense oligonucleotide. hCD73 mRNA expression values were normalized to expression of the housekeeping gene HPRTl. Residual hCD73 mRNA expression relative to untreated cells (set as 100) is depicted. Depicted is the mean of triplicate wells +/- SD.

Fig. 7A and 7B depict a concentration-dependent hCD73 mRNA and protein knockdown by A05008HM (SEQ ID No. 4), A05018HM (SEQ ID No. 3) and A05028HM (SEQ ID No. 5). Analysis of protein expression and cell viability by flow cytometry of CD73 (Fig. 7A), and 7-AAD staining of SKOV-3 cells (Fig. 7B) was performed after treatment with the indicated antisense oligonucleotides for 6 days. As a control, cells were similarly treated with negl. Median fluorescence intensity of CD73 protein expression (Fig. 7A) and total dead cells (7-AAD positive cells) (Fig. 7B) relative to the number of control cells that were not treated with any antisense oligonucleotide (=1) is depicted.

Fig. 8 shows knockdown of CD73 protein expression on SKOV3 and EFO-21 cells after antisense oligonucleotide treatment. CD73 protein expression on both tested cell lines was analyzed after treatment with A05018HM (SEQ ID No. 3) for six days. As a control, cells were similarly treated with negl. Median fluorescence intensity of CD73 protein expression relative to the expression of control cells that were not treated with any antisense oligonucleotide (=1) is depicted.

Fig. 9A-9D depict effects of hCD73 knockdown on extracellular pyrophosphate levels and cell viability by SKOV-3 and EFO-21 cells. Generation of pyrophosphate as an indirect measure for the production of adenosine was analyzed in cell supernatants from EFO-21 cells (Fig. 9A) and SKOV-3 cells (Fig. 9C), that were treated for 6 days with A05018HM (SEQ ID No. 3). Before measurement, exogenous AMP was added to the cells at 500μΜ at the indicated time points. Effect of the treatment with A05018HM (SEQ ID No. 3) on cell viability of EFO-21 cells (Fig. 9B) and SKOV-3 cells (Fig. 9D) was tested using the cell titer blue assay.

Fig. 10A to IOC shows EFO-21 cells treated with the CD73-specific antisense

oligonucleotide A05018HM (black column) or the control oligonucleotide S6 (white column) at 5 μΜ for a total treatment time of 6 days. hCD73 protein expression was analyzed by flow cytometry. Fig. 10A depicts residual hCD73 expression relative to untreated cells (dotted column; set as 1). To analyze the capacity to degrade extracellular AMP, 300 μΜ of AMP was added to cells or cell-free PBS (striped column) 6.5 h before termination. Fig. 10B shows relative AMP levels relative to cell-free AMP-supplemented PBS (set as 1) and Fig. IOC depicts absolute adenosine concentrations in cell

supernatants (cell-conditioned PBS). Depicted is the mean of 3-wells +/- SD or single values for conditions with cell free PBS.

Fig. 11A to 11C depict human CD4⁺ T cells labelled with cell proliferation dye, activated with anti-CD3 and treated with 5 μΜ of the CD73 specific antisense oligonucleotide A05018HM (black column) or the control oligonucleotide S6 (white column) for a total treatment time of 5 days. In the vehicle control (striped column), cells were activated with anti-CD3 only. Subsequently, 300 μΜ of AMP or vehicle were added to cells on day 3 and day 4 after start of oligonucleotide treatment. Fig. 11A shows CD73 protein expression, Fig. 11B depicts proliferation, and Fig. 11 C shows absolute cell numbers of CD4⁺ T cells which were analyzed using Flow Cytometry on day 5 after start of oligonucleotide treatment. Depicted is the mean of triplicate wells +/- SD.

Fig. 12 depicts in vivo effect of hmCD73 antisense oligonucleotide (A05027HM) treatment on mCD73 mRNA expression in mouse liver. The results depicted in Fig. 12 show mCD73 mRNA levels in livers of A05027HM- or vehicle- treated mice (white column).

Fig. 13 shows hCD73 mRNA of SEQ ID No. 1 (NM_002526.3). Detailed description

The present invention provides for the first time human and murine oligonucleotides which hybridize with mRNA sequences of the ectonucleotidase CD73 and inhibit the expression and activity, respectively, of CD73 for example on a tumor cell or a tumor - associated immune cell. In consequence, the level of ATP increases and the level of its degradation products such as ADP, AMP and immunosuppressive Adenosine decreases. All these effects result in an increase of antitumoral immune cells, immune activation (e.g., via cytotoxic T cells or NK cells) and recognition and elimination of tumor cells, respectively. Thus, the oligonucleotides of the present invention represent an interesting and highly efficient tool for use in a method of preventing and/or treating disorders, where the CD73 expression and activity, respectively, is increased.

In the following, the elements of the present invention will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. Throughout this specification and the claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be

understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as", "for example"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Oligonucleotides of the present invention are for example antisense oligonucleotides consisting of or comprising 10 to 25 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 12 to 18 nucleotides, or 14 to 17 nucleotides. The oligonucleotides for example consist of or comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 nucleotides. The oligonucleotides of the present invention comprise at least one nucleotide which is modified. The modified nucleotide is for example a bridged nucleotide such as a locked nucleic acid (LNA, e.g., 2',4'-LNA), cET, ENA, a 2 luoro modified nucleotide, a 2Ό- Methyl modified nucleotide or combinations thereof. In some embodiments, the oligonucleotide of the present invention comprises nucleotides having the same or different modifications. In some embodiments the oligonucleotide of the present invention comprises a modified phosphate backbone, wherein the phosphate is for example a phosphorothioate or methylphosphonate or both.

The oligonucleotide of the present invention comprises the one or more modified nucleotide at the 3'- and/or 5'- end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow in a row of 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more unmodified nucleotides. The following Table 1 presents embodiments of oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The oligonucleotides consisting of or comprising the sequences of Table 1 may comprise any other modified nucleotide and any other combination of modified and unmodified nucleotides. Oligonucleotides of Table 1 hybridize with mRNA of human and murine CD 73:

SEQ ID No. mRNA (Antisense)

Name Antisense Sequence 5 -3^' with PTO (*) and LNA (+)

Sequence 5 -3^"

3 A05018HM GATTTCCCAGTGCCAT +G^*+A*+T^*rrC^*C^*C^*A^*G*T^*G^*C^*+C^*+A*+T

4 A05008HM GCTGTGCACGTCGTT +G^*+C*+T^*G*T^*G^*C^*A^*C^*G*T^*C^*+G*+T*+T

5 A05028HM TGATTTCCCAGTGCCAT +T^*+G^*+A*T*T*T^*C^*C^*C^*A^*G*T^*G^*C^*+C^*+A*+T

6 A05009HM GGCTGTGCACGTCGT +G^*+G^*+C*T^*G*T^*G^*C^*A^*C^*G*T^*C^*+G*+T

7 A05019HM CTCAGAATTGGAAATT +C^*+r+C^*A^*G^*A^*A*T*T^*G^*G^*A^*A^*+A^*+r+T

8 A05026HM ACTCG AC ACTTG GTG C +A^*+C*+T^*C^*G^*A^*C^*A^*C*T^*rG^*G^*+r+G^*+C

9 A05001 HM CTGTGCACGTCGTT +C*+T^*+G*T^*G^*C^*A^*C^*G*T^*C^*+G*+T*+T

10 A05002HM AGCACGTTGGGTTC ^*+G^*C^*A^*C^*G G^*G^*G*T*+T^*+C

11 A05003HM ACGGTGAACCAGAT +A^*+C^*+G^*G*T^*G^*A^*A^*C^*C^*A^*+G^*+A*+T

12 A05004HM GCATAGGCCTGGAC +G^*+C^*+A*T^*A^*G^*G^*C^*C*T^*G^*+G^*+A^*+C

13 A05005HM CTCGACACTTGGTG +C*+T^*+C^*G^*A^*C^*A^*C G^*G*+T^*+G

14 A05006HM ACTCGACACTTGGT +A^*+C*+T^*C^*G^*A^*C^*A^*C*T*T^*+G^*+G*+T

15 A05007HM GGCACTCGACACTT +G^*+G^*+CWC*T^*C^*G^*A^*C^*A^*+C*+T*+T

16 A05010HM GTCCTCCCACCACGA +G*+T^*+C^*C*T^*C^*C^*C^*A^*C^*C^*A^*+C^*+G^*+A

17 A05011 HM GTCCTCCCACCACGA +G*+T^*C *T^*C^*C^*C^*A^*C^*C^*A^*C^*G^*+A

18 A05012HM TGTCCTCCCACCACG +T^*+G*T^*C^*C*T^*C^*C^*C^*A^*C^*C^*+A^*+C^*+G

19 A05013HM GAGTGTCCTCCCACC +G^*+A^*G*T^*G*T^*C^*C*T^*C^*C^*C^*A^*C^*+C

20 A05014HM TCGACACTTGGTGCA +T^*+C^*+G^*A^*C^*A^*C*T*T^*G^*G*T^*+G^*+C^*+A

21 A05015HM CTCGACACTTGGTGC +C*+T^*+C^*G^*A^*C^*A^*C^*T G^*G*+T^*+G^*+C

22 A05016HM ACTCGACACTTGGTG +A^*+C*+T^*C^*G^*A^*C^*A^*C*T*T^*+G^*G*T^*+G

23 A05017HM CACTCGACACTTGGT +C^*+A^*+C*T^*C^*G^*A^*C^*A^*C*T*T^*+G^*+G*+T

24 A05020HM TGTCCTCCCACCACGA +T^*+G*+T^*C^*C*T^*C^*C^*C^*A^*C^*C^*A^*+C^*+G^*+A

25 A05021 HM GTGTCCTCCCACCACG +G*+T^*G*T^*C^*C*T^*C^*C^*C^*A^*C^*C^*+A^*+C^*+G

26 A05022HM GAGTGTCCTCCCACCA +G^*+A^*G*T^*G*T^*C^*C*T^*C^*C^*C^*A^*C^*+C^*+A

27 A05023HM GTGTTGGAGTGTCCTC +G*T^*+G*T*T^*G^*G^*A^*G*T^*G*T^*C^*C*+T^*+C

28 A05024HM CTCG AC ACTTG GTG C A +C*+T^*+C^*G^*A^*C^*A^*C*T*T^*G^*G*T^*+G^*+C^*+A

29 A05025HM CTCG AC ACTTG GTG C A +C*+T^*+C^*G^*A^*C^*A^*C*T*T^*G^*G*T^*G^*+C^*+A

30 A05027HM GCACTCGACACTTGGT +G^*+C^*+A^*C*T^*C^*G^*A^*C^*A^*C*T*T^*+G^*+G*+T

31 A05029HM GTGTCCTCCCACCACGA +G*+T^*+G*T^*C^*C*T^*C^*C^*C^*A^*C^*C^*A^*+C^*+G^*+A

32 A05030HM AGTGTCCTCCCACCACG +A^*+G*+T^*G*T^*C^*C*T^*C^*C^*C^*A^*C^*C^*+A^*+C^*+G

33 A05031 HM GAGTGTCCTCCCACCAC +G^*+A^*G*T^*G*T^*C^*C*T^*C^*C^*C^*A^*C^*C^*A^*+C

34 A05032HM GAGTGTCCTCCCACCAC +G^*A^*G*T^*G*T^*C^*C*T^*C^*C^*C^*A^*C^*+C^*A^*+C

35 A05033HM GAGTGTCCTCCCACCAC +G^*A^*G*T^*G*T^*C^*C*T^*C^*C^*C^*A^*C^*C^*+A^*+C

36 A05034HM ACTCGACACTTGGTGCA +A^*+C*+T^*C^*G^*A^*C^*A^*C*T*T^*G^*G*T^*+G^*+C^*+A

37 A05035HM ACTCGACACTTGGTGCA +A^*+C*+T^*C^*G^*A^*C^*A^*C*T*T^*G^*G*T^*G^*+C^*+A

38 A05036HM GGCACTCGACACTTGGT +G^*+G^*+C^*A^*C*T^*C^*G^*A^*C^*A^*C*T*T^*+G^*+G*+T

39 A05037HM GGCACTCGACACTTGGT +G^*+G^*CA^*C*T^*C^*G^*A^*C^*A^*C*T*T^*+G^*+G*+T

42 A05038HM GCTGTGCACGTCGTT +G^*+C*T^*G*T^*G^*C^*A^*C^*G*T^*C^*+G*+T*+T

43 A05039HM CGGCTGTGCACGTCGTT +C^*G^*+G^*C*T^*G*T^*G^*C^*A^*C^*G*T^*C^*G*+T*+T

44 A05040HM GGCTGTGCACGTCGT +G^*+G^*+C*T^*G*T^*G^*C^*A^*C^*G*T^*C^*+G*+T

45 A05041 HM GATTTCCCAGTGCCAT +G^*+A*+T*T*T^*C^*C^*C^*A^*G*T^*G^*C^*C^*+A*+T 45 A05042HM GATTTCCCAGTGCCAT +G^*+A^*T*rrC^*C^*C^*A^*G^*T^*G^*C^*+C^*+A^*+T

46 A05043HM TGATTTCCCAGTGCCAT +T^*+G^*+A^*T^*T^*T^*C^*C^*C^*A^*G^*T^*G^*+C^*C^*+A^*+T

46 A05044HM TGATTTCCCAGTGCCAT +T^*+G^*+A^*T^*T^*T^*C^*C^*C^*A^*G^*T^*G^*C^*C^*+A^*+T

47 A05045HM CATGATTTCCCAGTGCC +C^*+A^*+T^*G^*A*rrrC^*C^*C^*A^*G^*T^*G^*+C^*+C

40 Neg1 +C^*+G^*+T^*T^*T^*A^*G^*G^*C^*T^*A^*T^*G^*T^*A^*+C^*+T^*+T

41 S6 +T^*+C^*+T^*A^*T^*C^*G^*T^*G^*A^*T^*G^*T^*T^*+T^*+C^*+T

Table 1: List of antisense oligonucleotides hybridizing with human and murine CD73 for example of SEQ ID No. 1 and SEQ ID No. 2, respectively. Negl is an antisense oligonucleotide representing a negative control which is not hybridizing with CD73 of SEQ ID No. 1 and SEQ ID No. 2.

The oligonucleotides of the present invention hybridize for example with mRNA of human or murine CD73 of SEQ ID No. 1 and/or SEQ ID No. 2. Such oligonucleotides are called CD73 antisense oligonucleotides.

In some embodiments, the oligonucleotide of the present invention inhibits at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of CD73 such as the, e.g., human or murine, CD73 expression. Thus, the

oligonucleotides of the present invention are immunosuppression-reverting

oligonucleotides which revert immunosuppression for example in a cell, tissue, organ, or a subject. The oligonucleotide of the present invention inhibits the expression of CD73 at a nanomolar or micromolar concentration for example in a concentration of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nM, or 1, 10 or 100 μΜ.

In some embodiments, the oligonucleotide of the present invention is used in a

concentration of 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or 1, 2.2, 3, 5, 6.6 or 10 μΜ.

In some embodiments the present invention refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically

acceptable carrier, excipient and/or dilutant. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic, another oligonucleotide, an antibody and/or a small molecule. In some embodiments, the oligonucleotide or the pharmaceutical composition of the present invention is for use in a method of preventing and/or treating a disorder. In some embodiments, the use of the oligonucleotide or the pharmaceutical composition of the present invention in a method of preventing and/or treating a disorder is combined with radiotherapy. The radiotherapy may be further combined with a chemotherapy (e.g., platinum, gemcitabine). The disorder is for example characterized by an CD73 imbalance, i.e., the CD73 level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The CD73 level is for example increased by an increased CD73 expression and activity, respectively. The CD73 level can be measured by any standard method such as immunohistochemistry, western blot, quantitative real time PCR or QuantiGene assay known to a person skilled in the art.

An oligonucleotide or a pharmaceutical composition of the present invention is

administered locally or systemically for example orally, sublingually, nasally,

subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumoral, intrathecal, transdermal and/or rectal. Alternatively or in combination ex vivo treated immune cells are administered. The oligonucleotide is administered alone or in

combination with another immunosuppression-reverting oligonucleotide of the present invention and optionally in combination with another compound such as another oligonucleotide, an antibody, a small molecule and/or a chemotherapeutic (e.g., platinum, gemcitabine). In some embodiments, the other oligonucleotide (i.e., not being part of the present invention), the antibody, and/or the small molecule are effective in preventing and/or treating an autoimmune disorder, for example autoimmune arthritis or

gastrointestinal autoimmune diseases such as inflammatory bowel disease (IBD) or colitis, an immune disorder, for example an immune exhaustion due to chronic viral infections such as HIV infection, a cardiovascular disorder, an inflammatory disorder for example a chronic airway inflammation, a bacterial, viral and/or fungal infection for example sepsis or a Mycobacterium bovis infection, a liver disorder, a chronic kidney disorder, a psychiatric disorder (e.g., schizophrenia, bipolar disorders, Alzheimer's disease) and/or cancer.

An oligonucleotide or a pharmaceutical composition of the present invention is used for example in a method of preventing and/or treating a solid tumor or a hematologic tumor. Examples of cancers preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention are breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small- cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia,

retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion,

rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma. In some embodiments two or more oligonucleotides of the present invention are administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In other embodiments, one or more oligonucleotides of the present invention are administered together with another compound such as another oligonucleotide (i.e., not being part of the present invention), an antibody, a small molecule and/or a chemotherapeutic, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In some embodiments of these combinations, the immunosuppression-reverting

oligonucleotide inhibits the expression and activity, respectively, of an immune suppressive factor and the other oligonucleotide (i.e., not being part of the present invention), the antibody and/or small molecule inhibits (antagonist) or stimulates

(agonist) the same and/or another immune suppressive factor and/or an immune stimulatory factor. The immune suppressive factor is for example selected from the group consisting of IDOl, ID02, CTLA-4, PD-1, PD-L1, LAG- 3, VISTA, A2AR, CD39, CD73, STAT3, TD02, TIM- 3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop, Xbpl and a combination thereof. The immune stimulatory factor is for example selected from the group consisting of 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and a combination thereof.

The immune suppressive factor is a factor whose expression and/or activity is for example increased in a cell, tissue, organ or subject. The immune stimulatory factor is a factor whose level is increased or decreased in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual conditions.

An antibody in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example an anti-PD-1 antibody, an anti-PD-Ll antibody, or a bispecific antibody. A small molecule in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example AMPCP (adenosine 5'-(a,6-methylene)diphosphate; e.g., Structure20, 2161-2173, December 5, 2012), which acts as an ADP analog and is therefore an competitive inhibitor of CD73 activity.

A subject of the present invention is for example a mammalian, a bird or a fish. Examples The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples. The following experiments are performed on cells endogenously expressing CD73, i.e., the cells do not represent an artificial system comprising transfected reporter constructs. Such artificial systems generally show a higher degree of inhibition and lower IC50 values than endogenous systems which are closer to therapeutically relevant in vivo systems. Further, in the following

experiments no transfecting agent is used, i.e., gymnotic delivery is performed.

Transfecting agents are known to increase the activity of an oligonucleotide which influences the IC50 value (see for example Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). As artificial systems using a transfecting agent are hard or impossible to translate into therapeutic

approaches and no transfection formulation has been approved so far for oligonucleotides, the following experiments are performed without any transfecting agent.

Example 1: Design of human CD73 antisense oligonucleotides

For the design of antisense oligonucleotides with specificity for human (h)CD73 the hCD73 mRNA sequence with SEQ ID No. 1 (NM_002526.3) and the mCD73 mRNA sequence with SEQ ID No. 2 ( M_0011851.4) was used. 14, 15, 16 and 17mers were designed according to in-house criteria, negl (described in WO2014154843 Al) was used as control antisense oligonucleotide in all experiments (Table 1). The distribution of hCD73 oligonucleotide binding sites on the hCD73 mRNA are shown in Fig. 1.

Example 2: Efficacy screen of hmCD73 antisense oligonucleotides in human cancer cell lines

In order to analyze the efficacy of hmCD73 antisense oligonucleotides of the present invention with regard to the knockdown of hmCD73 mRNA expression in cancer cell lines, A-172 (human glioblastoma, ATCC), EFO-21 (human ovary cystadenocarcinoma, DSMZ) and mouse 4T1 (mouse mammary gland cells, ATCC) cells were treated with a single dose (concentration: ΙΟμΜ without addition of any transfection reagent; this process is called gymnotic delivery) of the respective antisense oligonucleotide as shown in Fig. 2A, 2B, 2C and 2D. hCD73, hGAPDH and hHPRTl or mCD73 and mHPRTl mRNA expression were analyzed three days later using the QuantiGene Singleplex assay (Affymetrix). hmCD73 mRNA expression values were normalized to hGAPDH (A- 172), hHPRTl (EFO-21) or mHPRTl (4T1) mRNA expression values, respectively.

Strikingly, a knockdown efficiency of >80% and >60 %, respectively, was observed for 5 (A-172 cells); of >80 % for 7 (EFO-21 cells) and of >90% for 10 (4T1 cells) antisense oligonucleotides as indicated in Fig. 2A to 2D.

To further analyze the effects of hmCD73 antisense oligonucleotides on CD73 mRNA expression in cancer cell lines human SKOV-3 (human ovarian adenocarcinoma, ATCC) cells were treated for three days with 10 μΜ of the respective antisense oligonucleotide without addition of any transfection reagent. As control, cells were treated with negl, an oligonucleotide having no sequence complementarity to any human or mouse mRNA. As vehicle control, cells were treated with medium. CD73 expression values were normalized to HPRT1 values and are shown relative to untreated cells (set as 1).

Strikingly, hCD73 mRNA levels were reduced by >80 % by 2 of 33 tested antisense oligonucleotides in SKOV-3 cells (see Fig. 2E). Treatment with the control

oligonucleotide neg 1 did not reduce CD73 mRNA in the three cell lines.

Exact values of the mean normalized mRNA expression of hmCD73 compared to non- treated cells and the corresponding relative expression of the housekeeping gene mRNA is given below for A-172 cells (Table 2 and 3 for first and second screening rounds), EFO- 21 cells (Table 4), 4T1 cells (Table 5) and SKOV-3 cells (Table 6): Mean hCD73 mRNA expression

relative to untreated cells (set as 1)

A05027HM 0.28

A05018HM 0.30

A05008HM 0.31

A05028HM 0.34

A05009HM 0.38

A05024HM 0.41

A05014HM 0.46

A05001HM 0.47

A05025HM 0.48

A05020HM 0.50

A05026HM 0.50

A05022HM 0.52

A05011HM 0.52

A05015HM 0.55

A05012HM 0.58

A05016HM 0.59

A05017HM 0.60

A05010HM 0.64

A05005HM 0.65

A05019HM 0.68

A05029HM 0.70

A05013HM 0.70

negl 0.97

untreated

1.00

control

Table 2: List of the mean normalized hCD73 mRNA expression values in antisense oligonucleoti de-treated A- 172 cells compared to non- treated cells of a first screening round.

A05024HM 0,22

A05011HM 0,25

A05026HM 0,27

A05025HM 0,27

A05017HM 0,28

A05001HM 0,28

A05020HM 0,30

A05022HM 0,31

A05015HM 0,34

A05012HM 0,35

A05016HM 0,38

A05010HM 0,39

A05036HM 0,39

A05005HM 0,40

A05029HM 0,43

A05013HM 0,45

A05019HM 0,46

A05023HM 0,46

A05037HM 0,47

A05021HM 0,47

A05006HM 0,49

A05002HM 0,50

A05003HM 0,53

A05004HM 0,53

A05007HM 0,56

A05034HM 0,62

A05033HM 0,64

A05035HM 0,64

A05032HM 0,76

A05031HM 0,77

neg 1 0,94

A05030HM 1,05

Table 3: List of the mean normalized hCD73 mRNA expression values in antisense oligonucleoti de-treated A- 172 cells compared to non-treated cells of a second screening round.

A05008HM 0,12

A05009HM 0,13

A05025HM 0,17

A05026HM 0,17

A05001HM 0,18

A05002HM 0,20

A05023HM 0,21

A05015HM 0,22

A05017HM 0,22

A05003HM 0,22

A05036HM 0,22

A05019HM 0,22

A05006HM 0,24

A05037HM 0,25

A05022HM 0,25

A05034HM 0,27

A05005HM 0,27

A05032HM 0,28

A05024HM 0,29

A05010HM 0,29

A05033HM 0,30

A05016HM 0,30

A05014HM 0,31

A05027HM 0,31

A05031HM 0,31

A05029HM 0,37

A05013HM 0,38

A05035HM 0,40

A05011HM 0,42

A05007HM 0,51

A05004HM 0,55

A05020HM 0,56

A05012HM 0,56

A05021HM 0,61

A05030HM 0,71

negl 1,09

Table 4: List of the mean normalized hCD73 mRNA expression values in antisense oligonucleotide-treated EFO-21 cells compared to non- treated cells. Relative mCD73 mRNA expression (compared to

Oligo ID

non-treated cells in 4T1 cells)

A05018HM 0,01

A05020HM 0,02

A05035HM 0,03

A05010HM 0,04

A05001HM 0,05

A05027HM 0,06

A05011HM 0,06

A05036HM 0,06

A05012HM 0,08

A05008HM 0,10

A05017HM 0,10

A05007HM 0,15

A05031HM 0,15

A05022HM 0,15

A05029HM 0,15

A05016HM 0,16

A05015HM 0,16

A05033HM 0,16

A05013HM 0,18

A05030HM 0,20

A05009HM 0,23

A05034HM 0,28

A05005HM 0,28

A05002HM 0,29

A05025HM 0,35

A05032HM 0,37

A05028HM 0,41

A05019HM 0,44

A05003HM 0,47

A05021HM 0,48

A05006HM 0,52

A05014HM 0,70

A05026HM 0,79

A05023HM 0,97

A05024HM 1,17

negl 1,39

A05037HM 1,41

A05004HM 1,70 Table 5: List of the mean normalized mCD73 mRNA expression values in antisense oligonucleotide-treated 4T1 cells compared to non-treated cells.

Table 6: Mean normalized hmCD73 mRNA expression values in antisense

oligonucleotide-treated SKOV-3 cells relative to untreated cells (set as 1). Example 3: Correlation analysis of antisense oligonucleotide efficacy in human EFO-21 compared to A- 172 cells and human A172 compared to mouse 4T1 cells

To further select the candidates with the highest activity in the three tested cell lines, A- 172, EFO-21 and 4T1, a correlation analysis was performed (data derived from Fig. 2B to 2D). As depicted in Fig. 3A and Fig. 3B, 5 potent antisense oligonucleotides for determination of ICso in A- 172, EFO-21 and 4T1 cells, namely A05008HM (SEQ ID No. 4), A05009HM (SEQ ID No. 6), A05018HM (SEQ ID No. 3), A05026HM (SEQ ID No. 8), and A05028HM (SEQ ID No. 5) (marked in black) were selected. Importantly, the control antisense oligonucleotide negl had no negative influence on the expression of hmCD73 in all three tested cell lines.

Example 4: IC50 determination of selected hmCD73 antisense oligonucleotides in SKOV- 3 cells and EFO-21 cells, respectively (mRNA level)

In order to determine the ICso of the hmCD73 antisense oligonucleotides A05008HM (SEQ ID No. 4), A05009HM (SEQ ID No. 6), A05018HM (SEQ ID No. 3), A05026HM (SEQ ID No. 8), and A05028HM (SEQ ID No. 5), SKOV-3 cells (human ovarian cancer cells, ATCC) were treated with titrated amounts of the respective antisense

oligonucleotide (concentrations: 10 μΜ, 3.3 μΜ, 1.1 μΜ, 370 nM, 120 nM, 41 nM, 14 nM, or 4.5 nM (Fig. 4A). hmCD73 mRNA expression was analyzed three days later. As shown in Fig. 4A and in the following Table 7, the antisense oligonucleotides A05008HM (SEQ ID No. 4), A05018HM (SEQ ID No. 3) and A05028HM (SEQ ID No. 5) had the highest potency in SKOV-3 cells with regard to downregulation of hmCD73 mRNA compared to untreated cells with a maximal target inhibition of 86%, 88% and 75%, respectively.

Concentration-dependent target knockdown in SKOV-3 cells was used for calculation of IC50 values shown in Table 7:

Table 7: Overview of IC50 values and target inhibition of selected hmCD73 antisense oligonucleotides in SKOV-3 cells. IC50 values of selected hmCD73-specific antisense oligonucleotides were determined in a further test in EFO-21 cells. Therefore, EFO-21 cells were treated with 10 μΜ, 3.3 μΜ, 1.1 μΜ, 370 nM, 120 nM, 41 nM, 14 nM, or 4.5 nM of A05018HM (SEQ ID No.3), A05027HM (SEQ ID No.30), A05028HM (SEQ ID No.5), and A05037HM (SEQ ID No.39). hCD73 mRNA expression was analyzed after 3 days of treatment. Fig. 4 B depicts the concentration-dependent reduction of hmCD73 mRNA expression by hmCD73 antisense oligonucleotides. ICso-values were calculated by GraphPad Prism and are shown in Table 8:

Table 8: ICso-values and target inhibition of selected hmCD73 antisense oligonuleotides in EFO-21 cells

Example 5: Third screening round of hmCD73 antisense oligonucleotides in human and mouse cancer cell lines

For a third screening round, new antisense oligonucleotides were designed. These antisense oligonucleotides were based on efficient antisense oligonucleotides from the first screening round with modifications in length, exact position on mRNA and chemical modification pattern. Human EFO-21 (ovary cystadenocarcinoma) (Fig. 5A), human SKOV-3 (ovarian adenocarcinoma) (Fig. 5B) and mouse 4T1 (breast cancer) (Fig. 5C) cells were treated for three days with 10 μΜ of the respective antisense oligonucleotide without addition of any transfection reagent. As control, cells were treated with S6, an oligonucleotide having no sequence complementarity to any human or mouse mRNA. As vehicle control, cells were treated with medium. The antisense oligonucleotides A05008HM, A05018HM, and A05028HM that had shown potent activity in the first screening round were used as reference. Values of the mean mRNA expression of hmCD73 normalized to HPRT1 (Fig. 5A-C) relative to non-treated cells (set as 1) are listed for EFO-21 cells (Table 9), SKOV-3 cells (Table 10) and 4T1 cells (Table 11). Strikingly, CD73 mRNA levels were reduced by >80 % by 11 of 13 tested ASOs in EFO- 21 cells (see Fig. 5A), by >80 % by 7 of 13 tested ASOs in SKOV-3 cells (see Fig. 5B), and by >80 % by 9 of 13 tested ASOs in 4T1 cells (see Fig. 5C). Treatment with the control oligonucleotide S6 did not reduce CD73 mRNA potently in the three cell lines.

Table 9: Mean normalized hmCD73 mRNA expression values in antisense

oligonucleotide-treated EFO-21 cells relative to untreated cells (set as 1).

Table 10: Mean normalized hmCD73 mRNA expression values in antisense oligonucleotide-treated SKOV-3 cells relative to untreated cells (set as 1).

Table 11: Mean normalized hmCD73 mRNA expression values in antisense

oligonucleotide-treated 4T1 cells relative to untreated cells (set as 1).

Example 6: IC50 determination of selected hmCD73 antisense oligonucleotides of the third screening round in EFO-21 cells (mRNA level) The hmCD73 antisense oligonucleotides A05038HM (SEQ ID No.42), A05041HM (SEQ ID No.45), A05042HM (SEQ ID No.45), and A05044HM (SEQ ID No.46) had shown potent single-does activity in the three cell lines EFO-21, SKOV-3 and 4T1. In order to investigate the concentration- dependency of effects and to determine the IC50 values EFO-21- cells were treated with 10 μΜ, 3.3 μΜ, 1.1 μΜ, 370 nM, 120 nM, 41 nM, 14 nM or 4.5 nM of the respective antisense oligonucleotide. The antisense oligonucleotide A05018HM that showed potent activity in the first screening round was used as reference. hCD73 mRNA expression was analyzed after 3 days of treatment. Fig. 6 depicts the concentration-dependent reduction of hCD73 expression by hmCD73 antisense oligonucleotides. ICso-values calculated by GraphPad Prism are shown in Table 12: mRNA inhibition [%]

ASO IC50 (nM) ΙΟμΜ 3.33μΜ Ι.ΙΙμΜ 0.37μΜ 0.12μΜ 0.014μΜ 0.0045μΜ

A05018HM 288.8 86.49 83.98 76.60 59.51 34.11 20.73 22.17

A05038HM 558.2 89.14 79.80 59.80 42.38 23.60 5.80 6.81

A05041HM 357.5 87.42 80.26 64.10 47.95 23.04 12.08 2.90

A05042HM 631.0 80.40 73.49 57.03 43.47 24.71 25.01 1.49

A05044HM 345.2 89.70 83.29 73.75 54.18 30.39 18.10 16.20

Table 12: ICso-values and target inhibition of selected hmCD73 antisense oligonucleotides at titrated concentrations in EFO-21 cells

Example 7: Concentration dependent effect on CD73 protein expression and cell viability by A05008HM (SEQ ID No. 4), A05018HM (SEQ ID No. 3) and A05028HM (SEQ ID No. 5) The highly potent hmCD73 antisense oligonucleotides A05008HM (SEQ ID No. 4),

A05018HM (SEQ ID No. 3) and A05028HM (SEQ ID No. 5) were characterized in detail with regard to their knockdown efficacy on the hCD73 protein expression and their influence on cell viability at different concentrations. SKOV-3 cells were therefore treated with different concentrations of the respective antisense oligonucleotide for three days. Then, cells were cultivated for further three days in fresh DMEM medium containing the antisense oligonucleotide at the indicated concentration. Protein expression was analyzed by flow cytometry using the CD73 antibody (clone AD2) and 7- AAD to investigate viability. As shown in Fig. 7A, all three antisense oligonucleotides show potent inhibition of hCD73 protein at all indicated concentrations, whereas treatment with negl had no inhibitory effect. However, cell viability was partially affected by A05028HM (Fig. 7B). In contrast, A05008HM (SEQ ID No. 4) and

A05018HM (SEQ ID No. 3) did not affect viability of SKOV-3 cells in any of the conditions tested. Table 13 summarizes protein knockdown efficiency of the selected human CD73 antisense oligonucleotides A05008HM (SEQ ID No. 4), A05018HM (SEQ ID No. 3) and A05028HM (SEQ ID No. 5) in SKOV-3 cells:

Inhibition [%] (Protein/mRNA)

ASO ΙΟμΜ 5μΜ ΙμΜ Ο,δμΜ Ο,ΙμΜ

A05008HM 66,11 69,25 49,60 43,76 22,81

A05018HM 77,71 77,46 54, 12 43,87 9,61

A05028HM 87,43 77,66 53,40 54,78 23, 16 negl 13,20 -18,21 11,32 5,48 -7,38 Table 13: inhibition of CD73 protein expression of selected hmCD73 antisense oligonucleotides at titrated concentrations in SKOV-3 cells

Example 8: Effect of CD73 protein knockdown on extracellular pyrophosphate levels in human EFO-21 and SKOV-3 cells

Adenosine is one major immunosuppressive molecule generated during ATP degradation by hmCD73. Adenosine can be detected indirectly by the generation of pyrophosphate during the degradation of AMP to adenosine by a colorimetric phosphate assay kit (ab65622, abeam). Therefore, human SKOV-3 and EF021 cells were treated with 5μΜ antisense oligonucleotide A05018HM for 6 days (3+3). After 3 days, DMEM medium was replaced against fresh DMEM medium containing 5μΜ of the antisense oligonucleotide. Protein knockdown of CD73 in both tested cell lines was confirmed after 6 days by flow cytometry (Fig. 8). As a control, cells were treated with negl (Fig. 8). Generation of free phosphate by EFO-21 cells (Fig. 9A) and SKOV-3 cells (Fig. 9C) was analyzed in cell supernatants after addition of 500μΜ AMP to the cells at indicated time points. Treatment with the antisense oligonucleotides did not affect cell viability of EFO-21 cells (Fig. 9B) and SKOV-3 cells (Fig. 9D) as investigated by the cell titer blue assay. Strikingly, phosphate production efficacy was clearly reduced for EFO-21 and SKOV-3 cells after treatment with A05018HM (Fig. 9A, 9C) resulting in about 1,5 x lower phosphate concentrations compared to cells treated with the negative control neg 1 and compared to non-treated cells (Fig. 9A, 9C, Table 14). Therefore, adenosine production of cancer cells can be effectively blocked by anti-CD73 specific antisense oligonucleotide treatment. In Table 14, phosphate concentrations derived from cell supernatants of SKOV-3 and EF021 cells treated with A05018HM are given as well as concentrations derived from the corresponding control reactions:

Table 14: Determination of phosphate concentration as indirect detection method for adenosine in supernatants of human EFO-21 and SKOV-3 cells after CD73 protein knockdown and after addition of exogenous AMP to the cells. Example 9: Investigation of the effect of hmCD73-specific antisense oligonucleotide on degradation of extracellular AMP and the conversion to adenosine by a human ovarian cancer cell line

A05018HM had shown significant activity in suppressing hCD73 mRNA and protein expression in human cancer cells. In the following experiments, the effect of hmCD73 antisense oligonucleotide treatment on the capacity to degrade extracellular AMP to immunosuppressive adenosine was investigated in EFO-21 cells.

AMP- as well as adenosine levels can be determined by mass spectrometry. In order to analyze the effect of hmCD73 antisense oligonucleotide treatment on the capacity of human EFO-21 cells to convert extracellular AMP to adenosine, cells were treated with 5 μΜ of the antisense oligonucleotide A05018HM or the control oligonucleotide S6 for a total treatment time of 6 days. As vehicle control, cells were treated with medium only. CD73 protein expression was analyzed by flow cytometry after 6 days of treatment and was significantly suppressed in cells treated with A05018HM (black column) compared to cells treated with S6 (white column; Fig. 10A). To investigate the capacity to degrade extracellular AMP, cell culture media was replaced by PBS supplemented with 300 μΜ of AMP after 6 days of treatment and incubated for 6.5 h. To investigate the degradation of AMP in the absence of cells, 300 μΜ of AMP was added to cell-free PBS and was incubated for 6.5 h (dotted column).

Thereafter, AMP and adenosine levels were determined in cell-supernatants (cell- conditioned PBS) and cell-free PBS by mass spectrometry. AMP-levels in cell- supernatants of untreated cells (dotted column) were potently reduced compared with AMP-levels in cell-free PBS (striped column; Fig. 10B), while adenosine levels were increased (Fig. IOC). This indicates a strong cell-associated capacity for conversion of AMP to adenosine. Strikingly, in supernatants of A05018HM treated EFO-21 cells (black column) AMP levels were significantly increased and adenosine levels were significantly decreased, when compared to supernatants of S6-treated (white column), or vehicle- treated cells (dotted column; Fig. 10B and IOC, Table 15 and 16). Accordingly, these data clearly indicate that treatment of human EFO-21 cells with A05018HM,

significantly inhibits the capacity of cells to convert extracellular AMP to

immunosuppressive adenosine.

Table 15: Determination of relative AMP concentrations in cell supernatants relative to cell-free PBS (set as 1) after treatment of EFO-21 cells with AMP.

Table 16: Determination of absolute adenosine concentrations in cell supernatants after treatment of EFO-21 cells with AMP.

Example 10: Investigation of the effect of hmCD73-specific antisense oligonucleotide on T cell proliferation in the presence or absence of extracellular AMP

The previous results (see Example 9) in the present invention revealed that treatment of human cancer cell lines with A05018HM significantly inhibits their capacity to convert extracellular AMP to adenosine. Since the CD39-CD73 axis plays an important role for T cell function the effects of A05018HM on proliferation of human CD4⁺ T cells in the presence or absence of extracellular AMP was investigated. Therefore, human CD4⁺ T cells were labelled with cell proliferation dye, activated with anti-CD3 and treated with 5 μΜ of the antisense oligonucleotide A05018HM (black column) or the control oligonucleotide S6 (white column) for a total treatment time of 5 days. In the vehicle control (striped column), cells were activated with anti-CD3 only. Subsequently, 300 μΜ of AMP or vehicle were added to cells on day 3 and day 4 after start of oligonucleotide treatment. On day 5 after start of oligonucleotide treatment, CD73 protein expression, proliferation, and absolute cell numbers of CD4⁺ T cells were analyzed using Flow Cytometry.

A05018HM treatment of CD4⁺ T cells potently suppressed CD73 protein expression (Fig. 11A). In the absence of extracellular AMP, no differences in proliferation (Fig. 11B upper panel), or absolute cell numbers (Fig. 11C) were observed between A05018HM-, S6- and vehicle-treated CD4⁺ T cells. Strikingly, supplementation with 300 μΜ of AMP impaired proliferation (Fig. 11B lower panel) and significantly reduced absolute numbers (Fig. 11C) of CD4⁺ T cells treated with S6 or vehicle. In contrast, proliferation (Fig 11B in lower panel) of A05018HM treated CD4⁺ T cells was not impaired by supplementation of cell culture medium with AMP. Accordingly, absolute T cell numbers (Fig. 11C) were almost not reduced by AMP- supplementation in A05018HM-treated cells.

In summary, these results revealed that supplementation of cell culture medium with AMP significantly impaired proliferation and absolute cell numbers of CD73 expressing CD4⁺ T cells. Strikingly, CD73-protein knockdown by A05018HM-treatment reversed the inhibitory effects of supplemented AMP on T cell proliferation most probably by inhibition of formation of immunosuppressive adenosine.

Example 11: In vivo mCD73 mRNA knockdown by human / mouse cross-reactive CD73 antisense oligonucleotide (A05027HM) in mouse liver

The potent hmCD73 antisense oligonucleotide A05027HM was selected and its effects on mCD73 mRNA expression in livers of Balb/c mice after systemic administration of unformulated oligonucleotide were investigated. Therefore, Balb/c mice were treated with subcutaneous injections of A05027HM at a dose of 20 mg/kg on days 1,2,3,4,5,8,10, and 12 (5 mice / group). As control, Balb/c mice were treated with vehicle (saline, six mice / group). Three days after the last antisense oligonucleotide treatment (day 15), mice were sacrificed and livers were sampled for analysis of mCD73 mRNA levels. The results depicted in Fig. 12 show mCD73 mRNA levels in livers of A05027HM- or vehicle- treated mice (white column). Strikingly, mCD73 mRNA levels were significantly (p=0.0043) reduced upon treatment of mice with A05027HM when compared to vehicle controls.

Claims

Claims

1. An immunosuppression-reverting oligonucleotide comprising 12 to 18 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of an ectoenzyme (CD73) of SEQ ID NO.l (human) and/or of a sequence of SEQ ID NO.2 (mouse), wherein the oligonucleotide inhibits at least 50 % of the CD73 expression.

2. The oligonucleotide of claim 1, wherein the modified nucleotide is selected from the group consisting of a bridged nucleic acid such as LNA, cET, ENA, 2'Fluoro modified nucleotide, 2O-Methyl modified nucleotide and combinations thereof.

3. The oligonucleotide of claim 1 or 2 hybridizing with CD73 of SEQ ID. NO.l and/or SEQ ID NO.2 comprising a sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID N0.13, SEQ ID NO.14, SEQ ID N0.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID N0.19, SEQ ID NO.20, SEQ ID N0.21, SEQ ID N0.22, SEQ ID N0.23, SEQ ID N0.24, SEQ ID N0.25, SEQ ID N0.26, SEQ ID N0.27, SEQ ID N0.28, SEQ ID N0.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID N0.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID N0.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID N0.42, SEQ ID N0.43, SEQ ID N0.44, SEQ ID N0.45, SEQ ID N0.46, SEQ ID N0.47, and combinations thereof.

4. The oligonucleotide of any one of claims 1 to 3, wherein the oligonucleotide is selected from the group consisting of +G*+A*+T*T*T*C*C*C*A*G*T*G*C*+C*+A*+T

(A05018HM), +G*+C*+T*G*T*G*C*A*C*G*T*C*+G*+T*+T (A05008HM),

+T*+G*+A*T*T*T*C*C*C*A*G*T*G*C*+C*+A*+T (A05028HM),

+G*+G*+C*T*G*T*G*C*A*C*G*T*C*+G*+T (A05009HM),

+C*+T*+C*A*G*A*A*T*T*G*G*A*A*+A*+T*+T (A05019HM) ,

+A*+C*+T*C*G*A*C*A*C*T*T*G*G*+T*+G*+C (A05026HM),

+C*+T*+G*T*G*C*A*C*G*T*C*+G*+T*+T (A05001HM),

+A*+G*C*A*C*G*T*T*G*G*G*T*+T*+C (A05002HM),

+A*+C*+G*G*T*G*A*A*C*C*A*+G*+A*+T (A05003HM),

+G*+C*+A*T*A*G*G*C*C*T*G*+G*+A*+C (A05004H),

+c*+T*+C*G*A*C*A*C*T*T*G*G*+T*+G (A050005HM), +A*+C*+T*C*G*A*C*A*C*T*T*+G*+G*+T (A05006HM),

+G*+G*+C*A*C*T*C*G*A*C*A*+C*+T*+T (A05007HM),

+G*+T*+C*C*T*C*C*C*A*C*C*A*+C*+G*+A (A05010HM), +G*+T*C*C*T*C*C*C*A*C*C*A*C*G*+A (A05011HM),

+T*+G*T*C*C*T*C*C*C*A*C*C*+A*+C*+G (A05012HM), +G*+A*G*T*G*T*C*C*T*C*C*C*A*C*+C (A05013HM),

+T*+C*+G*A*C*A*C*T*T*G*G*T*+G*+C*+A (A05014HM), +C*+T*+C*G*A*C*A*C*T*T*G*G*+T*+G*+C (A05015HM), +A*+C*+T*C*G*A*C*A*C*T*T*+G*G*T*+G (A05016HM), +C*+A*+C*T*C*G*A*C*A*C*T*T*+G*+G*+T (A05017HM), +T*+G*+T*C*C*T*C*C*C*A*C*C*A*+C*+G*+A (A05020HM), +G*+T*G*T*C*C*T*C*C*C*A*C*C*+A*+C*+G (A05021HM), K}*+A*G*T*G*T*C*C*T*C*C*C*A*C*+C*+A (A05022HM), +G*T*+G*T*T*G*G*A*G*T*G*T*C*C*+T*+C (A05023HM), +C* T*+C*G*A*C*A*C*T*T*G*G*T*+G*+C*+A (A05024HM), +C*+T*+C*G*A*C*A*C*T*T*G*G*T*G*+C*+A (A05025HM), +G*+C*+A*C*T*C*G*A*C*A*C*T*T*+G*+G*+T (A05027HM), +G*+T*+G*T*C*C*T*C*C*C*A*C*C*A*+C*+G*+A (A05029HM), +A*+G*+T*G*T*C*C*T*C*C*C*A*C*C*+A*+C*+G (A05030HM), K}*+A*G*T*G*T*C*C*T*C*C*C*A*C*C*A*+C (A05031HM), +G*A*G*T*G*T*C*C*T*C*C*C*A*C*+C*A*+C (A05032HM), +G*A*G*T*G*T*C*C*T*C*C*C*A*C*C*+A*+C (A05033HM), +A*+C*+T*C*G*A*C*A*C*T*T*G*G*T*+G*+C*+A (A05034HM), +A*+C*+T*C*G*A*C*A*C*T*T*G*G*T*G*+C*+A (A05035HM), +G*+G*+C*A*C*T*C*G*A*C*A*C*T*T*+G*+G*+T (A05036HM), +G*+G*CA*C*T*C*G*A*C*A*C*T*T*+G*+G*+T (A05037HM), +G*+C*T*G*T*G*C*A*C*G*T*C*+G*+T*+T (A05038HM), +C*G*+G*C*T*G*T*G*C*A*C*G*T*C*G*+T*+T (A05039HM), +G*+G*+C*T*G*T*G*C*A*C*G*T*C*+G*+T (A05040HM), +G*+A*+T*T*T*C*C*C*A*G*T*G*C*C*+A*+T (A05041HM), +G*+A*T*T*T*C*C*C*A*G*T*G*C*+C*+A*+T (A05042HM), +T*+G*+A*T*T*T*C*C*C*A*G*T*G*+C*C*+A*+T (A05043HM), +T*+G*+A*T*T*T*C*C*C*A*G*T*G*C*C*+A*+T (A05044HM), +C*+A*+T*G*A*T*T*T*C*C*C*A*G*T*G*+C*+C (A05045HM), and combinations thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.

5. The oligonucleotide of any one of claims 1 to 4, wherein the oligonucleotide inhibits the expression of CD73 at a nanomolar concentration.

6. A pharmaceutical composition comprising an immunosuppression-reverting oligonucleotide of any one of claims 1 to 5 and a pharmaceutically acceptable carrier, excipient and/or dilutant.

7. The pharmaceutical composition of claim 6, further comprising a chemotherapeutic such as platinum, gemcitabine, another oligonucleotide, an antibody and/or a small molecule.

8. The pharmaceutical composition of claim 7, wherein the other oligonucleotide, the antibody and/or the small molecule inhibits or stimulates an immune suppressive factor and/or an immune stimulatory factor.

9. The pharmaceutical composition of claim 8, wherein the immune suppressive factor is selected from the group consisting of IDOl, ID02, CTLA-4, PD-1, PD-Ll, LAG-3, VISTA,

A2AR, CD39, CD73, STAT3, TD02, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD 160, Chop, Xbpl and a combination thereof.

10. The pharmaceutical composition of claim 8, wherein the immune stimulatory factor is selected from the group consisting of 4- IBB, Ox40, KIR, GITR, CD27, 2B4 and a combination thereof.

11. The immunosuppression-reverting oligonucleotide of any one of claims 1 to 5 or the pharmaceutical composition of any one of claims 6 to 10 for use in a method of preventing and/or treating a disorder, where a CD73 imbalance is involved.

12. The immunosuppression-reverting oligonucleotide or the pharmaceutical composition for use according to claim 11, wherein the disorder is an autoimmune disorder, an immune disorder, a psychiatric disorder and/or cancer.

13. The immunosuppression-reverting oligonucleotide or the pharmaceutical composition for use according to claim 11, wherein the cancer is breast cancer, lung cancer,

malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small- cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia,

retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion,

rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma.

14. The immunosuppression-reverting oligonucleotide or the pharmaceutical composition for use according to any one of claims 11 to 13, wherein the oligonucleotide or the composition is suitable to be administered locally or systemically.