Immunosuppression-Reverting Oligonucleotides Inhibiting the Expression of IDO

The present disclosure refers to an immunosuppression-reverting oligonucleotide hybridizing with a nucleic acid sequence of indoleamine-2,3-dioxygenase (IDO) such as IDO1 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-L1, LAG-3, VISTA, A2AR, BTLA, IDO, CD39, CD73, STAT3, TDO2, TIM-3, MICA, NKG2A, KIR, TIGIT, TGF-beta, Ox40, GITR, CD27, CD160, 2B4 and 4-1BB.

Tryptophan for example is an amino acid which is essential for cell proliferation and survival. It is required for the biosynthesis of the neurotransmitter serotonin, the synthesis of the cofactor nicotinamide adenine dinucleotide (NAD), and is an important component in the immune system response (“immune escape”) to tumors. Depletion of levels of tryptophan is associated with adverse effects on the proliferation and function of lymphocytes and diminished immune system response.

The enzyme indoleamine-2,3-deoxygenase (IDO) is an intracellular enzyme and it is overexpressed in many human tumors or in suppressive immune cells. IDO catalyzes the initial, rate-limiting step in the conversion of tryptophan to kynurenine resulting in lack of tryptophan and severe immunosuppressive effects of kynurenines. These effects result in suppression for example of T-cells and natural killer (NK) cells against tumor cells for example with regard to cell proliferation, cytokine secretion and/or cytotoxic reactivity. In addition, IDO expression results for example in dendritic cells in the induction of regulatory T-cells which represent a negative prognostic factor in tumor diseases. Thus, IDO is a highly relevant immunosuppressive factor for example in the tumor environment. Moreover, IDO has been implicated in neurologic and psychiatric disorders including mood disorders as well as other chronic diseases characterized by IDO activation and tryptophan degradation such as viral infections, for example, AIDS, Alzheimer's disease, cancers including T-cell leukemia and colon cancer, autoimmune diseases, diseases of the eye such as cataracts, bacterial infections such as Lyme disease, and streptococcal infections.

Small molecules such as 1-methyl-D-tryptophan have been developed and tested in clinical trials. However, 1-methyl-D-tryptophan for example shows an increase in the expression of IDO mRNA and protein due to a feedback mechanism by enzymatic inhibition of IDO. Thus, the activity of the small molecules and their in vivo half-life is limited.

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 IDO 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 IDO, 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.

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 indoleamine-2,3-dioxygenase (IDO1) of SEQ ID NO.1 (human) and/or a sequence of SEQ ID NO.2 (mouse/rat). 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, 2′O-Methyl modified nucleotide or a combination thereof). In some embodiments, the oligonucleotide inhibits at least 50% of the IDO1 expression and in some embodiments the oligonucleotide inhibits the expression of IDO1 at a nanomolar concentration.

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, dilutant or a combination thereof. In some embodiments, this pharmaceutical composition additionally comprises a chemotherapeutic such as platinum or gemcitabine, another oligonucleotide, an antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe 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 or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe and/or the small molecule inhibits or stimulates an immune suppressive factor such as IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop, and/or Xbp1. In addition or alternatively, the oligonucleotide, the antibody or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe and/or the small molecule inhibits or stimulates an immune stimulatory factor such as 4-1BB, 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 an IDO imbalance is involved. In some embodiments, the disorder is for example an autoimmune disorder, an immune disorder, a psychiatric disorder and/or cancer. 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 shows the mRNA sequence of human (h) IDO-1 (SEQ ID No. 1; reference NM_002164.5).

FIG. 2 depicts the distribution of hIDO-1 antisense oligonucleotide binding sites on the hIDO1 mRNA of SEQ ID No. 1 as well as their modification(s) and length. hIDO1 antisense oligonucleotides were aligned to the hIDO1 mRNA sequence. The different grayscales indicate the different LNA modifications and symbols indicate the different length of the antisense oligonucleotides.

FIGS. 3A and 3B depict hIDO1 mRNA knockdown efficacy of hIDO1 antisense oligonucleotides in human cancer cell lines EFO-21 (ovarian cystadenocarcinoma;

FIG. 3A parts 1 and 2) and SKOV-3 (ovarian adenocarcinoma; FIG. 3B parts 1 and 2). EFO-21 and SKOV-3 cells were treated for 3 days with 10 μM of the respective antisense oligonucleotide. As negative control, cells were treated with neg1, an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT (described in WO2014154843 A1). Residual hIDO1 mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1.

FIG. 4 shows a correlation analysis of the efficacy of antisense oligonucleotides in EFO-21 and SKOV-3 cells.

FIG. 5 shows concentration-dependent hIDO1 mRNA knockdown by selected hIDO1 antisense oligonucleotides in EFO-21 cells which were A06007H (SEQ ID No.4), A06008H (SEQ ID No.11), A06030H (SEQ ID No.3), A06043H (SEQ ID No.45), A06044H (SEQ ID No.46), A06045H (SEQ ID No.47) and A06046H (SEQ ID No.48). EFO-21 cells were treated for 3 days with the indicated concentration of the respective antisense oligonucleotide. Residual hIDO1 expression is depicted compared to untreated control cells. hIDO1 mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Concentration-dependent target knockdown was used for calculation of IC₅₀values shown in Table 10.

FIG. 6A-6C depict a concentration-dependent hIDO1 mRNA- and protein knockdown by A06007H (SEQ ID No.4) and A06030H (SEQ ID No.3). Analysis of protein expression by flow cytometry (FIG. 6A), mRNA expression by QuantiGene Singleplex assay (FIG. 6B) and cell viability by cell titer blue assay (FIG. 6C) was performed in EFO-21 cells after treatment with the indicated antisense oligonucleotides for 6 days. As a control, cells were treated with neg1 for 6 days at 304. Relative expression/viability compared to untreated control cells (=1) is depicted.

FIGS. 7A and 7B depict effects of hIDO1 knockdown on L-kynurenine production in EFO-21 cells. EFO-21 cells were treated with the indicated antisense oligonucleotides A06007H (SEQ ID No.4) and A06030H (SEQ ID No.3) for 3 days at 504. Medium was replaced and supplemented and hIDO1 protein knockdown efficacy was analyzed after 24 h by flow cytometry, residual hIDO1 expression is depicted compared to untreated cells (FIG. 7A). 24 h and 48 h after medium replacement supernatants were harvested and L-kynurenine concentration was determined by ELISA (FIG. 7B). As control, cells were treated with neg1 at 504.

FIG. 8 depicts a dose dependent effect of hIDO1 antisense oligonucleotides on the production of kynurenine in EFO-21 cells. EFO-21 cells were treated with A06007H (SEQ ID No. 4) and A06030H (SEQ ID No. 3) at different concentrations (10 nM, 30 nM, 100 nM, 300 nM, 1 μM and 3 μM, respectively).

FIG. 9 shows knockdown of hIDO1 in dendritic cells, wherein human dendritic cells (DC) were generated using a 6 day protocol. During the last 3 days cells were treated with different concentrations of the hIDO1 specific antisense oligonucleotide A06030H (SEQ ID No.3) and hIDO1 protein expression was analyzed by flow cytometry. The antisense oligonucleotide neg1 was used as a control. Residual hIDO1 protein expression compared to untreated DC is shown.

FIG. 10 depicts the effect of hIDO1 knockdown in EFO-21 cells on the proliferation of T cells in coculture. EFO-21 cells were treated with A06007H (SEQ ID No. 4) and A06030H (SEQ ID No. 3) in different concentrations (10 nM, 30 nM, 100 nM, 300 nM, 1 μM and 3 μM, respectively). T cells labeled with a proliferation dye were added three days later and activated. Proliferation was analyzed on day four of the coculture.

FIGS. 11A and 11B depict hIDO1 mRNA knockdown efficacy of hIDO1 antisense oligonucleotides in human cancer cell lines EFO-21 (ovarian cystadenocarcinoma; FIG. 11A) and SKOV-3 (ovarian adenocarcinoma; FIG. 11B). EFO-21 and SKOV-3 cells were treated for 3 days with 5 μM of the respective antisense oligonucleotide. As negative control, cells were treated with S6. Residual hIDO1 mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1.

FIGS. 12.1 to 12.3 show concentration-dependent hIDO1 mRNA knockdown by selected hIDO1 antisense oligonucleotides of a second screening round in EFO-21 cells which were A06057H (SEQ ID No.93), A06060H (SEQ ID No.96), A06062H (SEQ ID No.99), A06065H (SEQ ID No.102), A06066H (SEQ ID No.103) and A06068H (SEQ ID No.105) and the antisense oligonucleotides A06007H (SEQ ID No. 4), A06030H (SEQ ID No. 3) and A06035H (SEQ ID No. 37) of a first screening round. EFO-21 cells were treated for 3 days with the indicated concentration of the respective antisense oligonucleotide. Residual hIDO1 expression is depicted compared to untreated control cells. hIDO1 mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Concentration-dependent target knockdown was used for calculation of IC₅₀values shown in Table 16.

FIG. 13 shows the mRNA sequence of murine (m) IDO-1 (SEQ ID No. 2; reference NM_008324.2).

FIG. 14 shows the distribution of mIDO-1 antisense oligonucleotide binding sites on the mIDO-1 mRNA of SEQ ID No. 2 (NM_008324.2) as well as their modification(s) and length. mIDO1 antisense oligonucleotide sequences were aligned to the mIDO1 mRNA sequence. The different grayscales indicate the different LNA modifications and symbols indicate the different length of the antisense oligonucleotides.

FIGS. 15A and 15B show mIDO1 mRNA knockdown efficacy of mIDO1 antisense oligonucleotides in murine cancer cell lines Renca (renal adenocarcinoma; FIG. 15A) and 4T1 (mammary carcinoma; FIG. 15B). Renca and 4T1 cells were treated for 3 days with 10 μM of the respective antisense oligonucleotide. As negative control, cells were treated with neg1, an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Residual mIDO1 mRNA expression relative to untreated cells is depicted. Expression values were normalized to expression of the housekeeping gene HPRT1.

FIG. 16 shows a correlation analysis of the efficacy of antisense oligonucleotides in Renca and 4T1 cells.

FIGS. 17.1 to 17.3 shows concentration-dependent mIDO1 mRNA knockdown by selected mIDO1 antisense oligonucleotides in Renca cells which were A06013MR (SEQ ID No.74), A06019MR (SEQ ID No.80), A06020MR (SEQ ID No.81), A06021MR(SEQ ID No.82), A06026MR (SEQ ID No.87), A06031MR (SEQ ID No.60) and A06032MR (SEQ ID No.61). Renca cells were treated for 3 days with the indicated concentration of the respective ASO. Residual mIDO1 expression is depicted compared to untreated control cells. mIDO1 mRNA expression values were normalized to expression of the housekeeping gene HPRT1.

FIG. 18A to 18E depicts antisense oligonucleotide-mediated in vivo mIDO1 knockdown in a syngeneic mouse tumor model. A06032MR(SEQ ID No. 61) was tested in a mouse tumor model and its administration resulted in a knockdown of IDO1 in tumor cells (FIG. 18B), monocytic myeloid-derived suppressor cells (M-MDSC) (FIG. 18C), tumor-associated macrophages (FIG. 18D) and in granulocytic myeloid-derived suppressor cells (FIG. 18E).

DETAILED DESCRIPTION

The present invention provides for the first time human and murine oligonucleotides which hybridize with mRNA sequences of indoleamine-2,3-dioxygenase (IDO) such as IDO1 and inhibit the expression and activity, respectively, of IDO. In consequence, the level of tryptophan increases and the level of metabolites of tryptophan such as kynurenine decreases. 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 IDO such as the IDO1 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′Fluoro modified nucleotide, a 2′O-Methyl modified nucleotide or a combination 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 and/or methylphosophonate, i.e., the oligonucleotide comprise 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 Tables 1 to 3 present 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 Tables 1 to 3 may comprise any other modified nucleotide and any other combination of modified and unmodified nucleotides. Oligonucleotides of Tables 1 and 2 hybridize with mRNA of human IDO1:

TABLE 1

List of antisense oligonucleotides hybridizing with human IDO1 for example

of SEQ ID No. 1; Neg1 is an antisense oligonucleotide representing a

negative control which is not hybridizing with IDO1 of SEQ ID No. 1.

SEQ ID

mRNA (Antisense)
Antisense Sequence 5′-3′ with PTO

No.
Name
Sequence 5′-3′
(*) and LNA (+)

3
A06030H
AGGCGCTGTGACTTGT
+A*+G*+G*C*G*C*T*G*T*G*A*C*T*+T*+G*+T

4
A06007H
GATTGTCCAGGAGTT
+G*+A*+T*T*G*T*C*C*A*G*G*A*+G*+T*+T

5
A06001H
GGCGCTGTGACTTG
+G*+G*C*G*C*T*G*T*G*A*C*+T*+T*+G

6
A06002H
AGGCGCTGTGACTT
+A*+G*+G*C*G*C*T*G*T*G*A*C*T*+T

7
A06003H
ATGCGAAGAACACT
+A*+T*+G*C*G*A*A*G*A*A*C*+A*+C*+T

8
A06004H
TATATGCGAAGAAC
+T*+A*+T*A*T*G*C*G*A*A*G*+A*+A*+C

9
A06005H
ACTTAGTCACGATT
+A*+C*+T*T*A*G*T*C*A*C*G*+A*+T*+T

10
A06006H
GCCATTCTTGTAGTC
+G*+C*+C*A*T*T*C*T*T*G*T*A*+G*+T*+C

11
A06008H
CTCAACTCTTTCTCG
+C*+T*+C*A*A*C*T*C*T*T*T*C*+T*+C*+G

12
A06009H
GGCGCTGTGACTTGT
+G*+G*C*G*C*T*G*T*G*A*C*T*+T*+G*+T

13
A06010H
TTGGCAAGACCTTAC
+T*+T*+G*G*C*A*A*G*A*C*C*T*+T*+A*+C

14
A06011H
GTTGGCAGTAAGGAA
+G*+T*+T*G*G*C*A*G*T*A*A*G*+G*+A*+A

15
A06012H
GACACAGTCTGCATA
+G*+A*+C*A*C*A*G*T*C*T*G*C*+A*+T*+A

16
A06013HM
GTCAGGGGCTTATTA
+G*+T*+C*A*G*G*G*G*C*T*T*A*T*+T*+A

17
A06014H
GAGAACAAAACGTCC
+G*+A*G*A*A*C*A*A*A*A*C*G*+T*+C*+C

18
A06015H
AGTGTCCCGTTCTTG
+A*+G*+T*G*T*C*C*C*G*T*T*C*+T*+T*+G

19
A06016H
TATGCGAAGAACACT
+T*+A*+T*G*C*G*A*A*G*A*A*C*+A*+C*+T

20
A06017H
AGGACGTCAAAGCAC
+A*+G*+G*A*C*G*T*C*A*A*A*G*+C*+A*+C

21
A06018H
GAGCTGGTGGCATAT
+G*+A*+G*C*T*G*G*T*G*G*C*A*T*+A*+T

22
A06019H
GAGCTGGTGGCATAT
+G*+A*G*C*T*G*G*T*G*G*C*A*T*+A*+T

23
A06020HM
GACAAACTCACGGAC
+G*+A*+C*A*A*A*C*T*C*A*C*G*+G*+A*+C

24
A06021HM
GACAAACTCACGGAC
+G*+A*+C*A*A*A*C*T*C*A*C*G*G*+A*+C

25
A06022HM
GACAAACTCACGGAC
+G*+A*C*A*A*A*C*T*C*A*C*G*+G*+A*+C

26
A06023HM
TAAGCTTCCCGCAGG
+T*+A*+A*G*C*T*T*C*C*C*G*C*+A*+G*+G

27
A06024H
AGATGGTAGCTCCTC
+A*G*+A*T*G*G*T*A*G*C*T*C*+C*+T*+C

28
A06025H
GTACTTAGTCACGAT
+G*+T*+A*C*T*T*A*G*T*C*A*C*+G*+A*+T

29
A06026H
TGGCTTGCAGGAATC
+T*+G*+G*C*T*T*G*C*A*G*G*A*+A*+T*+C

30
A06027H
GTCTTCAGAGGTCTT
+G*+T*C*T*T*C*A*G*A*G*G*T*C*+T*+T

31
A06028H
CTTGTAGTCTGCTCCT
+C*+T*+T*G*T*A*G*T*C*T*G*C*T*+C*+C*+T

32
A06029H
GGCGCTGTGACTTGTG
+G*+G*C*G*C*T*G*T*G*A*C*T*T*+G*+T*+G

33
A06031H
GCAAGACCTTACGGAC
+G*+C*+A*A*G*A*C*C*T*T*A*C*G*+G*+A*+C

34
A06032H
GTTGGCAGTAAGGAAC
+G*+T*+T*G*G*C*A*G*T*A*A*G*G*A*+A*+C

35
A06033H
GAGAACAAAACGTCCA
+G*+A*+G*A*A*C*A*A*A*A*C*G*T+C*+C*+A

36
A06034H
CAGTCTCCATCACGAA
+C*+A*+G*T*C*T*C*C*A*T*C*A*C*+G*+A*+A

37
A06035H
AGTGTCCCGTTCTTGC
+A*+G*+T*G*T*C*C*C*G*T*T*C*T*+T*+G*+C

38
A06036H
AATATATGCGAAGAAC
+A*+A*+T*A*T*A*T*G*C*G*A*A*G*+A*+A*+C

39
A06037H
CAGGACGTCAAAGCAC
+C*+A*+G*G*A*C*G*T*C*A*A*A*G*+C*+A*+C

40
A06038H
TGAGCTGGTGGCATAT
+T*+G*+A*G*C*T*G*G*T*G*G*C*A*+T*+A*+T

41
A06039H
GACAAACTCACGGACT
+G*+A*C*A*A*A*C*T*C*A*C*G*G*+A*+C*+T

42
A06040HM
TTGCAGATGGTAGCTC
+T*+T*+G*C*A*G*A*T*G*G*T*A*G*+C*+T*+C

43
A06041H
GAGGTCTTTTGTATTG
+G*+A*+G*G*T*C*T*T*T*T*G*T*A*+T*+T*+G

44
A06042H
ATTCTTGTAGTCTGCTC
+A*+T*+T*C*T*T*G*T*A*G*T*C*T*G*+C*+T*+C

45
A06043H
CCAGACTCTATGAGATC
+C*+C*+A*G*A*C*T*C*T*A*T*G*A*G*+A*+T*+C

46
A06044H
GAGATGATCAATGCTGA
+G*+A*+G*A*T*G*A*T*C*A*A*T*G*C*+T*+G*+A

47
A06045H
AGGCGCTGTGACTTGTG
+A*+G*+G*C*G*C*T*G*T*G*A*C*T*T*+G*+T*+G

48
A06046H
GGTGATGCATCCCAGAA
+G*G*+T*G*A*T*G*C*A*T*C*C*C*A*+G*+A*+A

49
A06047H
GGCAAGACCTTACGGAC
+G*+G*+C*A*A*G*A*C*C*T*T*A*C*G*+G*+A*+C

50
A06048H
GTTGGCAGTAAGGAACA
+G*+T*+T*G*G*C*A*G*T*A*A*G*G*A*+A*+C*+A

51
A06049H
ACAAAACGTCCATGTTC
+A*+C*+A*A*A*A*C*G*T*C*C*A*T*G*+T*+T*+C

52
A06050H
AGTGTCCCGTTCTTGCA
+A*+G*+T*G*T*C*C*C*G*T*T*C*T*T*+G*+C*+A

53
A06051H
GAACTGAGCAGCATGTC
+G*+A*A*C*T*G*A*G*C*A*G*C*A*T*+G*T*+C

54
A06052H
GAGCTGGTGGCATATAT
+G*+A*+G*C*T*G*G*T*G*G*C*A*T*A*+T*+A*+T

55
A06053H
GTTCCTGTGAGCTGGTG
+G*+T*T*C*C*T*G*T*G*A*G*C*T*G*+G*+T*+G

56
A06054H
AGGACAAACTCACGGAC
+A*+G*+G*A*C*A*A*A*C*T*C*A*C*G*+G*+A*+C

57
A06055H
CCGCAGGCCAGCATCAC
+C*+C*G*C*A*G*G*C*C*A*G*C*A*T*C*A*+C

58
A06056H
TTGCAGATGGTAGCTCC
+T*+T*+G*C*A*G*A*T*G*G*T*A*G*C*T*+C*+C

59
Neg1

+C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T

Single-dose screens and dose-response investigations revealed the antisense oligonucleotides A06007H, A06008H, A06030H and A06035H as highly potent. To further explore their potential, 16 additional antisense oligonucleotides based on their basic sequences were designed and are shown in the following Table 2:

TABLE 2

List of antisense oligonucleotides hybridizing with human IDO1 for example

of SEQ ID No. 1; S6 is an antisense oligonucleotide representing a

negative control which is not hybridizing with IDO1 of SEQ ID No. 1.

SEQ ID

mRNA (Antisense)
Antisense Sequence 5′-3′ with

No.
Name
Sequence
PTO (*) and LNA (+)

93
A06057H
GCGCTGTGACTTGT
+G*+C*G*C*T*G*T*G*A*C*T*+T*+G*+T

94
A06058H
GCGCTGTGACTTGT
+T*+G*+T*C*C*C*G*T*T*C*T*+T*+G*+C

95
A06059H
GATTGTCCAGGAGTT
+G*+A*T*T*G*T*C*C*A*G*G*A*+G*+T*+T

96
A06060H
TGATTGTCCAGGAGT
+T*+G*+A*T*T*G*T*C*C*A*G*G*+A*+G*+T

97
A06061H
CTCAACTCTTTCTCG
+C*T*+C*A*A*C*T*C*T*T*T*C*+T*+C*+G

99
A06062H
AGGCGCTGTGACTTG
+A*+G*+G*C*G*C*T*G*T*G*A*C*T*+T*+G

100
A06063H
GTGTCCCGTTCTTGC
+G*+T*+G*T*C*C*C*G*T*T*C*T*+T*+G*+C

101
A06064H
GATTGTCCAGGAGTTT
+G*+A*T*T*G*T*C*C*A*G*G*A*G*+T*+T*+T

102
A06065H
TGATTGTCCAGGAGTT
+T*+G*+A*T*T*G*T*C*C*A*G*G*A*+G*+T*+T

103
A06066H
TCTCAACTCTTTCTCG
+T*+C*+T*C*A*A*C*T*C*T*T*T*C*+T*+C*+G

104
A06067H
TTCTCAACTCTTTCTC
+T*+T*+C*T*C*A*A*C*T*C*T*T*T*+C*+T*+C

105
A06068H
AGGCGCTGTGACTTGT
+A*+G*+G*C*G*C*T*G*T*G*A*C*T*T*+G*+T

106
A06069H
AGTGTCCCGTTCTTGC
+A*+G*+T*G*T*C*C*C*G*T*T*C*T*T*+G*+C

107
A06070H
GATTGTCCAGGAGTTTT
+G*+A*+T*T*G*T*C*C*A*G*G*A*G*T*+T*+T*+T

108
A06071H
CTCAACTCTTTCTCGAA
+C*+T*+C*A*A*C*T*C*T*T*T*C*T*C*+G*+A*+A

109
A06072H
CTCAACTCTTTCTCGAA
C*+T*+C*+A*A*C*T*C*T*T*T*C*T*C*+G*+A*+A

110
S6

+T*+C*+T*A*T*C*G*T*G*A*T*G*T*T*+T*+C*+T

The following Table 3 shows oligonucleotides hybridizing with mRNA of rat or murine IDO1:

TABLE 3

List of antisense oligonucleotides hybridizing with rat or murine IDO1 for

example of SEQ ID No. 2; Neg1 is an antisense oligonucleotide representing

a negative control which is not hybridizing with IDO1 of SEQ ID No. 2.

SEQ

mRNA (Antisense)
Antisense Sequence 5′-3′ with PTO

ID No.
Name
Sequence 5′-3′
(*) and LNA (+)

60
A06031MR
TGTATCTTTCACACTCC
+T*+G*+T*A*T*C*T*T*T*C*A*C*A*C*+T*+C*+C

61
A06032MR
GTTGTATCTTTCACACT
+G*+T*+T*G*T*A*T*C*T*T*T*C*A*C*+A*+C*+T

62
A06001MR
GGCGCTGTAACCTGT
+G*+G*+C*G*C*T*G*T*A*A*C*C*+T*+G*+T

63
A06002MR
TCGGTTCCACACATA
+T*+C*+G*G*T*T*C*C*A*C*A*C*+A*+T*+A

64
A06003MR
TCCCCTCGGTTCCAC
+T*+C*C*C*C*T*C*G*G*T*T*C*C*A*+C

65
A06004MR
GTCCATGTTCTCGTA
+G*+T*C*C*A*T*G*T*T*C*T*C*+G*+T*+A

66
A06005MR
TCGCAGTCCCCACCA
+T*C*+G*C*A*G*T*C*C*C*C*A*C*C*+A

67
A06006MR
GAGAAGCTGCGATTT
+G*+A*+G*A*A*G*C*T*G*C*G*A*+T*+T*+T

68
A06007MR
TCACGCATCCTCTTA
+T*+C*+A*C*G*C*A*T*C*C*T*C*+T*+T*+A

69
A06008MR
AAGTCACGCATCCTC
+A*+A*+G*T*C*A*C*G*C*A*T*C*+C*+T*+C

70
A06009MR
AGGCGCTGTAACCTGT
+A*G*+G*C*G*C*T*G*T*A*A*C*C*T*G*+T

71
A06010MR
TCGGTTCCACACATAC
+T*+C*+G*G*T*T*C*C*A*C*A*C*A*+T*+A*+C

72
A06011MR
CATCCCCTCGGTTCCA
+C*+A*+T*C*C*C*C*T*C*G*G*T*T*+C*+C*+A

73
A06012MR
GGCAGCACCTTTCGAA
+G*+G*+C*A*G*C*A*C*C*T*T*T*C*+G*+A*+A

74
A06013MR
GAGAGCTCGCAGTAGG
+G*+A*G*A*G*C*T*C*G*C*A*G*T*+A*+G*+G

75
A06014MR
TGTCCATGTTCTCGTA
+T*+G*+T*C*C*A*T*G*T*T*C*T*C*+G*+T*+A

76
A06015MR
TCGCAGTCCCCACCAG
+T*+C*G*C*A*G*T*C*C*C*C*A*C*C*A*+G

77
A06016MR
AAGCTGCGATTTCCAC
+A*+A*+G*C*T*G*C*G*A*T*T*T*C*+C*+A*+C

78
A06017MR
AGTCACGCATCCTCTT
+A*+G*+T*C*A*C*G*C*A*T*C*C*T*+C*+T*+T

79
A06018MR
TGACAAACTCACGGAC
+T*+G*+A*C*A*A*A*C*T*C*A*C*G*+G*+A*+C

80
A06019MR
GTTGTATCTTTCACAC
+G*+T*+T*G*T*A*T*C*T*T*T*C*A*+C*+A*+C

81
A06020MR
AGTGGATGTGGTAGAGC
+A*+G*+T*G*G*A*T*G*T*G*G*T*A*G*+A*+G*+C

82
A06021MR
AGGCGCTGTAACCTGTG
+A*+G*+G*C*G*C*T*G*T*A*A*C*C*T*+G*+T*+G

83
A06022MR
TCGGTTCCACACATACG
+T*+C*+G*G*T*T*C*C*A*C*A*C*A*T*+A*+C*+G

84
A06023MR
CCTCGGTTCCACACATA
+C*+C*+T*C*G*G*T*T*C*C*A*C*A*C*+A*+T*+A

85
A06024MR
ATGTCCATGTTCTCGTA
+A*+T*+G*T*C*C*A*T*G*T*T*C*T*C*+G*+T*+A

86
A06025MR
TCGCAGTCCCCACCAGG
+T*+C*+G*C*A*G*T*C*C*C*C*A*C*C*A*+G*+G

87
A06026MR
ATTGCTTTGATTGCAGG
+A*+T*+T*G*C*T*T*T*G*A*T*T*G*C*+A*+G*+G

88
A06027MR
GTCACGCATCCTCTTAA
+G*+T*+C*A*C*G*C*A*T*C*C*T*C*T*+T*+A*+A

89
A06028MR
AGTCACGCATCCTCTTA
+A*+G*+T*C*A*C*G*C*A*T*C*C*T*C*+T*+T*+A

90
A06029MR
GAAGGACATCAAGACTC
+G*+A*+A*G*G*A*C*A*T*C*A*A*G*A*+C*+T*+C

91
A06030MR
GCTGGAGGCATGTACTC
+G*+C*+T*G*G*A*G*G*C*A*T*G*T*A*+C*+T*+C

92
Neg1

+C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T

The oligonucleotides of the present invention hybridize for example with mRNA of human or murine IDO of SEQ ID No. 1 and/or SEQ ID No. 2. Such oligonucleotides are called IDO antisense oligonucleotides. In some embodiments, the oligonucleotides hybridize within a hybridizing active area which is one or more region(s) on the IDO mRNA, e.g., of SEQ ID NO.1, where hybridization with an oligonucleotide highly likely results in a potent knockdown of the IDO expression. In the present invention surprisingly several hybridizing active areas were identified for example selected from position 250 to 455, position 100 to 160, position 245 to 305, position 300 to 360, and/or position 650 to 710 (including the terminal figures of the ranges) of IDO1 mRNA for example of SEQ ID No. 1. Examples of antisense oligonucleotides hybridizing within the above mentioned positions of IDO1 mRNA for example of SEQ ID No. 1 are shown in the following Tables 4 to 7:

TABLE 4

Nucleotide position 100 to 160 of IDO1 mRNA of SEQ ID No. 1

SEQ ID

Binding site on hIDO1 mRNA
No . . . /ASO

(Position of the first nucleotide)
name

131
107/A06070H

132
101/A06064H

133
4/A06007H

133
95/A06059H

133
102/A06065H

134
96/A06060H

TABLE 5

Nucleotide position 245 to 305 of IDO1 mRNA of SEQ ID No. 1

SEQ ID

Binding site on hIDO1 mRNA
No . . . /ASO

(Position of the first nucleotide)
name

280
11/A06008H

280
97/A06061H

280
103/A06066H

281
104/A06067H

278
108/A06071H

278
109/A06072H

TABLE 6

Nucleotide position 300 to 360 of IDO1 mRNA of SEQ ID No. 1

SEQ ID

Binding site on hIDO1 mRNA
No . . . /ASO

(Position of the first nucleotide)
name

332
3/A06030H

332
93/A06057H

332
94/A06058H

333
99/A06062H

332
105/A06068H

TABLE 7

Nucleotide position 650 to 710 of IDO1 mRNA of SEQ ID No. 1

SEQ ID

Binding site on hIDO1 mRNA
No . . . /ASO

(Position of the first nucleotide)
name

684
37/A06035H

684
100/A06063H

684
106/A06069H

In Tables 4 to 7 “ASO” is the abbreviation for “antisense oligonucleotide” and the sequences and LNA patterns of the ASOs are specified in Tables 1 and 2.

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 IDO such as the, e.g., human, rat or murine, IDO1 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 IDO such as IDO1 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 μM.

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 μM.

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 or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe 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 IDO imbalance, i.e., the IDO level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The IDO level is for example increased by an increased IDO such as IDO1 expression and activity, respectively. The IDO 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, an immune 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 or a fragment thereof such as a Fab fragment, a HERA fusion protein, a ligand trap, a nanobody, a BiTe 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 IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop, Xbp1 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-L1 antibody, or a bispecific antibody. A small molecule in combination with the oligonucleotide or the pharmaceutical composition of the present invention are for example NLG919, Indoximod, or Epacadostat.

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 IDO1, 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 IDO1 Antisense Oligonucleotides

For the design of antisense oligonucleotides with specificity for human (h) IDO1 the hIDO1 mRNA sequence with SEQ ID No. 1 (seq. ref. ID NM_002164.5; FIG. 1) was used. 14, 15, 16 and 17mers were designed according to in-house criteria, neg1 (described in WO2014154843 A1) was used as control antisense oligonucleotide in all experiments (Table 1). The distribution of the antisense oligonucleotide binding sites on the hIDO1 mRNA is shown in FIG. 2.

Example 2: Efficacy Screen of hIDO1 Antisense Oligonucleotides in Human Cancer Cell Lines

In order to analyze the efficacy of hIDO1 antisense oligonucleotides of the present invention with regard to the knockdown of hIDO1 mRNA expression in cancer cell lines, EFO-21 (human Ovarian Cystadenocarcinoma, DSMZ) and SKOV-3 (human Ovary Adenocarcinoma, ATCC) cells were treated with a single dose (concentration: 10 μM without addition of any transfection reagent; this process is called gymnotic delivery) of the respective antisense oligonucleotide as shown in FIGS. 3A and 3B. hIDO1 and HPRT1 mRNA expression was analyzed three days later using the QuantiGene Singleplex assay (Affymetrix) and hIDO1 expression values were normalized to HPRT1 values. Strikingly, as shown in FIG. 3A (EFO-21 cells) and 3B (SKOV-3 cells), a knockdown efficiency of >90% with 29 and 12 antisense oligonucleotides, respectively, was observed. Values of the mean normalized mRNA expression of hIDO1 compared to non-treated cells are listed for EFO-21 (Table 8) and SKOV-3 cells (Table 9) in the following:

TABLE 8

List of the mean normalized hIDO1 mRNA expression

values in antisense oligonucleotide-treated

EFO-21 cells compared to non-treated cells.

Relative hIDO1 mRNA expression

ASO
(compared to non-treated cells)

A06029H
0.005

A06007H
0.006

A06045H
0.008

A06009H
0.01

A06030H
0.012

A06002H
0.012

A06043H
0.014

A06001H
0.015

A06008H
0.019

A06028H
0.019

A06044H
0.02

A06046H
0.02

A06011H
0.028

A06035H
0.037

A06050H
0.049

A06015H
0.058

A06021HM
0.061

A06020H
0.061

A06054H
0.063

A06042H
0.066

A06022HM
0.073

A06025H
0.074

A06018H
0.078

A06005H
0.08

A06012H
0.085

A06039H
0.085

A06047H
0.086

A06038H
0.089

A06027H
0.095

A06034H
0.102

A06019H
0.104

A06040HM
0.114

A06056H
0.116

A06053H
0.124

A06024H
0.127

A06052H
0.127

A06031H
0.132

A06041H
0.136

A06032H
0.136

A06033H
0.15

A06013HM
0.2

A06006H
0.207

A06010H
0.261

A06051H
0.263

A06036H
0.272

A06016H
0.303

A06004H
0.348

A06003H
0.369

A06023HM
0.406

A06026H
0.478

A06014H
0.541

A06048H
0.547

A06017H
0.592

A06037H
0.604

A06055H
0.647

A06049H
0.755

Neg1
1.30

TABLE 9

List of the mean normalized hIDO1 mRNA expression

values in antisense oligonucleotide-treated

SKOV-3 cells compared to non-treated cells.

Relative hIDO1 mRNA expression

ASO
(compared to non-treated cells)

A06009H
0.026

A06029H
0.037

A06030H
0.04

A06035H
0.041

A06045H
0.05

A06001H
0.051

A06050H
0.051

A06015H
0.054

A06007H
0.067

A06028H
0.072

A06046H
0.078

A06002H
0.091

A06008H
0.105

A06044H
0.109

A06043H
0.14

A06025H
0.143

A06021HM
0.149

A06011H
0.168

A06005H
0.178

A06041H
0.181

A06020H
0.185

A06054H
0.191

A06022HM
0.215

A06038H
0.246

A06039H
0.248

A06027H
0.251

A06056H
0.255

A06033H
0.261

A06052H
0.261

A06012H
0.262

A06018H
0.266

A06042H
0.292

A06016H
0.307

A06040HM
0.308

A06019H
0.308

A06053H
0.338

A06010H
0.357

A06024H
0.369

A06034H
0.378

A06004H
0.402

A06032H
0.402

A06047H
0.405

A06003H
0.417

A06013HM
0.443

A06031H
0.453

A06026H
0.532

A06006H
0.547

A06023HM
0.592

A06051H
0.607

A06014H
0.609

A06036H
0.659

A06017H
0.777

A06048H
0.789

A06037H
0.858

A06049H
0.89

A06055H
1.091

neg1
1.118

Example 3: Correlation Analysis of Antisense Oligonucleotide Efficacy in EFO-21 and SKOV-3 Cells

To further select the candidates with the highest activity in both tested cell lines EFO-21 and SKOV-3 a correlation analysis was performed (data derived from FIGS. 3A and 3B). As depicted in FIG. 4, 7 potent antisense oligonucleotides for determination of IC₅₀in EFO-21 cells, namely A06007H (SEQ ID No. 4), A06008H (SEQ ID No. 11), A06030H (SEQ ID No. 3), A06043H (SEQ ID No. 45), A06044H (SEQ ID No. 46), A06045H (SEQ ID No. 47) and A06046H (SEQ ID No. 48) (marked in black) were selected. Importantly, the control antisense oligonucleotide neg1 had no negative influence on the expression of hIDO1 in both cell lines.

Example 4: IC₅₀Determination of Selected hIDO1 Antisense Oligonucleotides in EFO-21 Cells (mRNA Level)

In order to determine the IC₅₀of the hIDO1 antisense oligonucleotides A06007H (SEQ ID No. 4), A06008H (SEQ ID No. 11), A06030H (SEQ ID No. 3), A06043H (SEQ ID No. 45), A06044H (SEQ ID No. 46), A06045H (SEQ ID No. 47) and A06046H (SEQ ID No. 48), EFO-21 cells were treated with titrated amounts of the respective antisense oligonucleotide (concentrations: 6.6 μM, 2.2 μM, 740 nM, 250 nM, 82 nM, 27 nM, 9 nM, 3 nM). hIDO1 mRNA expression was analyzed three days later. As shown in FIG. 5 and following Table 10, the antisense oligonucleotides A06007H and A06030H had the highest potency in EFO-21 with regard to downregulation of hIDO1 mRNA compared to untreated cells with a maximal target inhibition of 99.7% and 99.8%, respectively. Table 10 shows IC₅₀values and target inhibition of the above mentioned selected antisense oligonucleotides at titrated concentrations in EFO-21 cells:

TABLE 10

Inhibition (%)

IC₅₀
6.6
2.2
740
250
82
27
9
3

ASO
(nM)
μM
μM
nM
nM
nM
nM
nM
nM

A06007H
17
99.7
99.5
97.9
91.3
78.3
60.7
35.6
26.8

A06008H
36
99.4
98.4
95.8
86.4
58.1
51.9
31.4
20.8

A06030H
11
99.8
99.5
98.2
93
77.4
60.2
52.2
36.3

A06043H
49
99.5
99.2
97.2
90
76.7
25.2
5.9
15.4

A06044H
205
98.9
96.3
89.2
56.1
0
10.2
0
7.3

A06045H
78
99.3
98.9
96.5
85.6
44
17.5
0
0

A06046H
~246
97.6
94.1
81
26.9
0
0
0
0

Example 5: Detailed Characterization of Antisense Oligonucleotides A06007H and A06030H

The highly potent hIDO1 antisense oligonucleotides A06007H (SEQ ID No. 4) and A06030H (SEQ ID No. 3) were characterized in detail with regard to their knockdown efficacy on the hIDO1 protein and mRNA expression and their influence on cell viability at different concentrations. EFO-21 cell were therefore treated with different concentrations of the respective antisense oligonucleotide for three days, then splitted at a ratio of 1:3 and treated again with the respective antisense oligonucleotide at the indicated concentration. After another three days, protein expression was analyzed by flow cytometry using the hIDO1 antibody clone “eyedio”, mRNA expression was analyzed and cell viability was investigated using the CellTiter-Blue Cell Viability Assay (Promega). As shown in FIG. 6A, 6B, 6C and Table 11, both antisense oligonucleotides show potent inhibition of hIDO1 protein (FIG. 6A) and mRNA expression (FIG. 6B) after 6 days antisense oligonucleotide treatment in total whereas treatment with neg1 had no inhibitory effect. Furthermore, cell viability was only mildly affected, when cells were treated with 3 μM and 1 μM of A06030H, respectively (FIG. 6C). All other conditions had no influence on the viability of EFO-21 cells. Table 11 summarizes IC₅₀values and target inhibition on protein and mRNA level in EFO-21 cells:

TABLE 11

Overview of IC₅₀values of hIDO1 antisense oligonucleotides

Inhibition (%) (Protein/mRNA)

IC₅₀(nM)

ASO
(Protein/mRNA)
3 μM
1 μM
300 nM
100 nM
30 nM
10 nM

A06007H
80/34
94/99.6
95.2/98.5
85.3/91.6
53.4/71.9
0/47.3
0/29.4

A06030H
30/9
93/99.7
92.7/99.5
95.4/98.3
87.6/93.2
35.8/75.6
0/52.6

Example 6: Downstream Effect of hIDO1 Knockdown on Kynurenine Production in EFO-21 Cells

Kynurenines (L-kynurenine, kynurenic acid, 3-hydroxykynurenine) are the major immunosuppressive molecules that are generated during tryptophan degradation by hIDO1. The first kynurenine that is produced during tryptophan degradation is L-kynurenine which can be detected in cell culture supernatants by an enzyme linked immunosorbent assay (ELISA) (L-Kynurenine ELISA kit, ImmuSmol). EFO-21 cells were treated for three days with the antisense oligonucleotides A06007H (SEQ ID No. 4) and A06030H (SEQ ID No. 3) at 504. Medium was changed to RPMI-1640 and supplemented with fresh antisense oligonucleotide at the respective concentration. As RPMI-1640 has a defined tryptophan concentration of only 24.5 μM (according to sigmaaldrich.com) RPMI-1640 was supplemented with 200 μM L-tryptophan (L-trp) in an additional experimental condition.

Protein knockdown efficiency of both antisense oligonucleotides was verified after 24 hours (FIG. 7A, % target inhibition: A06007H: 94.3, A06030H: 91.4), the supernatants were harvested 24 and 48 hours after medium change and L-kynurenine concentrations were analyzed by ELISA. Strikingly, L-kynurenine production was nearly completely abolished when EFO-21 were treated with A06007H (SEQ ID No. 4) or A06030H (SEQ ID No. 3) (FIG. 7B, Table 12). In contrast, treatment with the control antisense oligonucleotide neg1 had no effect on L-kynurenine production. The addition of L-tryptophan to the medium resulted in an increased kynurenine production only after 48 hours compared to unmodified RPMI-1640 in untreated and neg1 treated EFO-21 cells. Table 12 presents the effect of hIDO1 knockdown on L-kynurenine production in EFO-21 cells:

TABLE 12

Determination of L-kynurenine concentration in

supernatants of EFO-21 cells after hIDO1 protein

knockdown and after addition of L-tryptophan

L-kynurenine (μM) (24 h/48)

Unmodified

ASO
RPMI-1640
RPMI-1640 + l-trp

No ASO
18.8/28.9
19.1/42.3

neg1
22.7/27
22.5/40.9

A06007H
2.6/1.7
2.1/2

A06030H

2/2.4
2.3/2.9

Medium

1/1.1
1.5/1.5

control

Example 7: Dose Dependent hIDO1 Knockdown on Kynurenine Production in EFO-21 Cells

In addition to the experiments described in Example 6, the effect of treatment of EFO-21 cells with hIDO1 antisense oligonucleotides, e.g., A06007H (SEQ ID No. 4) and A06030H (SEQ ID No. 3) at different concentrations was investigated. Therefore, EFO-21 cells were treated with 10 nM, 30 nM, 100 nM, 300 nM, 1 μM or 3 μM of the respective antisense oligonucleotide for three days. S6 was used as control antisense oligonucleotide (ASO). Medium was then changed to RPMI-1640 and supplemented with fresh antisense oligonucleotide at the respective concentration and 100 μM L-tryptophan. Supernatant was harvested 24 h later and L-kynurenine levels were determined by ELISA.

Strikingly, a potent reduction of 1-Kynurenine levels upon treatment of cells with both tested hIDO1 antisense oligonucleotides with a >50% reduction at concentrations as low as 100 nM compared to untreated cells was observed (FIG. 8).

Example 8: Efficient Knockdown of hIDO1 in Dendritic Cells

Monocytes were enriched from peripheral blood mononuclear cells by plastic adherence. Monocytes were differentiated into dendritic cells (DC) for three days, followed by maturation for three days. DC were treated with neg1 or antisense oligonucleotide A06030H at different concentrations during the maturation period. As shown in FIG. 9 and Table 13, hIDO1 could efficiently be knocked down on the protein level with an IC₅₀value of 1.204. Table 9 shows knockdown of hIDO1 in dendritic cells using the antisense oligonucleotide A06030H:

TABLE 13

Inhibition (%)

IC₅₀
10
5
1
500
100
50

ASO
(μM)
μM
μM
μM
nM
nM
nM

A06030H
1.2
84.8
73.6
50.7
30.9
9.6
6.9

Example 9: Effect of hIDO1 Knockdown in EFO-21 Cells on the Proliferation of T Cells in Coculture

Tryptophan starvation and the presence of kynurenines in the tumor microenvironment play an important role in the suppression of immune effector cells (e.g. T cells). The effect of hIDO knockdown in tumor cells on the proliferation of T cells is investigated in coculture in vitro. EFO-21 cells were treated with different concentrations of the respective antisense oligonucleotide, e.g., A06007H (SEQ ID No. 4) and A06030H (SEQ ID No. 3), respectively. S6 was used as control antisense oligonucleotide (ASO). T cells labeled with a proliferation dye were added three days later, activated with CD2/CD3/CD28 antibodies and proliferation was analyzed by flow cytometry four days after T cell activation.

Strikingly, upon knockdown of hIDO1 in EFO-21 cells, strong proliferation of activated CD45+ cells in a concentration dependent manner was observed (FIG. 10). The strongest effect was observed upon treatment with the hIDO1 antisense oligonucleotide A06030H at a concentration of 3 μM that resulted in a 7,8 fold increased proliferation of CD45+ cells compared to the control antisense oligonucleotide condition.

Example 10: Efficacy Screens of hIDO1 Antisense Oligonucleotides in EFO-21 and SKOV-3 Cells

The efficacy of additional hIDO1 antisense oligonucleotides with regard to the knockdown of hIDO1 mRNA expression in cancer cell lines was investigated in a further screening round. EFO-21 (human Ovarian Cystadenocarcinoma, DSMZ) and SKOV-3 (human Ovary Adenocarcinoma, ATCC) cells were treated with the respective antisense oligonucleotide at a single dose (concentration: 504) without addition of any transfection reagent (this process is called gymnotic delivery). hIDO1 and HPRT1 mRNA expression was analyzed after three days of treatment using the QuantiGene Singleplex assay (Affymetrix) hIDO1 expression values were normalized to HPRT1 values and are shown in FIGS. 11A and 11B relative to untreated cells (set as 1). Surprisingly, a knockdown efficiency of >90% was observed in EFO-21 cells with 15 of 16 newly designed antisense oligonucleotides and all three tested antisense oligonucleotides from the first screening round, namely A06007H (SEQ ID No. 4), A06030H (SEQ ID No. 3) and A06035H (SEQ ID No. 37) (FIG. 11A). Furthermore, an efficiency of >80% was observed in SKOV-3 cells with 8 of 16 newly designed antisense oligonucleotides and all four tested antisense oligonucleotides from the first screening round, namely A06007H (SEQ ID No. 4), A06008H (SEQ ID No. 11), A06030H (SEQ ID No. 3) and A06035H (SEQ ID No. 37) (FIG. 11B). Values of the mean normalized mRNA expression of hIDO1 compared to non-treated cells (set as 1) are listed for EFO-21 (Table 14) and SKOV-3 cells (Table 15) in the following:

TABLE 14

List of mean normalized hIDO1 mRNA expression

values in antisense oligonucleotide-treated

EFO-21 cells compared to non-treated cells.

Relative hIDO1 mRNA expression

ASO
(compared to non-treated cells (set as 1))

A06030H
0.002

A06007H
0.003

A06062H
0.003

A06057H
0.004

A06065H
0.004

A06060H
0.005

A06068H
0.005

A06066H
0.011

A06035H
0.015

A06059H
0.015

A06061H
0.016

A06070H
0.016

A06063H
0.019

A06058H
0.021

A06069H
0.023

A06064H
0.026

A06071H
0.028

A06067H
0.039

A06072H
0.153

S6
0.854

TABLE 15

List of mean normalized hIDO1 mRNA expression

values in antisense oligonucleotide-treated

SKOV-3 cells compared to non-treated cells.

Relative hIDO1 mRNA expression

ASO
(compared to non-treated cells (set as 1))

A06035H
0.054

A06030H
0.063

A06062H
0.084

A06007H
0.108

A06060H
0.120

A06065H
0.122

A06063H
0.139

A06057H
0.154

A06068H
0.155

A06069H
0.163

A06008H
0.187

A06058H
0.187

A06066H
0.203

A06059H
0.249

A06061H
0.259

A06071H
0.263

A06067H
0.287

A06070H
0.317

A06064H
0.372

A06072H
0.550

S6
1.069

Example 11: IC₅₀Determination of Selected hIDO1 Antisense Oligonucleotides in EFO-21 Cells (mRNA Level)

In order to determine the IC₅₀of the potent hIDO1 antisense oligonucleotides A06057H (SEQ ID No. 99), A06060H (SEQ ID No. 96), A06062H (SEQ ID No. 99), A06065H (SEQ ID No. 102), A06066H (SEQ ID No. 103) and A06068H (SEQ ID No. 105) that have been identified in the second screening round and the antisense oligonucleotides A06007H (SEQ ID No. 4), A06030H (SEQ ID No. 3) and A06035H (SEQ ID No. 37) that have been identified in the first screening round, EFO-21 cells were treated with different concentrations of the respective antisense oligonucleotides (concentrations: 304, 1 μM, 300 nM, 100 nM, 30 nM, 10 nM). hIDO1 mRNA expression was analyzed after three days of treatment. As shown in FIG. 12 and following Table 16 all tested antisense oligonucleotides had a high potency in EFO-21 cells with regard to downregulation of hIDO1 mRNA with a maximal target inhibition between 95.0% and 99.5% compared to untreated cells. Table 16 shows IC₅₀values and target inhibition of the above mentioned selected antisense oligonucleotides at titrated concentrations in EFO-21 cells:

TABLE 16

Overview of IC₅₀values of hIDO1 antisense

oligonucleotides in EFO-21 cells.

Inhibition (%)

IC₅₀
3
1
300
100
30
10

ASO
(nM)
μM
μM
nM
nM
nM
nM

A06007H
39
99.4
98.2
92.5
74.1
42.7
31.5

A06030H
21
99.5
98.2
94.7
83.6
60.2
35.3

A06035H
30
95.0
96.6
92.7
74.9
49.6
30.2

A06057H
46
98.7
96.6
91.1
68.5
42.0
11.0

A06060H
83
98.5
94.7
80.8
56.0
25.3
4.5

A06062H
58
99.4
97.6
88.5
62.5
39.4
17.0

A06065H
92
98.3
93.5
77.5
57.9
14.2
11.2

A06066H
129
97.0
86.6
75.4
37.2
35.1
4.0

A06068H
75
95.7
96.1
89.6
56.2
27.1
7.9

Example 12: Design of Mouse/Rat IDO1 Antisense Oligonucleotides

Due to the sequence differences between human and mouse(m)/rat(r) IDO1 only few hIDO1 antisense oligonucleotides are cross-reactive to mouse/rat IDO1. As they showed only limited knockdown efficacy in human cell lines, surrogate antisense oligonucleotides were designed with specificity for mouse/rat IDO1. The mouse IDO1 mRNA sequence with SEQ ID No. 2 (seq. ref. NM_008324; FIG. 13) was used as basis for the design of 15, 16 and 17mer antisense oligonucleotides, neg1 (described in WO2014154843 A1) served as control in all experiments (Table 3). The distribution of the antisense oligonucleotide binding sites on the mIDO1 mRNA is shown in FIG. 14.

Example 13: Efficacy Screen of mIDO1 Antisense Oligonucleotides in Murine Cancer Cell Lines

In order to analyze the efficacy of mIDO1 antisense oligonucleotides with regard to the knockdown of mIDO1 mRNA expression in cancer cell lines, Renca (mouse renal adenocarcinoma, ATCC) and 4T1 cells (tumor of the mammary gland, ATCC) cells were treated with murine interferon gamma (mIFNg) to induce mIDO1 expression and a single dose (concentration: 5 μM without addition of any transfection reagent; this process is called gymnotic delivery) of the respective antisense oligonucleotide as indicated in FIGS. 15A and 15B. mIDO1 and HPRT1 mRNA expression was analyzed three days later using the QuantiGene Singleplex assay (Affymetrix) and mIDO1 expression values were normalized to HPRT1 values. Strikingly, as shown in FIGS. 15A and 15B, treatment with 8 and 18 antisense oligonucleotides resulted in a knockdown efficacy of >80% in Renca (FIG. 15A) and 4T1 (FIG. 15B) cells, respectively. Values of the mean normalized mRNA expression of mIDO1 compared to non-treated cells are listed for Renca (Table 17) and 4T1 cells (Table 18) in the following:

TABLE 17

List of mean normalized mIDO1 mRNA expression

values in antisense oligonucleotide-treated

Renca cells compared to non-treated cells

Relative mIDO1 mRNA expression

ASO
(compared to non-treated cells)

A06031MR
0.026

A06013MR
0.033

A06032MR
0.049

A06019MR
0.069

A06014MR
0.13

A06020MR
0.162

A06021MR
0.17

A06026MR
0.177

A06008MR
0.204

A06025MR
0.209

A06007MR
0.252

A06011MR
0.266

A06030MR
0.285

A06015MR
0.289

A06004MR
0.292

A06028MR
0.3

A06022MR
0.301

A06017MR
0.307

A06018MR
0.314

A06029MR
0.347

A06027MR
0.368

A06009MR
0.403

A06023MR
0.439

A06005MR
0.461

A06010MR
0.503

A06006MR
0.513

A06016MR
0.537

A06001MR
0.566

A06002MR
0.576

A06012MR
0.769

A06024MR
0.839

neg1
0.931

A06003MR
1.058

TABLE 18

List of mean normalized mIDO1 mRNA expression values in antisense

oligonucleotide-treated 4T1 cells compared to non-treated cells

Relative mIDO1 mRNA expression

ASO
(compared to non-treated cells)

A06025MR
0.004

A06031MR
0.014

A06032MR
0.028

A06013MR
0.038

A06011MR
0.063

A06026MR
0.071

A06019MR
0.072

A06018MR
0.086

A06015MR
0.091

A06028MR
0.115

A06021MR
0.118

A06010MR
0.119

A06022MR
0.143

A06029MR
0.145

A06027MR
0.159

A06023MR
0.164

A06020MR
0.169

A06017MR
0.181

A06030MR
0.207

A06008MR
0.237

A06014MR
0.242

A06004MR
0.269

A06009MR
0.286

A06016MR
0.399

A06007MR
0.404

A06005MR
0.405

A06002MR
0.414

A06012MR
0.416

A06024MR
0.452

A06001MR
0.851

A06006MR
0.875

A06003MR
0.943

neg1
1.56

Example 14: Knockdown Efficacy of mIDO1 Antisense Oligonucleotides in Murine Cancer Cell Lines

To further select the candidates with the highest activity in both tested cell lines a correlation analysis was performed (data derived from FIG. 15). As depicted in FIG. 16 potent antisense oligonucleotides were selected for determination of IC₅₀in Renca cells, namely A06013MR (SEQ ID No. 74), A06019MR (SEQ ID No. 80), A06020MR (SEQ ID No. 81), A06021MR (SEQ ID No. 82), A06026MR (SEQ ID No. 87), A06031MR (SEQ ID No. 60) and A06032MR (SEQ ID No. 61) (marked in black). Importantly, the control antisense oligonucleotide neg1 had no negative influence on the expression of mIDO1 in both cell lines.

Example 15: IC₅₀Determination of Selected mIDO1 Antisense Oligonucleotides in Renca Cells (mRNA Level)

In order to determine the IC₅₀of the mIDO1 antisense oligonucleotides A06013MR (SEQ ID No. 74), A06019MR (SEQ ID No. 80), A06020MR (SEQ ID No. 81), A06021MR (SEQ ID No. 82), A06026MR (SEQ ID No. 87), A06031MR (SEQ ID No. 60) and A06032MR (SEQ ID No. 61), Renca cells were treated with mIFNg to induce mIDO1 expression and titrated amounts of the respective antisense oligonucleotides (concentrations: 10 μM, 3 μM, 1 μM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM). mIDO1 mRNA expression was analyzed three days later. As shown in FIG. 17 and in the following Table 15, the antisense oligonucleotides A06031MR (SEQ ID No. 60) and A06032MR (SEQ ID No. 61) had the highest potency in Renca cells with regard to downregulation of mIDO1 mRNA compared to untreated cells with a maximal target inhibition of 98.9% and 97.3%, respectively. Table 19 shows IC₅₀values and target inhibition of selected antisense oligonucleotides at titrated concentrations in Renca cells:

TABLE 19

Overview of IC₅₀values of mIDO1 antisense oligonucleotides.

IC₅₀
Inhibition (%)

ASO
(nM)
10 μM
3 μM
1 μM
300 nM
100 nM
30 nM
10 nM
3 nM

A06013MR
104
97.3
95.4
85.7
65.2
50.1
32.3
21.5
0

A06019MR
94
95.3
92.3
86.1
71.1
49.6
30.8
9.5
7

A06020MR
n/a
82.9
45.9
36.5
13
34.7
13.3
2.4
14.9

A06021MR
345
92.4
83.9
71.2
50
35.4
36.1
12.2
4.2

A06026MR
n/a
88.8
82.9
68.9
49.1
29.5
52.3
35.2
19.4

A06031MR
3
98.9
98.4
98
96.7
90.6
86.3
69.3
48.1

A06032MR
13
97.3
95.4
85.7
65.2
50.1
32.3
21.5
0

Example 16: ASO-Mediated In Vivo mIDO1 Knockdown in a Syngeneic Mouse Tumor Model

The in vivo knockdown capacity of mIDO1 antisense oligonucleotide A06032MR (SEQ ID No. 61) was analyzed in a subcutaneous syngeneic murine tumor model. Therefore, MC-38 cells were injected subcutaneously into C57BL/6 mice. After the tumors had reached a size of 50-70 mm³, mice were treated with the control antisense oligonucleotide neg1 or the mIDO1-specific antisense oligonucleotide A06032MR for 5 days by daily intraperitoneal injection of 20 mg/kg without the use of a delivery agent. Mice were sacrificed on day 8 and single cell suspensions of tumors were prepared after tumor resection (experimental setup: FIG. 18A).

The knockdown of mIDO1 on the protein level was investigated in different cells types by flow cytometry. Strikingly, a ˜50% knockdown of IDO1 was observed in tumor cells (FIG. 18B), monocytic myeloid-derived suppressor cells (M-MDSC) (FIG. 18C) and tumor-associated macrophages (FIG. 18D). Further, a knockdown of ˜30% was observed in granulocytic myeloid-derived suppressor cells (G-MDSC) (FIG. 18E).

Number	Date	Country	Kind
16192803.1	Oct 2016	EP	regional
17187775.6	Aug 2017	EP	regional

Immunosuppression-Reverting Oligonucleotides Inhibiting the Expression of IDO

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