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Title:
NUCLEIC ACID POLYMERS INHIBITING THE EXPRESSION OF XBP1
Document Type and Number:
WIPO Patent Application WO/2020/011909
Kind Code:
A1
Abstract:
The present invention relates to an oligonucleotide comprising 12 to 20 nucleotides. At least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of the X-box binding protein 1 (XBP1) of SEQ ID NO.1 (human (h)) and/or SEQ ID NO. 2 (murine (m)), wherein the oligonucleotide inhibits at least 50 % of the XBP1 expression. Further, the invention refers to a pharmaceutical composition comprising such oligonucleotide and to the use of the oligonucleotide and the pharmaceutical composition, respectively, for preventing and/or treating an autoimmune disorder, an inflammatory disorder, and/or cancer.

Inventors:
KLAR RICHARD (DE)
MICHEL SVEN (DE)
JASCHINSKI FRANK (DE)
Application Number:
PCT/EP2019/068654
Publication Date:
January 16, 2020
Filing Date:
July 11, 2019
Export Citation:
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Assignee:
SECARNA PHARMACEUTICALS GMBH & CO KG (DE)
International Classes:
C12N15/113; A61P35/00
Domestic Patent References:
WO2013134774A12013-09-12
WO2001049717A22001-07-12
WO2010088498A12010-08-05
WO2014154843A12014-10-02
Other References:
K. TANEGASHIMA ET AL.: "Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo", DEVELOPMENT, vol. 136, no. 21, 30 September 2009 (2009-09-30), GB, pages 3543 - 3548, XP055534218, ISSN: 0950-1991, DOI: 10.1242/dev.036715
LI YUAN ET AL.: "IRE1beta is required for mesoderm formation in Xenopus embryos", MECHANISMS OF DEVELOPMENT, ELSEVIER SCIENCE IRELAND LTD, IE, vol. 125, no. 3-4, 8 December 2007 (2007-12-08), pages 207 - 222, XP022509752, ISSN: 0925-4773, DOI: 10.1016/J.MOD.2007.11.010
CUBILLOS-RUIZ ET AL., CELL, vol. 161, no. 7, 18 June 2015 (2015-06-18), pages 1527 - 38
CONDAMINE ET AL., SCI IMMUNOL., vol. l, no. 2, August 2016 (2016-08-01)
CHEN ET AL., NATURE, vol. 508, no. 7494, 3 April 2014 (2014-04-03), pages 103 - 107
BETTIGOLE ET AL., NAT IMMUNOL., vol. 16, no. 8, August 2015 (2015-08-01), pages 829 - 37
ZHANG ET AL., GENE THERAPY, vol. 18, 2011, pages 326 - 333
STANTON ET AL., NUCLEIC ACID THERAPEUTICS, vol. 22, no. 5, 2012
Attorney, Agent or Firm:
WITMANS, H.A. (NL)
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Claims:
Claims

1. An oligonucleotide comprising 12 to 20 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of the X-box binding protein 1 (XBP1) of SEQ ID NO.l (human (h)) and/or SEQ ID NO. 2 (murine (m)), wherein the oligonucleotide inhibits at least 50 % of the XBP1 expression.

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

3. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide hybridizes within nucleotide position 1692-1777, nucleotide position 1170-1230, nucleotide position 125- 185, nucleotide position 870-931, nucleotide position 733-828, nucleotide position 408- 468, nucleotide position 267-358, and/or nucleotide position 511-648 of SEQ ID NO. 1 and/or SEQ ID NO.2.

4. The oligonucleotide of any one of claims 1 to 3 hybridizing with human (h) and/or murine (m) XBP1 of SEQ ID. NO.l and/or SEQ ID NO.2 comprising a sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID N0.22, SEQ ID NO.23, SEQ ID N0.24, SEQ ID N0.25, SEQ ID N0.26, SEQ ID N0.27, SEQ ID N0.28, SEQ ID NO.29, SEQ ID NO.30, and combinations thereof.

5. The oligonucleotide of any one of claims 1 to 4, wherein the oligonucleotide is selected from the group consisting of

+T*+C*+C*A*T*C*A*A*G*C*A*T*T*T*+A*+C*+A (A21041HM, SEQ ID NO.3), +G*+A*+A*G*C*T*A*C*A*C*T*A*G*C*+A*+G (A21042H; SEQ ID NO.4),

+G*+G*+A*T*C*A*T*T*C*C*T*T*A*+G*+A*+C (A21040H; SEQ ID NO.5),

+T*+G*+G*C*A*C*C*A*T*G*A*G*C*G*G*+C*+A (A21015H; SEQ ID NO.6),

+C*+A*+T*T*A*G*C*T*T*G*G*C*T*+C*+T*+C (A21035H; SEQ ID NO.7),

+T*+T*+C*A*C*T*A*C*C*A*C*A*T*+T*+A*+G (A2i036H; SEQ ID NO.8), +G*+A*+T*T*T*T*C*A*C*T*A*C*C*+A*+C*+A (A21037H; SEQ ID N0.9), +C*+T*+C*G*A*T*T*T*T*C*A*C*+T*+A*+C (A21038H; SEQ ID NO.10),

+T*+C*+C*T*C*G*A*T*T*T*T*C*A*+C*+T*+A (A21039H; SEQ ID NO.11),

+G*+G*+A*G*G*C*T*G*G*T*A*A*G*G*+A*+A*+C (A21032HM; SEQ ID NO.12), +T*+T*-i-G*G*c*T*G*A*T*G*A*c*G*-i-T*-i-C (A2IO33H; SEQ ID NO.13),

+G*+C*T*T*G*G*C*T*G*A*T*G*A*+C*+G*+T (A21034H; SEQ ID NO.14),

+T*+C*+T*T*A*A*C*T*C*C*T*G*G*T*+T*+C (A21018H; SEQ ID N0.15),

+C*+G*+C*T*G*T*C*T*T*A*A*C*+T*+C*+C (A21019H; SEQ ID NO.16),

+T*+C*+T*G*G*C*A*G*T*C*T*G*A*+G*+C*+T (A21016H; SEQ ID NO.17),

+C*+A*+G*C*T*C*A*C*T*C*A*T*+T*C*+G (A21017H; SEQ ID NO.18),

+G*+C*+G*G*A*C*T*C*A*G*C*A*+G*+A*+C (A21020HM; SEQ ID NO.19),

+T*+C*+T*G*A*G*T*G*C*T*G*C*G*G*A*+C*+T (A21021HM; SEQ ID NO.20), +G*+T*C*T*G*A*G*T*G*C*T*G*C*+G*+G*+A (A21022HM; SEQ ID N0.21),

+T*+A*+G*T*C*T*G*A*G*T*G*C*T*G*C*+G*+G (A21023HM; SEQ ID N0.22), +G*+G*+T*G*C*A*C*G*T*A*G*T*C*T*+G*+A*+G (A21024H; SEQ ID N0.23),

+G*+A*+G*G*T*G*C*A*C*G*T*A*G*T*+C*+T*+G (A21025H; SEQ ID N0.24),

+C*+A*+G*A*G*G*T*G*C*A*C*G*T*+A*+G*+T (A21026H; SEQ ID N0.25),

+G*+C*+A*G*A*G*G*T*G*C*A*C*G*T*+A*+G*+T (A21027H; SEQ ID N0.26), +G*+C*+A*G*A*G*G*T*G*C*A*C*+G*+T*+A (A21028H; SEQ ID N0.27),

+C*+A*+A*T*A*C*C*G*C*C*A*G*A*A*+T*+C*+C (A21029H; SEQ ID N0.28),

+A*+G*+T*C*A*A*T*A*C*C*G*C*C*A*+G*+A*+A (A21030H; SEQ ID N0.29),

+G*+A*+A*G*A*G*T*C*A*A*T*A*C*C*+G*+C*+C (A21031H; SEQ ID NO.30), and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.

6. The oligonucleotide of any one of claims 1 to 5, wherein the oligonucleotide inhibits the expression of XBP1 at a nanomolar concentration.

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

8. The pharmaceutical composition of claim 7, further comprising a chemotherapeutic such as platinum, gemcitabine, another oligonucleotide, an immune modulatory factor such as an immune stimulatory and/or an immune inhibitory factor, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTeand, and/or a small molecule.

9. The pharmaceutical composition of claim 8, wherein the immune modulatory factor is selected from the group consisting of suppressive factors such as CTLA-4, IDO, PD-1, PD-L1, LAG-3, VISTA, A2AR, BTLA, CD39, CD73, STAT3, TD02, TIM-3, NKG2A, KIR, TIGIT, TGF-beta, Chop or PERK and combinations thereof, or from the group consisting of stimulatory factors such as 0x40, GITR, CD27, CD 160, 2B4,4-1BB, or MICA and combinations thereof. CTLA-4, IDO, PD-1, PD-L1, LAG-3, VISTA, A2AR, BTLA, CD39, CD73, STAT3, TD02, TIM-3, MICA, NKG2A, KIR, TIGIT, TGF-beta, 0x40, GITR, CD27, CD 160, 2B4 and 4- IBB, and combinations thereof.

10. The oligonucleotide of any one of claims 1 to 6 or the pharmaceutical composition of any one of claims 7 to 9 for use in a method of preventing and/or treating a disorder, wherein a XBP1 imbalance is involved.

11. The oligonucleotide or the pharmaceutical composition for use according to claim 10, wherein the disorder is an autoimmune disorder for example diabetes, an inflammatory disorder such as eosinophilic oesophagitis, eosinophilic gastroenteritis and eosinophilic colitis, and/or cancer.

12. The oligonucleotide or the pharmaceutical composition for use according to claim 10 or 11, wherein the cancer is selected from the group consisting of breast cancer, adenocarcinoma such as ovary or renal adenocarcinoma, 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, polycythemia vera, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma.

13. The oligonucleotide according to any one of claims 1 to 6 or 10 to 12, or the pharmaceutical composition according to any one of claims 7 to 9 or for use according to any one of claims 10 to 12, wherein the oligonucleotide or the composition is suitable to be administered locally or systemically.

Description:
Nucleic acid polymers inhibiting the expression of XBP1

The present disclosure refers to a nucleic acid polymer such as an oligonucleotide hybridizing with a nucleic acid sequence of a X-box binding protein 1 (XBP1) and to a pharmaceutical composition comprising such nucleic acid polymer and a

pharmaceutically acceptable carrier, excipient and/or dilutant. The oligonucleotide or the pharmaceutical composition is for example for use in preventing and/or treating an autoimmune disorder, an inflammatory disorder and/or cancer.

Technical background

Immune cells play an important role in the complex microenvironment of a tumor. There are immune effector cells like e.g. T cells or NK cells that are capable of recognizing immunogenic structures on the surface of tumor cells and thereby can attack tumor cells by the release of certain cytokines like e.g. interferon gamma (IFN-g) or cytotoxic molecules like e.g. granzyme B. T cells for example can be stimulated by dendritic cells (DC) that also reside within the tumor. There are furthermore suppressive cells like e.g. regulatory T cells or myeloid- derived suppressor cells (MDSC), that produce cytokines like e.g. interleukin 10 (IL-10) or transforming growth factor beta (TGF-6) and immunosuppressive enzymes like e.g. arginase. Those cytokines have the capability to suppress the aforementioned immune effector cells.

Hostile conditions in the tumor microenvironment (e.g. reactive oxygen species (ROS) or nutrient starvation) can lead to induction of the endoplasmic reticulum (ER)-stress response. This response is tightly regulated by pathways like for example the enzyme inositol-requiring enzyme 1 (IREla) / X-box binding protein 1 (XBP1) axis. IREla catalyzes splicing of the XBP1 mRNA which generates the active form of XBP1, spliced XBP1 (sXBPl), a transcription factor that regulates different downstream genes. In tumor resident DC in ovarian cancer for example, sXBPl is responsible for the aberrant accumulation of triglycerides which leads to a reduced antigen-presenting capacity and thereby reduced stimulation of T cells. Conditional knockout of XBP1 in those cells converted them into immunostimulatory cells in animal models (Cubillos-Ruiz et al.

2015, Cell, 2015 Jun 18;161(7):1527-38). Furthermore, in neutrophils for example, sXBPl is involved in the transformation of those cells into immunosuppressive cells (Condamine et al. 2016, Sci Immunol. 2016 Aug;l(2)).

In triple negative breast cancer (TNBC) for example, the IRE la / XBP1 pathway is a key regulator of the function of tumor initiating cells (TIC). Silencing of XBP1 markedly reduced tumorigenicity of different TNBC cell lines (Chen et al. 2014, Nature. 2014 Apr 3;508(7494):103-107).

In addition, sXBPl is an important factor in the differentiation of myeloid progenitors to eosinophils (Bettigole et al. 2015, Nat Immunol. 2015 Aug;16(8):829-37). Eosinophils play an important role in the pathology of different diseases like for example eosinophilic oesophagitis, eosinophilic gastroenteritis and eosinophilic colitis.

As XBP1 is an intracellular factor with no enzymatic activity, inhibition of XBP1 by antibodies or small molecules is not or hardly suitable. Accordingly, an agent which is safe and effective in inhibiting the function or expression of an intracellular mediator of immunosuppression and tumorigenic functions like XBP1 would be an important improvement for the treatment of patients suffering from diseases or conditions affected for example by the activity of this suppressive factor.

Oligonucleotides of the present invention are very successful in the inhibition of the expression of XBP1. The mode of action of an oligonucleotide differs from the mode of action of an antibody or small molecule, and oligonucleotides are highly advantageous regarding for example

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

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

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

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

Therefore, targeting XBP1 expression for example in cancer cells, myeloid progenitors, eosinophils and/or other immune cells on mRNA-level by antisense-oligonucleotides is a promising state-of-the-art approach to develop and improve for example immunotherapies against different cancers, and therapies against inflammatory and immune diseases, respectively.

Summary

The present invention refers to an oligonucleotide comprising 12 to 20 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of the X-box binding protein 1 (XBP1) of SEQ ID NO.l (human mRNA) or SEQ ID NO. 2 (murine mRNA) or SEQ ID NO.69 (human pre-mRNA) or SEQ ID NO.70 (murine pre-mRNA) or the oligonucleotides hybridizes with two or more of these sequences, for example wherein the oligonucleotide inhibits at least 50 % of the XBP1 expression. The modified nucleotide is for example selected from the group consisting of a bridged nucleic acid such as LNA, cET, ENA, 2'Fluoro modified nucleotide, 2O-Methyl or 2’O-Methoxyethyl modified nucleotide and combinations thereof.

An oligonucleotide of the present invention hybridizes for example with nucleotide position 125-185, nucleotide position 267-358, nucleotide position 408-468, nucleotide position 511-648, nucleotide position 733-828, nucleotide position 870-931, nucleotide position 1170-1230, and/or nucleotide position 1692-1777 of SEQ ID NO. 1, and/or within nucleotide position 0 or 1-100, nucleotide position 101-200, and/or nucleotide position 201-300, of SEQ ID NO.2.

The oligonucleotide of the present invention hybridizes for example with human (h) and/or murine (m) XBP1 of SEQ ID.NO.l and/or SEQ ID NO.2 comprising a sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ

ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID N0.22, SEQ ID NO.23, SEQ ID N0.24, SEQ ID N0.25, SEQ ID N0.26, SEQ ID N0.27, SEQ ID NO.28, SEQ ID N0.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID N0.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID N0.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID N0.41, SEQ ID N0.42, SEQ ID N0.43, SEQ ID N0.44, SEQ ID NO.45, SEQ ID N0.46, SEQ ID N0.47, SEQ ID N0.48, SEQ ID N0.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52, SEQ ID NO.53, SEQ ID N0.54, SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID N0.58, SEQ ID NO.59, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, SEQ ID N0.65, SEQ ID NO.66, SEQ ID NO.67 and combinations thereof. Examples of these oligonucleotides and potential modifications are shown in Tables 1 and 2.

Further, the oligonucleotide of the present invention hybridizes for example

alternatively or in adition with human (h) and/or murine (m) XBP1 of SEQ ID. NO.69 and/or SEQ ID NO.70 comprising a sequence selected from the group consisting of SEQ ID NO.71, SEQ ID NO.72, SEQ ID NO.73, SEQ ID N0.74, SEQ ID NO.75, SEQ ID NO.76, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, SEQ ID N0.84, SEQ ID N0.85, SEQ ID NO.86, SEQ ID NO.87, SEQ ID NO.88, SEQ ID NO.89, SEQ ID NO.90, SEQ ID NO.91, SEQ ID N0.92, SEQ ID NO.93, SEQ ID N0.94, SEQ ID N0.95, SEQ ID N0.96, SEQ ID N0.97, SEQ ID NO.98, SEQ ID N0.99, SEQ ID NO.100, SEQ ID NO.101, SEQ ID NO.102, SEQ ID NO.103, SEQ ID NO.104, SEQ ID NO.105, SEQ ID NO.106, SEQ ID NO.107, SEQ ID

NO.108, SEQ ID NO.109, SEQ ID NO.110, SEQ ID NO. Ill, SEQ ID NO.112, SEQ ID

NO.113, SEQ ID NO.114, SEQ ID NO.115, SEQ ID NO.116, SEQ ID NO.117, SEQ ID

NO.118, SEQ ID NO.119, SEQ ID NO.120, SEQ ID NO.121, SEQ ID NO.122, SEQ ID

NO.123, SEQ ID NO.124, SEQ ID NO.125, SEQ ID NO.126, SEQ ID NO.127, SEQ ID

NO.128, SEQ ID NO.129, SEQ ID NO.130, SEQ ID NO.131, SEQ ID NO.132, SEQ ID

NO.133, SEQ ID NO.134, SEQ ID NO.135, SEQ ID NO.136, SEQ ID NO.137, SEQ ID

NO.138, SEQ ID NO.139, SEQ ID NO.140, SEQ ID NO.141, SEQ ID NO.142, SEQ ID

NO.143, SEQ ID NO.144, SEQ ID NO.145, SEQ ID NO.146, SEQ ID NO.147, SEQ ID

NO.148, SEQ ID NO.149, SEQ ID NO.150, SEQ ID NO.151, SEQ ID NO.152, SEQ ID

NO.153, SEQ ID NO.154, SEQ ID NO.155, SEQ ID NO.156, SEQ ID NO.157, SEQ ID

NO.158, SEQ ID NO.159, SEQ ID NO.160, SEQ ID NO.161, SEQ ID NO.162, SEQ ID

NO.163, SEQ ID NO.164, SEQ ID NO.165, SEQ ID NO.166, SEQ ID NO.167, SEQ ID

NO.168, SEQ ID NO.169, SEQ ID NO.170, SEQ ID NO.171, SEQ ID NO.172, SEQ ID

NO.173 and combinations thereof. Examples of these oligonucleotides and potential modifications are shown in Tables 7 and 8.

The oligonucleotide of the present invention inhibits the expression of XBP1 at a nanomolar or micromolar concentration. The present invention further refers to a pharmaceutical composition comprising an oligonucleotide, i.e., one or more, of the present oligonucleotides and a pharmaceutically acceptable carrier, excipient and/or dilutant.

In some embodiments the pharmaceutical composition of the present invention comprises a chemotherapeutic such as platinum or gemcitabine, another oligonucleotide, an immune modulatory factor such as an immune stimulatory and/or an immune inhibitory factor, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTeand and/or a small molecule. An immune modulatory factor is for example selected from the group consisting of suppressive factors like for example CTLA-4, IDO, PD-1, PD-L1, LAG-3, VISTA, A2AR, BTLA, CD39, CD73, STAT3, TD02, TIM-3, NKG2A, KIR, TIGIT, TGF-beta, Chop or PERK and combinations thereof, or from the group consisting of stimulatory factors like for example 0x40, GITR, CD27,

CD 160, 2B4,4-1BB, or MICA and combinations thereof.

The oligonucleotide or the pharmaceutical composition of the present invention is for example for use in a method of preventing and/or treating a disorder, wherein a XBP1 imbalance is involved. Such disorder is for example an autoimmune disorder such as diabetes (type I or II), an inflammatory disorder such as eosinophilic oesophagitis, eosinophilic gastroenteritis and eosinophilic colitis, and/or cancer. The cancer is for example selected from the group consisting of breast cancer, adenocarcinoma such as ovary or renal adenocarcinoma, 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, polycythemia vera, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma. The oligonucleotide or the pharmaceutical composition is for example suitable to be 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 sequence of human XBP1 mRNA (NM_005080.3; SEQ ID NO. 1), which was used for the selection of 15mer, 16mer or 17mer human antisense oligonucleotides (ASO) according to the present invention. Some of these antisense oligonucleotides also hybridize with murine XBP1.

Fig. 2 depicts the sequence of murine XBP1 mRNA (NM_013842.3; SEQ ID NO. 2), which was used for the selection of 15mer, 16mer or 17mer murine antisense

oligonucleotides (ASO) according to the present invention. Some of these antisense oligonucleotides also hybridize with rat XBP1.

Fig. 3 depicts distribution of XBP1 antisense oligonucleotide binding sites on the human (h) XBP1 mRNA as well as length and modification pattern of XBP1 antisense

oligonucleotides.

Fig. 4A and 4B show screening of the knockdown efficacy of human XBP1 mRNA by XBP1 antisense oligonucleotides of the present invention in human ovarian

cystadenocarcinoma cells (EFO-21 cells; Fig. 4A) and human ovary adenocarcinoma cells (SKOV-3 cells; Fig. 4B).

Fig. 5 shows dose-dependent knockdown of XBP1 mRNA expression by selected XBP1 antisense oligonucleotides of the present invention in EFO-21 cells. Fig. 6 depicts distribution of XBP1 antisense oligonucleotide binding sites on the murine (m) XBP1 mRNA as well as length and modification pattern of XBP1 antisense

oligonucleotides.

Fig. 7A and 7B show screening of the murine knockdown efficacy of murine XBP1 mRNA by XBP1 antisense oligonucleotides of the present invention in murine breast cancer cells (4T1 cells; Fig. 7A) and murine renal adenocarcinoma cells (Renca cells;

Fig. 7B).

Detailed description

The present invention provides for the first time human, murine and rat oligonucleotides, respectively, which hybridize with mRNA sequences of a X-box binding protein 1 (XBP1) and inhibit the expression and activity, respectively, of XBP1 such as human, murine and/or rat XBP1. 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 XBP1 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 2Ό- Methyl modified nucleotide or a 2’O-Methyloxyethyl modified nucleotide or combinations thereof. In some embodiments, the oligonucleotide of the present invention comprises nucleotides having the same or different modifications. In some embodiments the oligonucleotide of the present invention comprises a modified phosphate backbone, wherein the phosphate is for example a phosphorothioate.

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, 2 7 and 8, respectively, 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, 2, 7 and 8, respectively, may comprise any other modified nucleotide and any other combination of modified and unmodified nucleotides. Oligonucleotides of Table 1 hybridize for example with mRNA of human (H) and/or murine (M) XBP1:

Table 1: List of antisense oligonucleotides hybridizing with human (H) and some also with murine (HM) XBP1 mRNA for example of SEQ ID NO. 1; Negl is an antisense oligonucleotide representing a negative control which is not hybridizing with XBP1 of SEQ ID NO. 1. The following Table 2 shows oligonucleotides hybridizing for example with mRNA of murine (M) and/or rat (R) XBP1:

Table 2: List of antisense oligonucleotides hybridizing with murine (M) and some also with rat (MR) XBP1 for example of SEQ ID NO. 2; Negl is an antisense oligonucleotide representing a negative control which is not hybridizing with XBP1 of SEQ ID NO. 2. The oligonucleotides of the present invention hybridize for example with mRNA of human, murine and/or rat XBP1 of SEQ ID NO. 1 and/or SEQ ID NO. 2. Such

oligonucleotides are called XBP1 antisense oligonucleotides. In some embodiments, the oligonucleotides hybridize within a hybridizing active area which comprises for example one or more region(s) on the XBP1 mRNA, e.g., of SEQ ID NO.l and/or SEQ ID NO.2, where hybridization with an oligonucleotide highly likely results in a potent knockdown of XBP1 expression. In the present invention surprisingly several hybridizing active areas were identified which are for example shown in the following Tables 3 to 6. The position indicated in the Tables for the oligonucleotides of the present invention refers to the first nucleotide of the oligonucleotide hybridizing with the mRNA of the active area. In case of closely adjacent active areas for example the binding of the first nucleotide of the oligonucleotide lies in one active area and the binding of further nucleotides of the same oligonucleotide lies in another adjacent area.

Hybridizing active areas of SEQ ID NO.l are for example selected from position 125-185, position 267-358, position 408-468, position 511-648, position 733-828, position 870-931, position 1170-1230 and position 1692-1777 of XBP1 mRNA for example of SEQ ID NO.l. Examples of antisense oligonucleotides hybridizing within the above mentioned positions of XBP1 mRNA for example of SEQ ID NO.l are shown in the following Table 3:

Nucleotide position 267 - 358 Binding site on human XBP1

mRNA

(position of the first nucleotide SEQ ID NO. / ASO

position) name

Alternative hybridizing active areas of SEQ ID NO.l are for example selected from position 125-185, position 267-327, position 298-358, position 408-468, position 511-571, position 587-648, position 733-793, position 767-828, position 870-931, position 1170- 1230, position 1692-1752, and position 1717-1777 of XBP1 mRNA for example of SEQ ID

NO.l. These areas comprise about 60 nucleotides and thus, the active areas are more limited than the active areas of Table 3. Examples of antisense oligonucleotides hybridizing within the above mentioned positions of XBP1 mRNA for example of SEQ ID NO.l are shown in the following Table 4:

Hybridizing active areas of SEQ ID NO.2 are for example selected from position 0 or 1 to 100, position 101 to 200, and/or position 201 to 300 of XBP1 mRNA for example of SEQ ID NO.2. Examples of antisense oligonucleotides hybridizing within the above mentioned positions of XBP1 mRNA for example of SEQ ID NO.2 are shown in the following

Table 5:

Nucleotide 200 - 300

Alternative hybridizing active areas of SEQ ID NO.2 are for example selected from position 0 or 1 to 60, position 23 to 83, position 51 to 111, position 80 to 140, position 116 to 176, position 140 to 200, position 193 to 253, position 220 to 281, position 230 to 290, position 304 to 365, position 469 to 530, position 509 to 569, position 545 to 605, position 673 to 733, position 699 to 759, position 724 to 784, position 981 to 1041, position 1091 to 1151, position 1157 to 1217, position 1169 to 1229, position 1548 to 1608, position 1617 to 1677, position 1635 to 1695, position 1710 to 1770 and/or position 1775 to 1835 of XBP1 mRNA for example of SEQ ID NO.2. These areas comprise about 60 nucleotides and thus, the active areas are more limited than the active areas of Table 5. Examples of antisense oligonucleotides hybridizing within the above mentioned positions of XBP1 mRNA for example of SEQ ID NO.2 are shown in the following Table 6:

Binding site on murine

XBP1 mRNA

(position of the first SEQ ID NO. / ASO nucleotide position) name

The following Table 7 refers to further antisense oligonucleotides of the present invention, which hybrizide with human (H) and some also with murine (HM) XBP1 RNA for example of SEQ ID NO.69. Human (H) sequences without“I” hybridize also for example with SEQ ID NO.l, and murine (HM) sequences also hybridize for example with SEQ ID NO.2:

Table 7: List of antisense oligonucleotides hybridizing with (H) and some also with murine (HM) XBP1 pre-mRNA for example of SEQ ID NO.69. (I) indicates that the antisense oligonucleotide hybridizes with an intronic region for example of the pre- mRNA of XBP1 of SEQ ID NO. 69.

Table 8 relates to further antisense oligonucleotides of the present invention, which hybrizide with (M) mouse XBP1 RNA for example of SEQ ID NO.70. (I) indicates that the antisense oligonucleotide hybridizes with intronic regions on the pre-mRNA of XBP1:

Table 8: List of antisense oligonucleotides hybridizing with (M) mouse XBP1 RNA for example of SEQ ID NO.70. (I) indicates that the antisense oligonucleotide hybridizes with intro nic regions on the pre-mRNA of XBP1

In Tables 3 to 6“ASO” is the abbreviation for“antisense oligonucleotide” and the sequences and LNA patterns of the ASOs (ASO name) are specified in Table 1, 2, 7 and 8, respectively. In some embodiments, the oligonucleotide of the present invention inhibits for example at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of XBP1 such as the, e.g., human, murine or rat, XBP1 expression. Thus, the oligonucleotides of the present invention are for example oligonucleotides which inhibit and stimulate the immune system, inhibit or stimulate differentiation of myeloid progenitors, or prevent or inhibit tumor growth for example in a cell, tissue, organ, or a subject. The oligonucleotide of the present invention inhibits the expression of XBP1 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 mM. 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 mM.

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 such as platinum or gemcitabine, another oligonucleotide, an immune modulatory factor such as an immune stimulatory and/or an immune inhibitory factor, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or a small molecule which is for example effective in tumor treatment, treatment of immune diseases and auto immune diseases, respectively, and/or treatment of inflammatory diseases such as eosinophilic

oesophagitis, eosinophilic gastroenteritis or eosinophilic colitis. An immune modulatory factor is for example selected from the group consisting of suppressive factors like for example CTLA-4, IDO, PD-1, PD-L1, LAG-3, VISTA, A2AR, BTLA, CD39, CD73, STAT3, TD02, TIM-3, NKG2A, KIR, TIGIT, TGF-beta, Chop or PERK and combinations thereof, or from the group consisting of stimulatory factors like for example 0x40, GITR, CD27, CD 160, 2B4,4-1BB, or MICA and combinations thereof.

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 a XBP1 imbalance, i.e., the XBP1 level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The XBP1 level is for example increased by an increased XBP1 expression and activity, respectively. The XBP1 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 (suitable to be) 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 antisense oligonucleotide of the present invention and optionally in combination with another compound such as another oligonucleotide, an immune modulatory factor such as an immune stimulatory and/or an immune inhibitory factor, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, 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, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, and/or the small molecule are effective in preventing and/or treating an autoimmune disorder, an immune disorder, diabetes (type I and II, respectively), artheriosclerosis, a nephrological disorder 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, polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, 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 immune modulatory factor such as an immune stimulatory and/or an immune inhibitory factor, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, 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 antisense 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, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or small molecule inhibits (antagonist) or stimulates (agonist) the same and/or another immune

modulatory, i.e., suppressive factor and/or an immune stimulatory factor. The immune modulatory factor is for example selected from the group consisting of suppressive factors like for example CTLA-4, IDO, PD-1, PD-L1, LAG-3, VISTA, A2AR, BTLA, CD39, CD73, STAT3, TD02, TIM-3, NKG2A, KIR, TIGIT, TGF-beta, Chop or PERK and combinations thereof, or from the group consisting of stimulatory factors like for example 0x40, GITR, CD27, CD160, 2B4,4-1BB, or MICA and combinations thereof.

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

An antibody in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example an anti-PD-1 antibody, an anti-PD-Ll antibody, or a bispecific antibody. A small molecule in combination with the oligonucleotide or the pharmaceutical composition of the present invention 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. 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 XBP1 antisense oligonucleotides

For the design of antisense oligonucleotides with specificity for human (h) XBP1 the hXBPl mRNA sequence with SEQ ID NO. 1 (RefSeq. ID NM_005080.3) was used. 15, 16 and 17mers, respectively, were designed according to in-house criteria, negl (described, e.g., in WO2014154843 Al) was used as control antisense oligonucleotide in all experiments (Table 1). The distribution of the antisense oligonucleotide binding sites on the hXBPl mRNA is shown in Fig. 3.

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

In order to investigate the knockdown efficacy of the in silico designed XBP1 antisense oligonucleotides, two efficacy screening rounds were performed in the cancer cell lines EFO-21 (human Ovarian Cystadenocarcinoma, DSMZ) and SKOV-3 (human Ovary Adenocarcinoma, ATCC). Cells were treated with the respective antisense

oligonucleotide at a concentration of 5 mM for three days without the addition of a transfection reagent. Cells were lyzed after the three days treatment period, XBP1 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay

(ThermoFisher) and the XBP1 expression values were normalized to HPRT1 values. The results for the first screening round of antisense oligonucleotides are shown in Fig. 4 and Tables 9 and 10. As depicted in Fig. 4A and Table 9, treatment of EFO-21 cells with the antisense oligonucleotides A21035H, A21041HM, A21040H, A21036H,

A21015H, A21034H, A21037H and A21042H resulted in a residual XBP1 mRNA expression of <0.5 compared to untreated cells. The control antisense oligonucleotide negl showed no effect on the XBP1 mRNA expression in this experiment. Antisense oligonucleotides were furthermore screened in SKOV-3 cells with regard to their XBP1 knockdown efficacy. As shown in Fig. 4B and Table 10, treatment with the antisense oligonucleotides A21040H, A21015H and A21041HM resulted in a residual XBP1 mRNA expression of <0.5, whereas the control antisense oligonucleotide negl had no effect. Tables 9 and 10 are shown in the following:

Table 9: List of the mean XBP1 mRNA expression values in XBP1 antisense oligonucleotide-treated EFO-21 cells compared to untreated cells. Expression values are normalized to HPRT1.

Table 10: List of the mean XBP1 mRNA expression values in XBP1 antisense

oligonucleotide-treated SKOV-3 cells compared to untreated cells. Expression values are normalized to HPRT1.

Example 3: Investigation of the dose-dependent knockdown by selected XBP1 antisense oligonucleotides in EFO-21 cells

The dose-dependent knockdown of XBP1 mRNA expression by XBP1 antisense oligonucleotides in EFO-21 cells was investigated and the respective IC50 values were calculated. Therefore, EFO-21 cells were treated for three days with the respective antisense oligonucleotide at the following concentrations: 10mM, 3mM, ImM, 300nM, 100nM, 30nM, lOnM, 3nM (all antisense oligonucleotides except A21036H) or 6mM, 2mM, 600nM, 200nM, 60nM, 20nM, 6nM, 2nM (antisense oligonucleotide A21036H). After the treatment period, cells were lyzed, XBP1 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the XBP1 expression values were normalized to HPRT1 values. A dose-dependent knockdown of XBP1 mRNA with all tested XBP1 antisense oligonucleotides (Fig. 5) with IC50 values below 600nM (Table 11 and 12) was observed. In the following Tables 11 and 12 are shown:

Table 11: Dose-dependent inhibition of XBP1 mRNA expression in EFO-21 cells by selected XBP1 antisense oligonucleotides and respective IC50 values (part 1).

Table 12: Dose-dependent inhibition of XBP1 mRNA expression in EFO-21 cells by selected XBP1 ASOs and respective IC50 values (part 2).

Example 4: Design of murine XBP1 antisense oligonucleotides

For the design of antisense oligonucleotides with specificity for murine (m) XBP1 the hXBPl mRNA sequence with SEQ ID NO. 2 (RefSeq. ID NM_013842.3) was used. 17mers were designed according to in-house criteria, negl (described, e.g., in WO2014154843 Al) was used as control antisense oligonucleotide in all experiments (Table 2). The distribution of the antisense oligonucleotide binding site on the mXBPl mRNA is shown in Fig. 6.

Example 5: Efficacy screen of hXBPl antisense oligonucleotides in murine cancer cell lines

In order to investigate the knockdown efficacy of the in silico designed murine XBP1 antisense oligonucleotides, two efficacy screening rounds were performed in the murine cancer cell lines 4T1 (Breast Cancer, ATCC) and Renca (Renal Adenocarcinoma, ATCC). Cells were treated with the respective antisense oligonucleotide at a concentration of 5mM for three days without the addition of a transfection reagent. Cells were lyzed after the three days treatment period, XBP1 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the XBP1 expression values were normalized to HPRT1 values. The results for the first screening round of antisense oligonucleotides are shown in Fig.7A, 7B as well as Tables 13 and 14. As depicted in Fig. 7A and Table 13, treatment of 4T1 cells with the antisense oligonucleotides A21035M, A21032M, A21031M, A21028M, A21034M, A21027MR and A21019MR resulted in a residual XBP1 mRNA expression of <0.2 compared to untreated cells. The control antisense oligonucleotide negl showed no effect on the XBP1 mRNA expression in this experiment. Antisense oligonucleotides were furthermore screened in Renca cells with regard to their XBP1 knockdown efficacy. As shown in Fig. 7B and Table 14, treatment with the antisense oligonucleotides A21031M, A21035M, A21034M, A21032M, A21028M and A21027MR resulted in a residual XBP1 mRNA expression of <0.3, whereas the control antisense oligonucleotide negl had no effect. In the following Tables 13 and 14 are shown:

Table 13: List of the mean XBP1 mRNA expression values in XBP1 antisense oligonucleotide-treated 4T1 cells compared to untreated cells. Expression values are normalized to HPRT1.

Table 14: List of the mean XBP1 mRNA expression values in XBP1 antisense oligonucleotide-treated Renca cells compared to untreated cells. Expression values are normalized to HPRT1.