Immunosuppression-Reverting Oligonucleotides Inhibiting the Expression of IDO

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 indoleamine-2,3-dioxygenase (IDO-1) of SEQ ID NO.1 (human) in a hybridizing active area, wherein the oligonucleotide inhibits at least 50% of the IDO-1 expression. The invention is further directed to a pharmaceutical composition comprising such oligonucleotide.

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 indoleamine-2,3-dioxygenase (IDO-1) of SEQ ID NO.1 (human) in a hybridizing active area wherein the oligonucleotide inhibits at least 50% of the IDO-1 expression.

2. The oligonucleotide of claim 1, wherein the hybridizing active area is selected from position 300 to 360, position 250 to 455, position 100 to 160, position 245 to 305, and/or position 650 to 710 of SEQ ID NO. 1.

3. 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, 2Fluoro modified nucleotide, 2O-Methyl modified nucleotide and a combination thereof.

4. The oligonucleotide of claim 1 hybridizing with IDO-1 of SEQ ID NO.1 comprising a sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.93, SEQ ID NO.94, SEQ ID NO.99, SEQ ID NO.105, SEQ ID NO.107, SEQ ID NO.101, SEQ ID NO.4, SEQ ID NO.95, SEQ ID NO.102, SEQ ID NO.96, SEQ ID NO.11, SEQ ID NO.97, SEQ ID NO.103, SEQ ID NO.104, SEQ ID NO.108, SEQ ID NO.109, SEQ ID NO.37, SEQ ID NO.100, SEQ ID NO.106 and a combination thereof.

5. The oligonucleotide of claim 1, wherein the oligonucleotide is selected from the group consisting of TABLE-US-00020 +A*+G*+G*C*G*C*T*G*T*G*A*C*T*+T*+G*+T(A06030H), +G*+C*G*C*T*G*T*G*A*C*T*+T*+G*+T(A06057H), +T*+G*+T*C*C*C*G*T*T*C*T*+T*+G*+C(A06058H), +A*+G*+G*C*G*C*T*G*T*G*A*C*T*+T*+G(A06062H), +A*+G*+G*C*G*C*T*G*T*G*A*C*T*T*+G*+T(A06068H), +G*+A*+T*T*G*T*C*C*A*G*G*A*G*T*+T*+T*+T(A06070H), +G*+A*T*T*G*T*C*C*A*G*G*A*+G*+T*+T(A06059H), +T*+G*+A*T*T*G*T*C*C*A*G*G*A*+G*+T*+T(A06065H), +T*+G*+A*T*T*G*T*C*C*A*G*G*+A*+G*+T(A06060H), +C*+T*+C*A*A*C*T*C*T*T*T*C*+T*+C*+G(A06008H), +C*T*+C*A*A*C*T*C*T*T*T*C*+T*+C*+G(A06061H), +T*+C*+T*C*A*A*C*T*C*T*T*T*C*+T*+C*+G(A06066H), +T*+T*+C*T*C*A*A*C*T*C*T*T*T*+C*+T*+C(A06067H), +C*+T*+C*A*A*C*T*C*T*T*T*C*T*C*+G*+A*+A(A06071H), C*+T*+C*+A*A*C*T*C*T*T*T*C*T*C*+G*+A*+A(A06072H), +A*+G*+T*G*T*C*C*C*G*T*T*C*T*+T*+G*+C(A06035H), +G*+T*+G*T*C*C*C*G*T*T*C*T*+T*+G*+C(A06063H), +A*+G*+T*G*T*C*C*C*G*T*T*C*T*T*+G*+C(A06069H), and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.

6. The oligonucleotide of claim 1, wherein the oligonucleotide inhibits the expression of IDO-1 at a nanomolar concentration.

7. A pharmaceutical composition comprising an immunosuppression-reverting oligonucleotide of claim 1 and a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof.

8. The pharmaceutical composition of claim 7, further comprising a chemotherapeutic agent selected from the group consisting of platinum, gemcitabine, another oligonucleotide, an antibody, a small molecule, and a combination thereof.

9. 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.

10. The pharmaceutical composition of claim 9, wherein the immune suppressive factor is 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.

11. The pharmaceutical composition of claim 9, wherein the immune stimulatory factor is selected from the group consisting of 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and a combination thereof.

12. A method of preventing and/or treating a disorder, where an IDO imbalance is involved, comprising administering to a subject in need thereof the immunosuppression-reverting oligonucleotide of claim 1.

13. The method according to claim 12, wherein the disorder is an autoimmune disorder, an immune disorder, a psychiatric disorder and/or cancer.

14. The method according to claim 13, 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.

15. The method according to claim 12, wherein the oligonucleotide or the composition is suitable to be administered locally or systemically.

Description

DESCRIPTION OF FIGURES

[0014] FIG. 1 shows the mRNA sequence of human (h) IDO-1 (SEQ ID No. 1; reference NM_002164.5).

[0015] 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.

[0016] FIGS. 3A and 3B depict hIDO1 mRNA knockdown efficacy of hIDO1 antisense oligonucleotides in human cancer cell lines EFO-21 (ovarian cystadenocarcinoma;

[0017] 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 M 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.

[0018] FIG. 4 shows a correlation analysis of the efficacy of antisense oligonucleotides in EFO-21 and SKOV-3 cells.

[0019] 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 IC.sub.50 values shown in Table 10.

[0020] 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.

[0021] 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.

[0022] 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 M and 3 M, respectively).

[0023] 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.

[0024] 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 M and 3 M, respectively). T cells labeled with a proliferation dye were added three days later and activated. Proliferation was analyzed on day four of the coculture.

[0025] 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 M 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.

[0026] 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 IC.sub.50 values shown in Table 16.

[0027] FIG. 13 shows the mRNA sequence of murine (m) IDO-1 (SEQ ID No. 2; reference NM_008324.2).

[0028] 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.

[0029] 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 M 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.

[0030] FIG. 16 shows a correlation analysis of the efficacy of antisense oligonucleotides in Renca and 4T1 cells.

[0031] 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.

[0032] 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

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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 2Fluoro modified nucleotide, a 2O-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.

[0037] 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-US-00001 TABLE1 ListofantisenseoligonucleotideshybridizingwithhumanIDO1forexample ofSEQIDNo.1;Neg1isanantisenseoligonucleotiderepresentinga negativecontrolwhichisnothybridizingwithIDO1ofSEQIDNo.1. SEQID mRNA(Antisense) AntisenseSequence5-3 withPTO No. Name Sequence5-3 (*)andLNA(+) 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

[0038] 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-US-00002 TABLE2 ListofantisenseoligonucleotideshybridizingwithhumanIDO1forexample ofSEQIDNo.1;S6isanantisenseoligonucleotiderepresentinga negativecontrolwhichisnothybridizingwithIDO1ofSEQIDNo.1. SEQID mRNA(Antisense) AntisenseSequence5-3 with No. Name Sequence PTO(*)andLNA(+) 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

[0039] The following Table 3 shows oligonucleotides hybridizing with mRNA of rat or murine IDO1:

TABLE-US-00003 TABLE3 ListofantisenseoligonucleotideshybridizingwithratormurineIDO1for exampleofSEQIDNo.2;Neg1isanantisenseoligonucleotiderepresenting anegativecontrolwhichisnothybridizingwithIDO1ofSEQIDNo.2. SEQ mRNA(Antisense) AntisenseSequence5-3 withPTO IDNo. Name Sequence5-3 (*)andLNA(+) 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

[0040] 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-US-00004 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-US-00005 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-US-00006 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-US-00007 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

[0041] 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.

[0042] 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 M.

[0043] 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 M.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] A subject of the present invention is for example a mammalian, a bird or a fish.

EXAMPLES

[0050] 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

[0051] 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

[0052] 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 M 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-US-00008 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-US-00009 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

[0053] 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 IC.sub.50 in 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: IC.SUB.50 .Determination of Selected hIDO1 Antisense Oligonucleotides in EFO-21 Cells (mRNA Level)

[0054] In order to determine the IC.sub.50 of 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 M, 2.2 M, 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 IC.sub.50 values and target inhibition of the above mentioned selected antisense oligonucleotides at titrated concentrations in EFO-21 cells:

TABLE-US-00010 TABLE 10 Inhibition (%) IC.sub.50 6.6 2.2 740 250 82 27 9 3 ASO (nM) M M 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

[0055] 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 M and 1 M of A06030H, respectively (FIG. 6C). All other conditions had no influence on the viability of EFO-21 cells. Table 11 summarizes IC.sub.50 values and target inhibition on protein and mRNA level in EFO-21 cells:

TABLE-US-00011 TABLE 11 Overview of IC.sub.50 values of hIDO1 antisense oligonucleotides Inhibition (%) (Protein/mRNA) IC.sub.50 (nM) ASO (Protein/mRNA) 3 M 1 M 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

[0056] 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 M (according to sigmaaldrich.com) RPMI-1640 was supplemented with 200 M L-tryptophan (L-trp) in an additional experimental condition.

[0057] 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-US-00012 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 (M) (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.sup. 22.5/40.9 A06007H 2.6/1.7 2.1/2.sup. A06030H .sup.2/2.4 2.3/2.9 Medium .sup.1/1.1 1.5/1.5 control

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

[0058] 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 M or 3 M 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 M L-tryptophan. Supernatant was harvested 24 h later and L-kynurenine levels were determined by ELISA.

[0059] 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

[0060] 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 IC.sub.50 value of 1.204. Table 9 shows knockdown of hIDO1 in dendritic cells using the antisense oligonucleotide A06030H:

TABLE-US-00013 TABLE 13 Inhibition (%) IC.sub.50 10 5 1 500 100 50 ASO (M) M M M 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

[0061] 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.

[0062] 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 M 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

[0063] 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-US-00014 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-US-00015 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: IC.SUB.50 .Determination of Selected hIDO1 Antisense Oligonucleotides in EFO-21 Cells (mRNA Level)

[0064] In order to determine the IC.sub.50 of 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 M, 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 IC.sub.50 values and target inhibition of the above mentioned selected antisense oligonucleotides at titrated concentrations in EFO-21 cells:

TABLE-US-00016 TABLE 16 Overview of IC.sub.50 values of hIDO1 antisense oligonucleotides in EFO-21 cells. Inhibition (%) IC.sub.50 3 1 300 100 30 10 ASO (nM) M M 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

[0065] 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

[0066] 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 M 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-US-00017 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-US-00018 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

[0067] 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 IC.sub.50 in 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: IC.SUB.50 .Determination of Selected mIDO1 Antisense Oligonucleotides in Renca Cells (mRNA Level)

[0068] In order to determine the IC.sub.50 of 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 M, 3 M, 1 M, 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 IC.sub.50 values and target inhibition of selected antisense oligonucleotides at titrated concentrations in Renca cells:

TABLE-US-00019 TABLE 19 Overview of IC.sub.50 values of mIDO1 antisense oligonucleotides. IC.sub.50 Inhibition (%) ASO (nM) 10 M 3 M 1 M 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

[0069] 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 mm.sup.3, 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).

[0070] 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).