EP2545070A1

EP2545070A1 - Stat3 activation inhibitors

Info

Publication number: EP2545070A1
Application number: EP11707809A
Authority: EP
Inventors: Corina Heinz; Bernd Groner; Axel Weber
Original assignee: Chemotherapeutisches Forschungsinstitut Georg Speyer Haus
Current assignee: Chemotherapeutisches Forschungsinstitut Georg Speyer Haus
Priority date: 2010-03-10
Filing date: 2011-03-10
Publication date: 2013-01-16
Also published as: WO2011110359A1; WO2011110359A8

Abstract

The present invention relates to novel Stat3 activation inhibitors, especially to (poly)peptides capable to inhibit Stat3 signaling. The invention furthermore concerns nucleic acids encoding for these (poly)peptides and vectors comprising said nucleic acids. In addition, the present invention relates to respective uses of the Sta3 activation inhibitors and pharmaceutical compositions comprising same. The invention also relates to the isolated coiled-coil domain of Stat3 and screening methods for identifying compounds having affinity to this domain.

Description

STAT3 ACTIVATION INHIBITORS

The present invention relates to novel Stat3 activation inhibitors, especially to (poly)peptides capable of inhibiting Stat3 signaling. The invention furthermore concerns nucleic acids encoding for these (poly)peptides and vectors comprising said nucleic acids. In addition, the present invention relates to respective uses of the Stat3 activation inhibitors and pharmaceu- tical compositions comprising same. The invention also relates to the isolated coiled-coil domain of Stat3 and screening methods for identifying compounds having affinity to this domain.

Signal transducer and activator of transcription 3 (Stat3) is an essential mediator of cytokine and growth factor signaling. The activation of latent Stat3 is dependent on its tyrosine phosphorylation by tyrosine kinases. Phosphorylation induces dimerization and translocation into the nucleus, where P-Stat3 (activated Stat3) binds to response elements present in the promoters of various target genes (Levy and Darnell, 2002; Bowman et al., 2000). Several counteracting proteins tightly regulate the extent and the duration of this signal. They in- elude the suppressors of cytokine signaling (Socs), the Src-homology 2 domain containing phosphatases (SHPs), the translocation inhibitor Grim-19 and the protein inhibitor of activated Stat3 (Pias3) (Chen et al., 2004; Frank, 2007).

In contrast to the transient Stat3 activation in normal cells, Stat3 is persistently activated in many cancers including leukemia, melanoma and cancers of the breast, head and neck, brain and pancreas (reviewed in (Frank, 2007; Haura et al., 2005). P-Stat3 promotes tumor development by the induction of genes encoding apoptosis inhibitors (e.g. Bcl_XL, Survivin, Mcl-1 ), cell-cycle regulators (e.g. cyclin D1/D2 and c-Myc), regulators of angiogenesis (e.g. VEGF, HIF1 a ) and genes promoting migration and invasion (e.g. MMPs). The multitude of molecules involved in the formation and progression of cancer represents new opportunities for drug discovery and development. The most promising drug targets are molecules which are functionally indispensible for the survival of cancer cells, whereas their temporary inhibition does not cause cell death in normal cells. Such components have been identified and include signaling molecules or transduction pathways which impose an "addiction phenotype" on cancer cells (Luo et al., 2009).

Transcription factor Stat3, regulating genes causing central hallmarks of cancer, plays such an indispensable role in cancer development and is constitutively activated in various can- cers. For instance, the down-regulation of Stat3 by siRNA, dominant negative variants Stat3- β), oligonucleotide decoys and chemical compounds showed that Stat3 signalling is required to maintain a distinct subset of tumor cells in vitro and in vivo (reviewed in Jing and Tweardy, 2005; Deng et al., 2007). The important role of activated Stat3 in cellular transformation has triggered the search for specific Stat3 inhibitors. Although considerable effort has been invested, only a few specific and selective Stat3 inhibitors have been described. Kinase inhibitors which interfere with the activation step, e.g. the Jak-kinase inhibitor AG490, Jak-lnhibitor I, and WP1066, or compounds which affect multiple other targets in addition to Stat3, e.g. curcumin, resvera- trol and platinum compounds, have been investigated. Several other compounds might be rather Stat3 specific, among them are hpSt3dODN, Stattic, S3I-M2001 , cucurbitacin, peptide P1 a and the previously identified peptide aptamer hTrx-3.8 which targets the Stat3-SH2 domain (Borghouts et al., 2008; Blaskovich et al., 2003; Dourlat et al., 2009; Schust et al., 2006; Siddiquee et al., 2007; Tadlaoui et al., 2009). However, these inhibitors need to be characterized further, in particular, it will be necessary to understand their mechanisms of action, to compare their EC₅₀-values, to improve their efficacy and to identify possible toxic side effects.

Thus, there is still a need to provide novel drugs suitable to interact with Stat3 and thereby inhibiting Stat3 signalling. However, the design of a potent and specific Stat3 inhibitor is not trivial since the development of therapeutic strategies targeting Stat3 is very limited. Stat3 is an intracellular protein, which does not exhibit any enzymatic activity. Therefore, Stat3 does not display any substrate binding pockets inhibitable by small molecules. Stat3 merely functions through protein-protein and protein-DNA interactions.

Thus, it was an object of the present invention to provide Stat3 activation inhibitors suitable to bind to Stat3 with high affinity and specificity, exhibiting no general toxicity, and, thus, suitable to be used as therapeutically active agent for the treatment or prevention of a disease, of symptoms of a disease or causes for a disease connected to Stat3, as, for example, cancer. Further, it was an object of the invention to provide a new pharmaceutical composition and a new method for the treatment of a disease, of symptoms of a disease or causes for a disease connected to Stat3, as, for example, cancer. A further related object of the invention was directed to identifying a targetable part or domain of Stat3 through which such novel drugs would cause their interaction. This part or domain could then allow to be used as the target for screening methods for identifying compounds having affinity to this domain and thus - by inhibiting Stat3 - would be useful in the treatment of cancer.

The main object of the present invention is solved by the subject-matter of the attached claims. The present invention provides (poly)peptides efficiently binding to functional domains of Stat3, and, therefore, having the potential to block essential interactions. In particular, the present invention is based on the unexpected findings that (poly)peptides de- rived from the carboxyl-terminal acidic region of Pias3 are suitable to bind strongly to the coiled-coil domain of Stat3, without exhibiting general toxicity, resulting in the specific suppression of Stat3 target gene expression, inhibition of migration and cellular growth.

Opposed to Stat3 the role of the negative Stat3 regulator, Pias3, has not been extensively studied in tumor cells harboring constitutively activated Stat3 molecules. Pias3 belongs to the Pias protein family also comprising Piasl , Pias-xa and Pias-χβ and Piasy (Chung et al., 1997; Shuai, 2006). These molecules positively or negatively regulate the activity of over 60 proteins, many of which are transcription factors e.g. Statl , Stat5, p53, NFKB, TIF2, Gfi1 , Smad and AR (Schmidt and Muller, 2003). Modulation of transcription factor activity by Pias proteins most likely involves multiple molecular mechanisms, including the recruitment of HDACs, the promotion of the sumoylation of transcription factors, the induction of the dissociation of dimers, or the sequestration of transcription factors to subnuclear structures (Schmidt and Muller, 2003; Shuai and Liu, 2005). Pias3 is comprised of distinct functional domains. The ami no-terminal is highly conserved among members of the Pias protein family and involved in binding e.g. to the p65 subunit of N FKB (Jang et al., 2004). The central domain, amino acid positions 327-369, is predicted to form a structure, which is similar to the RING domain in E3 ligases. It displays SUMO-1 E3 ligase activity towards e.g. p53, AR, LEF1 , Septin, and IFN regulatory factor-1 (IRF-1 ) (reviewed in Rytinki et al., 2009). The carboxyl-terminal regions of the Pias proteins are highly diverse. The C-terminal domain of Piasl was found to be involved in Statl binding (Liao et al., 2000).

A few studies have addressed the effects of exogenous Pias3 over-expression in cancer cells: i) About 89% of glioblastoma samples were found to be Pias3 negative and P-Stat3 positive and the ectopic expression of Pias3 in a glioblastoma cell line caused the inhibition of the transcriptional activity of Stat3 (Brantley et al., 2008). ii) Over-expression of Pias3 in melanoma and lung cancer cell lines inhibited cell growth and suppressed Stat3 activity (Ogata et al., 2006; Yagil et al., 2009). iii) Over-expression of Pias3 in v-Src over-expressing tumor cells suppressed Stat3 target gene expression (Herrmann et al., 2007). iv) Exogenous expression, mediated by infection with a adenovirus encoding kChaP/Pias3 in prostate cancer cells, induced apoptosis and reduced growth of prostate tumor xenografts in nude mice (Wible et al., 2002). These studies indicate that Pias3 might counteract the function of con- stitutively active Stat3.

Previous published studies indentified the N-terminus of mPias3 as the major Stat3 interaction domain (Levy et al., 2006; Sonnenblick et al., 2004). Following in line with this, also a further recent report disclosed that the N-terminal part of Pias3 would be involved in the interaction with Stat3 (Yagil et al., 2009).

Opposed to these findings expressed in the art, it was found that the C-terminal acidic domain of Pias3 interacts with the N-terminal coiled-coil domain of Stat3. Therefore, the finding of the present invention was totally surprising and overcame a prejudice deeply in- grained in the art following the recent research results. Therefore, in a first aspect of the present invention, the object of the present invention is solved by a (poly)peptide comprising a sequence of at least 10 contiguous amino acids selected from a basic amino acid sequence selected from

a) SEQ ID NO: 2, or

b) an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 2,

with the proviso that a (poly)peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is excluded.

In the context of the present invention the term "(poly)peptide" means a peptide, a protein, or a (poly)peptide which encompasses amino acid chains of a given length, wherein the amino residues are linked by covalent peptide bonds. However, peptidomimetics of such proteins/(poly)peptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention. Preferably, the (poly)peptide according to the present invention is a recombinant (poly)peptide.

In order to determine the percentage to which two sequences (amino acid sequences or the nucleic acid sequences encoding those sequences, e.g. DNA or RNA sequences) are identical, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. gaps can be inserted into the sequence of the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position. The percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions. The percentage to which two sequences are identical can be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et a/. (1993), PNAS USA, 90:5873-5877 or Altschul et a/. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program or, alternatively, for nucleic acid sequences, in the NBLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program. As already stated above, Pias3 is a natural binding partner of Stat3 and a regulator of Stat3 signaling. The binding affinity and specificity of their interacting domains has probably been optimized in an evolutionary process. The observation that the Pias3 protein is present in normal cells, which are capable of expressing high levels of P-Stat3 upon cytokine induc- tion (Fig. 1 A), emphasizes its essential function in the temporal regulation of Stat3 function. Furthermore, it was found that sumoylated Pias3 appeared during later stages of involution in the mammary gland (Fig. 1 B). The induction of involution in the mammary gland is accompanied by a strong P-Stat3 activation and results in the massive apoptosis of alveoli. Sumoylation results in increased protein stability and possibly points at a role for Pias3 to limit P-Stat3 activity at the end of the involution process (Chapman et al., 2000). It was further shown that the regulation of Pias3 expression is independent from Stat3 activation (Example 1 .2) and, in addition, that Pias3 is post-transcriptionally regulated (Example 1 .3).

Example 1 (described below) clearly showed that Pias3 protects normal cells from P-Stat3 addiction by fine-tuning P-Stat3 activity and preventing its oncogenic effects. This protection mechanism seems lost in tumor cells. Tumor cells seem to down-regulate Pias3 protein expression and thus allow the persistent action of P-Stat3. This then results in uncontrolled proliferation (Fig. 1 C, D). Similar observations were recently described for primary brain tumor samples and ALK7ALK^" lymphoblastic lymphomas (Brantley et al., 2008; Zhang et al., 2002), assigning a role as a tumor suppressor to Pias3.

Since the Pias3 gene is constitutively transcribed (Fig. 2), the down-regulation of the Pias3 protein might involve factors regulating its stability. PI3K AKT has been linked to Pias3 activity (Ogata et al., 2006), nitric oxide has been found to destabilize Pias3 and regulate its sumoylation (Qu et al., 2007). A crosstalk between hSiah2 (seven in absentia) and Pias3 modulates Pias dependent activation. hSiah2 is involved in the proteasome-dependent degradation of Pias3 (Depaux et al., 2007). It was found that treatment of 4T1 and Tu-9648 cells with a proteasome inhibitor led to an increase in Pias3 protein levels. This also confirms previous data (Brantley et al., 2008). In addition to these regulatory mechanisms, other processes affecting translation, e.g. miRNA-mediated translational inhibition, might be involved (Filipowicz, et al., 2008). Therefore, derivation of a Stat3-specific inhibitor from Pias3, which can be delivered into tumor cells, suppress Stat3 dependent transcription and the associated cellular phenotype would be of great interest. In this respect and as stated above, previously published studies described the N-terminus of mPias3 as the major Stat3 interaction domain (Sonnenblick et al., 2004; Levy et al., 2006; Yagil et al., 2009)

By the present invention, it was surprisingly found that (poly)peptides derived from the C- terminal of Pias3 bind to Stat3 (Example 2). In addition, it could be shown that (poly)peptides derived from the C-terminal of Pias3 do not exhibit any general toxicity (Example 7). Thus, the present invention provides for the first time specific Stat3 activation inhibitors. Therefore, according to one embodiment, the inventive (poly)peptide comprises a sequence of about 10 to about 300 contiguous amino acids selected from a basic amino acid sequence selected from

a) SEQ ID NO: 2, or

b) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 2,

wherein the entire (poly)peptide has a length of about 10 to about 500 amino acids. The inventive (poly)peptide typically comprises a sequence of about 10 to about 300 contiguous amino acids selected from a basic amino acid sequence represented by a) SEQ ID NO: 2, or b) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 2.

Preferably, the inventive (poly)peptide comprises a sequence of about 10 to about 300, of about 15 to about 300, of about 20 to about 300, of about 25 to about 300, of about 30 to about 300, of about 35 to about 300, of about 40 to about 300, of about 45 to about 300, of about 50 to about 300, of about 55 to about 300, of about 60 to about 300, of about 65 to about 300, of about 70 to about 300, or, alternatively, of about 10 to about 275, of about 15 to about 275, of about 20 to about 275, of about 25 to about 275, of about 30 to about 275, of about 35 to about 275, of about 40 to about 275, of about 45 to about 275, of about 50 to about 275, of about 55 to about 275, of about 60 to about 275, of about 65 to about 275, of about 70 to about 275, or, alternatively, of about 10 to about 250, of about 15 to about 250, of about 20 to about 250, of about 25 to about 250, of about 30 to about 250, of about 35 to about 250, of about 40 to about 250, of about 45 to about 250, of about 50 to about 250, of about 55 to about 260, of about 60 to about 250, of about 65 to about 250, of about 70 to about 250, or, alternatively, of about 10 to about 225, of about 15 to about 225, of about 20 to about 225, of about 25 to about 225, of about 30 to about 225, of about 35 to about 225, of about 40 to about 225, of about 45 to about 225, of about 50 to about 225, of about 55 to about 225, of about 60 to about 225, of about 65 to about 225, of about 70 to about 225, or, alternatively, of about 10 to about 200, of about 15 to about 200, of about 20 to about 200, of about 25 to about 200, of about 30 to about 200, of about 35 to about 200, of about 40 to about 200, of about 45 to about 200, of about 50 to about 200, of about 55 to about 200, of about 60 to about 200, of about 65 to about 200, of about 70 to about 200, or, alternatively, of about 10 to about 1 75, of about 15 to about 1 75, of about 20 to about 1 75, of about 25 to about 1 75, of about 30 to about 1 75, of about 35 to about 1 75, of about 40 to about 1 75, of about 45 to about 1 75, of about 50 to about 1 75, of about 55 to about 1 75, of about 60 to about 1 75, of about 65 to about 1 75, of about 70 to about 1 75, or, alternatively, of about 10 to about 1 50, of about 15 to about 150, of about 20 to about 150, of about 25 to about 150, of about 30 to about 150, of about 35 to about 150, of about 40 to about 150, of about 45 to about 1 50, of about 50 to about 150, of about 55 to about 1 50, of about 60 to about 150, of about 65 to about 1 50, of about 70 to about 1 50 contiguous amino acids selected from the amino acid sequence represented by a) SEQ ID NO: 2, or b) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 2. Further, the entire inventive (poly)peptide typically has a length of about 10 to about 500 amino acids. Preferably, the entire inventive (poly)peptide has a length of about 10 to about 500, of about 20 to about 500, of about 30 to about 500, of about 40 to about 500, of about 50 to about 500, of about 60 to about 500, of about 70 to about 500, of about 80 to about 500, of about 90 to about 500, of about 100 to about 500, or, alternatively, of about 10 to about 450, of about 20 to about 450, of about 30 to about 450, of about 40 to about 450, of about 50 to about 450, of about 60 to about 450, of about 70 to about 450, of about 80 to about 450, of about 90 to about 450, of about 100 to about 450, or, alternatively, of about 10 to about 400, of about 20 to about 400, of about 30 to about 400, of about 40 to about 400, of about 50 to about 400, of about 60 to about 400, of about 70 to about 400, of about 80 to about 400, of about 90 to about 400, of about 100 to about 400, or, alternatively, of about 10 to about 350, of about 20 to about 350, of about 30 to about 350, of about 40 to about 350, of about 50 to about 350, of about 60 to about 350, of about 70 to about 350, of about 80 to about 350, of about 90 to about 350, of about 100 to about 350, or, alterna- tively, of about 10 to about 300, of about 20 to about 300, of about 30 to about 450, of about 40 to about 300, of about 50 to about 300, of about 60 to about 300, of about 70 to about 300, of about 80 to about 300, of about 90 to about 300, of about 100 to about 275, or, alternatively, of about 10 to about 275, of about 20 to about 275, of about 30 to about 275, of about 40 to about 275, of about 50 to about 275, of about 60 to about 275, of about 70 to about 275, of about 80 to about 275, of about 90 to about 275, of about 100 to about 275, or, alternatively, of about 10 to about 250, of about 20 to about 250, of about 30 to about 250, of about 40 to about 250, of about 50 to about 250, of about 60 to about 250, of about 70 to about 250, of about 80 to about 250, of about 90 to about 250, of about 100 to about 250, or, alternatively, of about 10 to about 225, of about 20 to about 225, of about 30 to about 225, of about 40 to about 225, of about 50 to about 225, of about 60 to about 225, of about 70 to about 225, of about 80 to about 225, of about 90 to about 225, of about 100 to about 225, or, alternatively, of about 10 to about 200, of about 20 to about 200, of about 30 to about 200, of about 40 to about 200, of about 50 to about 200, of about 60 to about 200, of about 70 to about 200, of about 80 to about 200, of about 90 to about 200, of about 100 to about 200, or, alternatively, of about 10 to about 1 75, of about 20 to about 1 75, of about 30 to about 1 75, of about 40 to about 1 75, of about 50 to about 1 75, of about 60 to about 1 75, of about 70 to about 1 75, of about 80 to about 1 75, of about 90 to about 1 75, of about 100 to about 1 75, or, alternatively, of about 10 to about 1 50, of about 20 to about 1 50, of about 30 to about 150, of about 40 to about 150, of about 50 to about 1 50, of about 60 to about 150, of about 70 to about 150, of about 80 to about 150, of about 90 to about 150, of about 100 to about 150 amino acids.

According to a preferred embodiment of the inventive (poly)peptide, the (poly)peptide comprises an amino acid sequence selected from

a) SEQ ID NO: 3, or

b) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 3.

According to a specifically preferred embodiment of the inventive (poly)peptide, the (poly)peptide comprises an amino acid sequence selected from

a) SEQ ID NO: 2,

b) SEQ ID NO: 4,

c) SEQ ID NO: 5,

d) SEQ ID NO: 6, or

e) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

According to another specifically preferred embodiment of the inventive (poly)peptide, the (poly)peptide comprises an amino acid sequence selected from

a) SEQ ID NO: 4,

b) SEQ ID NO: 5,

c) SEQ ID NO: 6, or

d) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. According to still another specifically preferred embodiment of the inventive (poly)peptide, the (poly)peptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75, or a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75.

The (poly)peptide according to the present invention may further comprise a transduction domain, a signaling domain, and/or a scaffold protein. Preferably, the inventive (poly)peptide further comprises a transduction domain and/or a signaling domain. A "transduction domain" in the context of the present invention means a domain leading to the cellular uptake of the inventive (poly)peptide. Preferably, the transduction domain of the inventive (poly)peptide is selected from the group comprising oligo-arginin, oligo-lysin, TAT, HIV-rev, MAP, penetratin (antennapedia, lsl-1 homeodomain), chimeric CPPs (trans- portan, transportan-10, KALA, MPG peptides Ρβ and Pa) ppTG-1 , Pep-1 , antimicrobial de- rived CPPs (Buforin 2, Bac715-24, SynB), pVEC, VP22, calcitonin derived peptides (hCT(9- 32), SAP, and protamin.

"Oligo" in the context of the present invention means 25 or less, but more than one amino acids, i.e. 25, 24, 23, 22, 21 , 20, 19, 18, 1 7, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids.

A "signaling domain" in the context of the present invention means a domain for affinity purification. Preferably, the signaling domain of the inventive (poly)peptide is selected from the group comprising 6, 7, 8, 9 or 10 Histidine repeats, GST-tags, calmodulin binding pep- tide (TAP-tag), GS-TAP-tag Strep ll-tag, CYD-tag, CHH-MAFT-tag, myc-tag, HA-tag, PTP-tag, or LHH-tag. A "scaffold domain" in the context of the present invention means a domain for increasing the stability and purification characteristics, e.g. Thioredoxin, GFP, Lipocalins, GST, scFv, or protease inhibitors. According to a particular preferred embodiment the inventive (poly)peptide consists of an amino acid sequence selected from the amino acid sequence represented by

a) SEQ ID NO: 2,

b) SEQ ID NO: 3,

c) SEQ ID NO: 4,

d) SEQ ID NO: 5,

e) SEQ ID NO: 6, or

f) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences ac- cording to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ

ID NO: 6.

According to a still particular preferred embodiment the inventive (poly)peptide consists of an amino acid sequence selected from the amino acid sequence represented by any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75, or a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75.

According to another particular preferred embodiment the inventive (poly)peptide consists of an amino acid sequence selected from the amino acid sequence represented by

a) SEQ ID NO: 2,

b) SEQ ID NO: 3,

c) SEQ ID NO: 4,

d) SEQ ID NO: 5, e) SEQ ID NO: 6, or

f) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6,

and a transduction domain, a signaling domain, and/or scaffold domain.

According to a specifically preferred embodiment the inventive (poly)peptide consists of an amino acid sequence selected from the amino acid sequence represented by

a) SEQ ID NO: 2,

b) SEQ ID NO: 3,

c) SEQ ID NO: 4,

d) SEQ ID NO: 5, or

e) SEQ ID NO: 6.

Preferably, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 2. More preferably, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 4. Alternatively, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 5. Alternatively, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 6.

According to another specifically preferred embodiment the inventive (poly)peptide consists of an amino acid sequence selected from the amino acid sequence represented by

a) SEQ ID NO: 2,

b) SEQ ID NO: 3,

c) SEQ ID NO: 4,

d) SEQ ID NO: 5, or

e) SEQ ID NO: 6,

and a transduction domain, a signaling domain, and/or a scaffold domain.

Preferably, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 2 and a transduction domain, a signaling domain, and/or a scaffold domain. More preferably, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 4 and a transduction domain, a signaling domain and/or a scaffold domain. Alternatively, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 5 and a transduction domain, a signaling domain, and/or a scaffold domain. Alternatively, the inventive (poly)peptide consists of an amino acid sequence represented SEQ ID NO: 6 and transduction domain, a signaling domain, and/or a scaffold domain.

According to a further specifically preferred embodiment the inventive (poly)peptide comprises an amino acid sequence selected from the amino acid sequence represented by

a) SEQ ID NO: 8,

b) SEQ ID NO: 9,

c) SEQ ID NO: 10, or

d) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

Preferably, the inventive (poly)peptide consists of an an amino acid sequence selected from the amino acid sequence represented by

a) SEQ ID NO: 8,

b) SEQ ID NO: 9,

c) SEQ ID NO: 10, or

Especially the recombinant Pias-3 derived rPP-C8 (SEQ ID NO: 8) or rPP-C5 (SEQ ID NO: 9) peptides, preferably the rPP-C8 peptide, of this invention targeting the coiled-coil domain of Stat3 (SEQ ID No: 7), were not toxic to non-cancer cells, with Stat3 inhibit an essential signalling molecule in tumor cells, and thus offer new perspectives for both oncological treatment and translational oncology research. In this respect it could be shown that rPP-C8 does not affect the tyrosine phosphorylation of Stat3. Therefore, rPP-C8 probably acts after the activation step. In contrast to Pias3, which is only found in the nucleus of cells, rPP-C8 is also localized in the cytoplasm of transduced cells. This implies that the peptides bind to activated Stat3 molecules and prevent the translocation of these complexes to the nucleus. Additionally, rPP-C8 in dot-like structures was found in the nucleus.

According to another embodiment, the inventive (poly)peptide comprises

• optionally, a sequence (I),

• a sequence (II) of at least 1 0 contiguous amino acids selected from an amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 2, and

• optionally, a sequence (III),

• with the proviso that the (poly)peptide does not comprise an amino acid sequence (I) comprising a sequence located N-terminally from sequence (II) and represented by SEQ ID NO: 76 (corresponding to residues 31 9-328 of SEQ ID NO: 1 ), or having identity of more than about 99%, or of more than about 95%, or of more than about 90%, or of more than about 80% with the amino acids of the sequence represented by SEQ ID NO: 76, in case sequence (II) comprises the amino acid sequence represented by SEQ ID NO: 77 (corresponding to residues 1 -10 of SEQ ID NO: 2 and residues 329-338 of SEQ ID NO: 1 ).

Accordingly, the inventive (poly)peptide comprises a sequence (II) of at least 1 0 contiguous amino acids selected from a basic amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 2.

The inventive (poly)peptide optionally comprises a sequence (I) and/or a sequence (III). Sequence (I) and (III) may be any natural or synthetic sequence. Sequence (I), if present in the inventive (poly)peptide, is located N-terminally of sequence (II). Sequence (III), if present in the inventive (poly)peptide, is located C-terminally of sequence (II). Preferably, the sequence (II) is at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, or at least 124 contiguous amino acids in length. The sequence (II) may further com- prise of about 10 to about 300, or the number of contiguous amino acids, as defined herein above, of the sequence represented by SEQ ID NO:2, or of an amino acid sequence having particular identity with the sequence represented by SEQ ID NO:2, as defined herein above. Sequence (II) may further comprise of about 10 to about 20, of about 10 to about 30, of about 10 to about 40, of about 10 to about 50 of about 10 to about 60, of about 10 to about 70, of about 10 to about 80, of about 10 to about 90, of about 10 to about 100, of about 10 to about 210, of about 10 to about 120, of about 10 to about 124, of about 10 to about 130, of about 20 to about 30, of about 20 to about 40, of about 20 to about 50, of about 20 to about 60, of about 20 to about 70, of about 20 to about 80, of about 20 to about 90, of about 20 to about 100, of about 20 to about 210, of about 20 to about 120, of about 20 to about 124, of about 20 to about 130, of about 30 to about 40, of about 30 to about 50, of about 30 to about 60, of about 30 to about 70, of about 30 to about 80, of about 30 to about 90, of about 30 to about 100, of about 30 to about 210, of about 30 to about 120, of about 30 to about 124, of about 30 to about 130, of about 40 to about 50, of about 40 to about 60, of about 40 to about 70, of about 40 to about 80, of about 40 to about 90, of about 40 to about 100, of about 40 to about 210, of about 40 to about 120, of about 40 to about 124, of about 40 to about 130, of about 50 to about 60, of about 50 to about 70, of about 50 to about 80, of about 50 to about 90, of about 50 to about 100, of about 50 to about 210, of about 50 to about 120, of about 50 to about 124, or of about 50 to about 130 contiguous amino acids of the sequence represented by SEQ ID NO: 2, or an amino acid sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 2. The entire length of the inventive (poly)peptide may be as defined herein above. The length of the inventive (poly)peptide may further be identical with the length of sequence (II), and may be as defined herein above.

According to a further embodiment, the inventive (poly)peptide comprises a sequence, wherein the sequence (II) of at least 10 contiguous amino acids is selected from the amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 5. According to another embodiment, the inventive (poly)peptide comprises a sequence, wherein the sequence (II) is represented by the amino acid sequence of SEQ ID NO: 5, or a sequence having identity of at least about 80%, preferably of at least about 90% even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 5.

According to still another embodiment, the inventive (poly)peptide comprises a sequence, wherein the sequence (II) is represented by the amino acid sequence of any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75, or a sequence having identity of at least about 80%, preferably of at least about 90% even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequences according to any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75. According a specific embodiment, the inventive (poly)peptide does not comprise an amino acid sequence (III) comprising a sequence located C-terminally from sequence (II) and represented by SEQ ID NO: 78 (corresponding to residues 196-205 of SEQ ID NO: 2 and residues 524-533 of SEQ ID NO: 1 ), or having identity of more than about 99%, or of more than about 95%, or of more than about 90%, or of more than about 80% with the amino acids represented by SEQ ID NO: 78, in case sequence (II) comprises the amino acid sequence represented by SEQ ID NO: 55 (corresponding to residues 186-195 of SEQ ID NO: 2 and residues 514-523 of SEQ ID NO: 1 ); and/or the (poly)peptide does not comprise an amino acid sequence (I) comprising a sequence located N-terminally from sequence (II) and represented by SEQ ID NO: 79 (corresponding to residues 62-71 of SEQ ID NO: 2 and resi- dues 390-399 of SEQ ID NO: 1 ), or having identity of more than about 99%, or of more than about 95%, or of more than about 90%, or of more than 80% with the amino acids represented by SEQ ID NO: 79, in case (II) comprises the amino acid sequence represented by SEQ ID NO: 40 (corresponding to residues 72-81 of SEQ ID NO: 2 and residues 400-409 of SEQ ID NO: 1 ).

The (poly)peptide according to the present invention may further comprise a transduction domain, a signaling domain, and/or a scaffold protein, as described herein above. Preferably, the inventive (poly)peptide further comprises a transduction domain and/or a signaling domain.

According to a further embodiment, the inventive (poly)peptide comprises the amino acid sequence of any one of SEQ ID NO: 8, SEQ ID NO: 10, or a sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequences according to SEQ ID NO: 8, or SEQ ID NO: 10. The (poly)peptide of the present invention is capable to inhibit Stat3 signaling, preferably by interacting with SEQ ID NO: 7, wherein the interaction is preferably characterized by autonomously binding to SEQ ID NO: 7.

In this respect, it could be shown that Stat3 is inhibited in a post-activation stage and that the coiled-coil domain of Stat3 (SEQ ID NO: 7) serves as a drug target site. The recombinant Pias3 fragment, rPP-C8, specifically recognizes this domain of Stat3; it is not toxic in normal cells and offers new perspectives for structure based drug design.

Accordingly, in a further embodiment the (poly)peptide according to any of the embodi- ments of the (poly)petides according to the invention is capable of inhibiting Stat3 signalling, preferably by interacting with SEQ ID NO: 7, wherein the interaction preferably is characterized by an autonomous binding to an amino acid sequence of SEQ ID NO: 7, or to an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with SEQ ID NO: 7. The identification of the coiled-coil domain of Stat3 (SEQ ID NO: 7) as the binding region for Pias3 provides interesting implications for the mechanism by which rPP-C8 (SEQ ID NO: 8) inhibits Stat3 function. Another aspect of the present invention relates to a nucleic acid comprising a nucleic acid sequence encoding a (poly)peptide according to the present invention. The nucleic acids of the invention can be DNA, cDNA, genomic DNA, synthetic DNA, PNA, CNA, RNA or combinations thereof, and can be double-stranded or single-stranded, the sense and/or an antisense strand. Additionally, the present invention also concerns a nucleic acid hybridiz- ing under stringent conditions with the nucleic acid according as defined above.

A further aspect of the present invention is a recombinant vector comprising the nucleic acid(s) of the present invention or a peptide encoded by a nucleic acid of the present invention. The term "vector" refers to a protein or a nucleic acid or a mixture thereof which is capable of being introduced or of introducing the proteins and/or nucleic acid comprised into a cell. In one preferred embodiment the recombinant vector of the invention is a recombinant expression vector.

Another aspect of the present invention is a host cell comprising a nucleic acid or vector according to the present invention.

A "host cell" according to this invention is a cell that harbours foreign molecules, viruses or microorganisms. Especially it refers to cells containing a target nucleic acid molecule, for example a heterologous nucleic acid molecule such as a vector or plasmid or other low molecular weight nucleic acid, in which case the host cell is typically suitable for replicating the nucleic acid molecule of interest. Examples of suitable host cells useful in the present invention include, bacterial, yeast, insect and mammalian cells. Specific examples of such cells include, SF9 insect cells, Chinese Hamster Ovary (CHO) cells, E. Coli cells, as for example the E. Coli strains: DH10b cells, XU Blue cells, XL2Blue cells, Top10 cells, HB101 cells, and DH12S cells.

A further aspect of the present invention relates to a (poly)peptide, a nucleic acid or vector according to the present invention for use in medicine. As mentioned above Stat3 regulates the expression of various target genes involved in proliferation and survival, migration and angiogenesis. The (poly)peptides according to the present invention are suitable to inhibit Stat3 activation, and thereby inhibit the different processes regulated by P-Stat3 (Example 4).

Therefore, one preferred embodiment of the present invention concerns the inventive (poly)peptide, nucleic acid or vector for use in the treatment or prevention of a disease, of symptoms of a disease or causes for a disease connected to the transcription factor Stat3. Another preferred embodiment of the present invention relates to the use of a (poly)peptide, a nucleic acid or vector according to the present invention for the preparation of a medicament for the treatment or prevention of a disease, of symptoms of a disease or causes for a disease connected to the transcription factor Stat3. Diseases connected to the transcription factor Stat3 are, for example, cancer, an auto- immuno-disease, chronic inflammation, psoriasis, a liver disease, preferably, cancer.

It was shown that Pias3 protein expression is lost in cancer cell lines and tumor tissues (Example 1 .2), and, therefore, protection afforded by Pias3 in normal tissues against detrimen- tal effects of P-Stat3 is lost as well. Additionally, it was shown that the treatment of cancer cells with (poly)peptides according to the present invention inhibits different processes regulated by P-Stat3 (Examples 4 and 5.). In this respect it was shown that the (poly)peptides of the present invention are suitable to inhibit Stat3 activation, and, thereby, inhibit the expression levels of the anti-apoptotic target genes BclXL, Survivin and Cyclin D1 , a gene regulat- ing proliferation (Example 4). In addition, it was shown that the (poly)peptides of the present invention are suitable to prevent migration of cancer cells, such as, for example glioblastoma cells, and therefore, infiltration into surrounding normal tissues (Example 5). Finally, it was found that the (poly)peptides of the present invention are suitable to inhibit proliferation and to induce apoptosis in cancer cells (Example 7).

Therefore, the (poly)peptides of the present invention represents novel drugs for the treatment of cancer. Thus, in another preferred embodiment of the use according to the invention the treatment is suppression of tumor cells, suppression of the metastasing potential of tumor cells, activation of immuno cells interacting with tumor cells, removing the block of differentiation of dendritic cells, or inhibition of Stat3 functions.

One great advantage of the (poly)peptides of the present invention is that they do not exhibit general toxicity (Example 7).

Prevention or treatment of the disease and induction or enhancement of the immune response may be carried out by using different inventive medicaments as defined above in a time staggered manner.

A specific aspect of the present invention relates to a (poly)peptide, a nucleic acid or vector according to the present invention for use in medicine, as described above, together with a chemotherapeutic agent, i.e. co-administered with a chemotherapeutic agent. As used herein, the term "co-administration" or "co-administering" refers to administration of one component of the method, e.g., (poly)peptide, nucleic acid or vector of the present invention, with a chemotherapeutic agent, concurrently, i.e., simultaneously in time, or sequentially, i.e., administration of one component, followed by administration of the other component. That is, after administration of one component, the second component can be administered substantially immediately after the first component, or the second component can be administered after an effective time period after the first component, the effective time period being the amount of time given for realization of maximum benefit from the administration of the first component. The administration of the (poly)peptide, nucleic acid or vector of the present invention may be separated in time from the administration of the chemotheraptic agent by up to several weeks, and may precede it or follow it, but more commonly the administration of the (poly)peptide, nucleic acid or vector of the present invention will accompany the chemotherapy (such as the administration of one dose of chemotheraptic agent and and one dose (poly)peptide, nucleic acid or vector of the present invention within up to 48 hours, and most commonly within less than 24 hours).

As used herein, "chemotherapeutic agent" refers to naturally occurring or synthetic chemical compounds which are used for selective damage of cells, in particular cancer cells. The term "chemotherapeutic agent" is used synonymously with the terms "anti-neoplastic agent" and "anti-prol iterative agent", herein.

Examples of chemotherapeutic agents which may be used according to the invention are:

• alkylating agents, i.e. agents which generally exert cytotoxic activity by alkylating DNA, thus directly interfering with the reproductive cycle of the cell alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes). Exemplary alkylating agents may include, but are not limited to, Uracil mustard, Chlormethine, Cyclophosphamide (Cy- toxan&commat;), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylene- melamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Strep- tozocin, Dacarbazine, and Temozolomide;

• antimetabolites, i.e. agents which are inserted into nucleic acid sequences leading to altered cell division and metabolism (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors). Exemplary antimetabolites may include, but are not limited to, Methotrexate, Azacitidine, Decitabine, 5-Fluorouracil, Capecitabine, Doxifluridine, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine, Cladribine, Azathio- prine, Pentostatine, and Gemcitabine;

• mitosis inhibitors / anti-microtubule agents which interferes with cell division by disrupting the normal functionality of the cellular microtubules. Exemplary antimicro- tubule agents may include, but are not limited to, Taxanes, such as Taxol, Plaxitaxel, Docetaxel and Taxotere, and Vinca alkaloids, such as Vincristine and Vinblastine, Vinorelbine or Vindesine;

• platinum analogues, i.e. platinum compound which cross-linked nucleic acid strands. Exemplary platinum analogues may include, but are not limited to, Cis- platin, Carboplatin, Oxaliplatin, or Satraplatine;

• intercalating agents, i.e. agents which bind to DNA and prevent interaction with polymerases. Exemplary intercalating agents may include, but are not limited to, Anthracycline, Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, Mitoxantrone, or Amsacrine; • topoisomerase I and II inhibitors. . Exemplary topoisomerase inhibitors may include, but are not limited to, topoisomerase I inhibitors: Camptothecin, Topotecan, or Irinotecan; topoisomerase II inhibitors: Etoposide or Teniposide;

• other natural products and their derivatives (for example, antitumor antibiotics, en- zymes, lymphokines and epipodophyllotoxins), such as Actinomycin, Bleomycin,

Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Ara-C, Mithra- mycin, Deoxyco-formycin, Mitomycin-C, L- Asparaginase, Interferons (especially IFN-a); and

• other anti-proliferative cytotoxic agents, such as Navelbene, CPT-1 1 , Anastrazole, Letrazole, Capecitabine, Reloxafine, and Droloxafine.

The amount of the chemotherapeutic agent used may be variable. In a suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapeutic agent is adminis- tered with a (poly)peptide, a nucleic acid or vector of the present invention. Chemotherapy doses and dosing schedules may be determined by one of ordinary skill in the art.

Some exemplary doses of chemotherapeutic agents are given below:

• Irinotecan: 25-2500 mg/m²;

· Cisplatin: 75-120 mg/m² administered every three weeks;

• Carboplatin: within the range of 200-600 mg/m²;

• Paclitaxel: 1 30-225 mg/m² every 21 days;

• Gemcitabine are within the range of 80-1500 mg/m² administered weekly; and

• Leucovorin are 10-600 mg/m² administered weekly.

According to the invention, also multiple combinations of chemotherapeutic agents with the (poly)peptide, nucleic acid or vector of the present invention are likewise possible. For example, triple and quadruple combinations can provide greater efficacy. As is shown in example 8, combined administration of a (poly)peptide of the present invention and an chemotherapeutic agent according to the present invention leads to enhanced effects in the treatment of cancer cells (Figures 12 and 13). The development of resistances against chemotherapeutic agents is often being observed in the course of therapy of tumors. The development of drug resistance, e.g. in colon, prostate, ovarian and cervical carcinomas, has frequently been found to be accompanied by the inappropriate activation of the transcription factor Stat3. Stat3 induces strong expression of genes which counteract proliferation stop signals and induction of apoptosis. Stat3 also supports the repair of damaged DNA and thus promotes survival of tumor cells upon treatment with chemotherapeutic agents.

For these reasons it is advantageous to treat tumor cells with a combination of Stat3 inhibi- tors and chemotherapeutic agents. This might enhance the efficacy of chemotherapeutic agents, i.e. sensitizing chemotherapeutic treatment.

Preferably, a (poly)peptide, a nucleic acid or vector of the present invention may be used as a sensitizer. As used herein, "sensitizer" refers to a substance sensitizing chemotherapeutic treatment, i.e. allowing the usages of lower doses of chemotherapeutic agent(s), when coadministering the (poly)peptide, nucleic acid or vector of the present invention with the chemotherapeutic agent(s) as compared to administration of the chemotherapeutic agent(s) alone, in order to obtain the same therapeutic effect. It is anticipated that (poly)peptides, nucleic acids or vectors according to the present invention used in combination with chemotherapeutic agents may give rise to a significantly enhanced cytotoxic effect on cancerous cells, thus providing an increased therapeutic effect. Specifically, as an increased inhibitory effect is obtained with the above disclosed combinations on cancerous cells (Example 8), lower concentrations of the chemotherapeutic agents compared to the treatment regimes in which the agents are used alone, may be administered.

There is the potential to provide therapy wherein adverse side effects associated with the anticancer agents are considerably reduced which are normally observed when chemo- therapeutic agents are used alone in higher doses. By reducing the incidence of adverse effects, an improvement in the quality of life of a patient undergoing treatment for cancer is contemplated. Further, lowering the incidence of adverse effects may improve patient com- pliance and reduce the number of hospitalizations needed for the treatment of adverse effects.

Another aspect of the present invention concerns a pharmaceutical composition comprising a (poly)peptide according, a nucleic acid or vector according to the present invention and optionally a pharmaceutically acceptable carrier, adjuvant, and/or vehicle.

According to one embodiment, the pharmaceutical composition of the present invention may comprise a chemotherapeutic agent, as described above.

The inventive medicament, preferably a pharmaceutical composition, typically comprises a safe and effective amount of the compounds according to the invention (the (poly)peptides, the nucleic acids, the vectors) as defined above. As used here, "safe and effective amount" means an amount of the compounds as defined above, that is sufficient to significantly in- duce a positive modification of a condition to be treated, for example of a tumor, autoimmune diseases, etc. At the same time, however, a "safe and effective amount" is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment. A "safe and effective amount" of the compounds according to the invention as defined above will vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying doctor. The medicament according to the invention can be used according to the invention for human and also for veterinary medical purposes, as a pharmaceutical composition.

The medicament according to the invention typically contains a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable carrier" as used herein preferably includes the liquid or non-liquid basis of the inventive medicament. If the inventive medicament is provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions. Particularly for injection of the inventive medicament, a buffer, preferably an aqueous buffer, may be used, containing a sodium salt. The injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are e.g. in "in vivd' methods occurring liquids such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well, which are suitable for administration to a person. The term "compatible" as used here means that the constituents of the inventive medicament are capable of being mixed with the compound according to the invention as defined above in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive medicament under usual use conditions. Pharmaceutically acceptable carriers must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated. Some examples of compounds which can be used as pharmaceutically acceptable carriers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

The choice of a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive medicaments are administered. The inventive medicaments can be administered, for example, systemically. Routes for administration include, for example, transdermal, oral, parenteral, including subcutaneous or intravenous injections, topical and/or intranasal routes. The suitable amount of the inventive medicament to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams,-gels and the like. If the inventive medicament is to be administered perorally, tablets, capsules and the like are the preferred unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.

Also included in the present invention are methods of treating a disease connected to the transcription factor Stat3 by administering to a patient in need thereof a pharmaceutically effective amount of an inventive medicament, or a pharmaceutically effective amount of an inventive compound ((poly)peptide, nucleic acid or vector). Such a method typically comprises an optional first step of preparing the inventive medicament, or the inventive compound, and a second step, comprising administering a pharmaceutically effective amount of said inventive medicament, or said inventive compound.

As mentioned above Stat3 is a transcription factor closely related to tumor cells. In glioma cells constitutively activated Stat3 promotes cell-cycle progression and survival, stimulates angiogenesis and impairs immunological responses to cancer cells. Stat3 activation is ac- companied by an increased expression of cell-cycle and survival regulators, such as cyclin D1 , c-myc, Bcl-XL and survivin. Therefore, Stat3 is a preferred target for therapeutic intervention. It does not seem to be essential for the survival of many normal cells, but it is indispensable for many different tumor types. Efficient inhibition of Stat3 provides an attractive option, especially for targeted cancer therapy.

In addition, it has been found that the oncogenic effects of activated Stat3 are enhanced in cancer cells by down-regulating Pias3 expression. Loss of this endogenous inhibitor serves as a surrogate marker for predicting the sensitivity of tumor cells towards Stat3-directed therapies.

It was found in this invention that (poly)peptides derived from the C-terminal of Pias3 inter- act specifically with the coiled-coil region of Stat3 (Example 2). Therefore, the present invention identified the coiled-coil domain as a novel drug target site offering new perspectives for the design of potent inhibitors. The finding that the C-terminal acidic domain of Pias3 interacts with N-terminal coiled-coil domain of Stat3 came as a real surprise because again the art was reporting differently here, where it had been described that Pias3 interferes with the DNA-binding activity of Stat3 (Chung et al., 1977). Still, this was proven to be misleading as Pias3 does not directly recognize this Stat3 domain (Fig. 3D).

Therefore, another aspect of the present invention relates to a targeted (poly)peptide comprising or consisting of an amino acid sequence selected from the amino acid sequence represented by

a) SEQ ID NO: 7, or

b) an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 7,

with the proviso that a (poly)peptide consisting of the amino acid sequence represented by SEQ ID NO: 39 is excluded.

Another aspect of the present invention relates to a targeted (poly)peptide consisting of an amino acid sequence selected from

a) the amino acid sequence represented by SEQ ID NO: 7, or

b) an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 7. Another aspect of the present invention relates to a targeted (poly)peptide consisting of a maximum of 700, or 600, or 500, or 400, or 300, or 250, or 200 contiguous amino acids comprising an amino acid sequence selected from

a) the amino acid sequence represented by SEQ ID NO: 7, or

b) an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 7. In another embodiment this targeted (poly)peptide consists of a maximum of 600 contiguous amino acids, or of between 200 and 600 contiguous amino acids. In another embodiment this targeted (poly)peptide consists of a maximum of 500 contiguous amino acids, or of between 200 and 500 contiguous amino acids. In another embodiment this targeted (poly)peptide consists of a maximum of 400 contiguous amino acids, or of between 200 and 400 contiguous amino acids. In another embodiment this targeted (poly)peptide consists of a maximum of 300 contiguous amino acids, or of between 200 and 300 contiguous amino acids. In another embodiment this targeted (poly)peptide consists of a maximum of 200 contiguous amino acids. This targeted polypeptide of the invention is the target of the polypeptide according to the present invention described above.

The targeted polypeptide of the invention may be used as in a method for measuring the binding affinity of a molecule or for identifying a molecule - by various screening methods- capable of being used for the treatment of cancer. Accordingly, another aspect of the invention relates to a method for measuring the binding affinity of a (at least one) molecule to the targeted (poly)peptide of the invention comprising the steps of (a) bringing the - at least one - molecule in contact with the targeted (poly)peptide of the invention and (b) measuring the affinity of the molecule to the targeted (poly)peptide of the invention. Most preferably, this method for measuring the affinity is used to identify a, or at least one molecule capable of being used for the treatment of various diseases preferably cancer from among a group of molecules, preferably with an unknown capability of being used for the treatment of cancer. Another aspect of the invention relates to a Screening Method for the identification of a molecule for the treatment of cancer, wherein the molecule is identified by its binding affinity to the targeted (poly)peptide of the invention comprising the steps of (a) bringing the molecule in contact with the targeted (poly)peptide of the invention and (b) measuring the affinity of the molecule to the targeted (poly)peptide of the invention.

Figures: The following Figures are only illustrative to the present invention and shall describe particular embodiments of the present invention in further detail. However, these Figures are not intended to limit the subject matter of the present invention thereto.

Figure 1 : Expression of Stat3, P-Stat3 (Y705) and Pias3 in normal mouse tissues, breast and brain tumor cell lines and transplanted tumor tissues.

Figure 1 A) depicts a western blot analysis with antibodies specific for Stat3, P-Stat3 (activated Stat3 phosphorylated at Y⁷⁰⁵) and Pias3 of protein extracts obtained from normal mouse tissues. The antibodies recognize the respective human and murine proteins. The Pias3 antibody detects Pias3 (68 kDa) as well as its sumoylated form (85 kDa), both indicated by arrows.

Figure 1 B) depicts a western blot analysis of protein extracts obtained from mammary gland tissue at different developmental stages were analyzed with antibodies specific for Stat3, P-Stat3 and Pias3. Tissues were obtained at virgin (puberty), pregnant, early lactation, late lactation, early involution and late invo- lution stages.

Figure 1 C) depicts a western blot analysis of protein extracts obtained from human breast cancer cells (MCF-7, SK-BR-3, MDA-MB-231 , MDA-MB-453, MDA- MB-468, T47D) and murine mammary cancer cells (4T1 ).

Figure 1 D) depicts a western blot analysis of protein extracts obtained from human glioblastoma cell lines (MZ-54, MZ-18, U-87, U-373, Tu-2449, Tu-9649, Hs-

683). Stat3, P-Stat3 and Pias3 expression was determined with specific antibodies. The WHO-classifications of the glioblastomas from which the cell lines were derived are indicated below the blot. Figure 1 E) depicts a comparison of the expression levels of P-Stat3 in protein extracts obtained from tumor cell lysates (PC3 cells, lanel ; B16 cells, lane 3; U-87 cells, lane 5; 4ΤΊ cells, lane 7; SK-BR-3 cells, lane 9) or protein extracts obtained from tumors obtained upon transplantation of these tumor cell lines into NMRI-nu/nu mice (PC3, lane 2; U-87, lane 6; SK-BR-3, lane 10); Balb/c mice (4T1 , lane 8) or C57BI/6 (B1 6, lane 4). Extracts were prepared from the cancer cell lines (CL) grown in culture and from tumors (T) grown for two weeks after subcutaneously transplantation of the cells into the recipient mice.

Figure 1 F) depicts a western blot analysis of Pias3 expression in the tumor samples (T) obtained as described in (E). Protein extracts obtained from HepG2 lysates were included as a positive control. In all blots actin and β-tubulin antibodies were used to control the loading of the gels.

Pias3 is post-transcriptionally repressed in breast and glioma tumor cell lines and regulated by proteasomal degradation.

depicts Pias3 protein and mRNA expression in breast tumor cell lines (MCF- 7, SK-BR-3, MDA-MB-453, MDA-MB-231 , MDA-MB-468, 4T1 ) and in the hepatoma cell line HepG2. The upper part of the figure shows a Western blot of protein extracts from the indicated breast cancer cell lines probed with an antibody specific for Pias3. Extracts from Pias3 expressing HepG2 liver carcinoma cells served as a positive control. Relative Pias3 mRNA- levels are shown in the lower diagram. After reverse transcription of-mRNA, the cDNA samples were used for Q-PCR with Pias3 specific primers. The relative mRNA levels are shown in the lower diagram. They represent a ratio of the Ct values obtained by amplifying 18S rRNA in the same samples and were related to mRNA amounts detected in HepG2 cells. The experiment was performed in triplicate. Error bars represent +SD of the mean., depicts a Q-PCR analysis of P/AS3-mRNA in samples derived from virgin and pregnant mice or from mice at different stages after parturition. mRNA levels were related to the levels observed in breast cells of virgin mice. The experiment was performed in triplicate and error bars represent +SD of the mean. depicts a western blot analysis of Pias3 protein levels in extracts obtained from HepG2, 4T1 and Tu-9648 cells. The cells were incubated in the presence of MG-132 (50 μΜ) for 0, 1 and 4 hours before the protein extracts were prepared. To verify equal loading of the gels, the levels of actin expression were determined in the extracts with a specific antibody.

Mapping of the protein domains mediating the interaction between Pias3 and Stat3: The carboxyl-terminal acidic domain of Pias3 specifically recognizes the coiled-coil domain of Stat3.

depicts an interaction analysis of Stat3 and Stati with hPias3 and mPias3 in the yeast two hybrid system. Stat3 or Stati were fused into the Gal4 DBD bait constructs. Full-length hPias3, the two isoforms of mPias3 and the N- and C-terminal fragments of hPias3 shown in B were fused into the Gal4 TAD prey constructs. Yeast cells, transformed with the bait and prey plas- mids were grown overnight in selective medium and 10 fold serial dilutions of the cultures were prepared. 5 μΙ of each dilution was applied to plates with selection medium lacking tryptophan and leucine (-LT), tryptophan, leucine and histidine (-LTH) or medium lacking the three amino acids and containing 30 mM 3-aminotriazole (3-AT).

depicts a schematic presentation of the hPias3 protein and the subfragments used for the mapping of the interaction with Stat3 in yeast two hybrid experiments. The domain structure of the protein comprises: L-insert, amino acid position 80-121 ; PINIT domain, amino acid position 1 33-315; RING finger domain, amino acid position 327-369; acidic domain A, amino acid position 446-460; low-homology-domain LI , amino acid position 418-439 and L2, amino acid position 461 -494. The arrows indicate the positions of three cysteine residues present in the C and C8 fragments. Figure 3C) depicts a yeast two hybrid analysis with a Stat3-bait construct and the full length hPias3 or the hPias3 subfragments, shown in B, as prey constructs. The interaction strengths were determined in -galactosidase assays. Error bars represent +SD of the mean of nine independent measurements. Asterisks indicate p>0.1 compared to the interaction of Stat3 with hPias3. Figure 3D) depicts a domain structure of Stat3 showing the subfragments which were used to map the interaction with hPias3 in yeast two hybrid experiments. The numbers indicate the amino acid positions delimiting the functional domains of Stat3 used in the bait constructs. The yeast two hybrid analyses of the dif- ferent Stat3 domains as baits with different fragments of hPias3L are shown in the diagram below, -galactosidase assays were performed in triplicate and repeated at least three times. Error bars represent +SD of the mean. Asterisks indicate p values (* = p>0.1 , ** = p<0.05) of interactions compared to the results obtained for the interaction of full-length hPias3 with the coiled-coil domain of Stat3.

The carboxyl terminal fragments C5 and C8 of hPias3 interact with Stat3 in co-immunoprecipitation experiments.

depicts a coomassie blue stained gel and western blot analysis.The amino terminal fragment of hPias3, amino acid positions 9-326, (N), and the carboxyl terminal fragment of hPias3, amino acid positions 329-628, (C), were expressed as His-tagged recombinant proteins and purified by FPLC. The two purified fragments are shown on the left in a Coomassie blue stained gel. The recombinant proteins were incubated with 100 g of protein extracts obtained from Tu-9648 cell lysates for 4 h at 4°C. Stat3 was isloated from the protein mixture with Stat3 specific antibodies and protein A coated beads. The immunoabsorbed proteins were analyzed by Western blotting with a His-tag specific antibody. The C-terminal, but not the N-terminal Pias3 fragment interacts with Stat3 (right).

depicts a coomassie blue stained gel and western blot analysis. The carboxyl terminal hPias3 fragments C5, amino acid positions 474-543, and C8, amino acid positions 400-523, were expressed as His-tagged recombinant proteins and purified by FPLC. The two purified fragments are shown on the left in a Coomassie blue stained gel. The purified fragments were incubated with protein extracts from Tu-9648 cell lysates, Stat3 antibodies were added and the immune complexes were isolated on protein A coated beads. The immunoabsorbed proteins were analyzed by Western blotting with a His-tag specific antibody. Both hPias3 fragments, C5 and C8, interact with Stat3 (right). depicts a yeast two hybrid interaction analysis of a Stat3-bait construct with the C5 and C8 prey constructs. Serial dilutions of transformed yeast cells transformed with both constructs were grown on plates containing selection medium. The interaction of C8 with Stat3 allows the growth of the yeast cells under slightly more stringent conditions when compared to C5. The analysis of a p53-bait and a SV40 Large T antigen-prey construct served as a positive control.

Cellular uptake of the purified, recombinant carboxyl terminal hPias3 fragment, rPP-C8, via protein transduction and inhibition of Stat3 target gene expression.

depicts a schematic presentation of the transducible rPP-C8 peptide. rPP-C8 is fused to a protein transduction domain, PTD, comprising 9 arginines residues (9R) and two terminal His tags. In the Flag-rPP-C8 construct, the N- terminal His-tag has been replaced by a Flag-tag. This epitope binds a Flag specific antibody used to detect the protein by immunofluorescence. The cysteine residues present in rPP-C8 are indicated. Numbers above the constructs refer to the amino acid positions in hPias3.

depicts a western blot analysis. Therefore, purified His-tagged rPP-C8 peptides were added to the medium (1 μΜ) of growing 4T1 cells. Cytoplasmic and nuclear fractions of the cells were prepared at different times after rPP- C8 addition and the presence of rPP-C8 in these fractions was analyzed by western blotting. A His-tag specific antibody was used. The quality of the subcellular fractionations was confirmed by demonstrating the presence of lamin B1 in the nuclear fraction and β-tubulin in the cytoplasmic fractions. These proteins also served as controls for the equal loading of the lanes, depicts an immunofluorescence analysis of the intracellular localization of Flag-rPP-C8 in SK-BR-3 cells 4 hours after its addition to the medium. Before fixation of the cells, they were washed with 0.1 M acetic acid in PBS to remove peptides attached to the cell surface. Flag-rPP-C8 was detected with an antibody directed against the Flag epitope and an Alexa488 coupled secondary antibody. All cells show an even distribution of the peptide in the cytoplasm. In the nucleus the peptides accumulate in dotted structures. Nuclei were stained with DAPI. confocal laser scanning microscopy (CLSM) of SK-BR-3 cells stained with anti-Flag antibodies to visualize rPP-C8 in nuclear bodies, 4 hours after addition of Flag-rPP-C8 to the cells. Nuclei were stained with TO-PRO-3-iodide (Molecular Probes).

depicts the rPP-C8 effects on the mRNA expression of Stat3 target genes. MZ- 54 and Tu-9648 cells were pre-incubated with peptides for 24 hours. After that period, peptides were again added to the medium and the cells were harvested at the indicated time points. mRNA was isolated, reverse transcribed and the transcripts of target genes were amplified using specific primer sequences. The PCR products obtained were analyzed by agarose gel electrophoresis and the results of the analysis of MZ-54 cells are shown. The band intensities were quantified using the ImageQuant program (GE- Healthcare) and the value observed at 0 time was considered as 1 00%. The quantitation of the transcripts present in MZ-54 cells and Tu-9648 cells is shown below the gel bands.

depicts the rPP-C8 effects on the protein expression of Stat3 target genes. 4T1 cells were pre-treated for 48h with rPP-C8. After this period 2 μΜ rPP-C8 was added for the times indicated and protein extracts were prepared. The expression of the Stat3 target genes, BclxL, Survivin, and CyclinDI was detected with specific antibodies. The detection of actin was used to verify the equal loading of the gels. The results were quantified and the band intensities were measured. The signal intensity obtained at 0 time was considered as a 1 00%. The relative expression levels obtained are indicated below each blot, depicts the expression of Stat3 and P-Stat3 in Tu-9648 cells treated for 4 hours with rPP-C8. The levels of P-Stat3 and Stat3 were determined in nuclear and cytoplasmic extracts by western blotting of protein extracts with specific antibodies. Expression of Actin was determined to verify equal loading of the gel.

Treatment of Tu-2 49 glioblastoma cells with rPP-C8 inhibits their motility. . depicts a cellular migration assay, a.k.a. wound healing assay, with invasive Tu-2449 glioblastoma cells. Cells were grown for 2 days in the presence of 2 μΜ rPP-C8. The addition of PBS served as a negative control. The cell monolayer was scratched with a yellow pipette tip and photographed (0 time). The cells were incubated overnight with mitomycin c (10 pg/ml) to inhibit further proliferation of all cells. Additionally, cells were grown in the presence or absence of the peptides. After 1 6 hours the gaps between the cells were photographed again (20x magnification).

Figure 6B) depicts a cellular migration assay. The cells were treated for 2 days with rPP- C8 and then seeded into transwell inserts at a density of 1 x10⁶ cells/ml. They were grown overnight in the presence or absence of 2 μΜ peptides. After 16 h the percentage of cells that migrated to the bottom of the insert was deter- mined (* = p<0.1 , ** = p<0.05).

Figure 7: rPP-C8, but not rPP-C8 cys, effectively inhibits cell growth of Stat3 dependent cancer cells.

Figure 7A) 2 μΜ rPP-C8 was added to the medium of growing glioblastoma cells (Tu- 9648, Tu-2449, MZ-54), breast cancer cells (T47D, 4T1 ), immortalized normal cells (NIH-3T3, HC1 1 ) and normal primary cells (MEC, HUVEC) seeded in 6-well plates. The medium containing rPP-C8 was replaced every 24 hours for four days. Then the cells were photographed (right) at a 20x magnification and stained with crystal violet to visualize the overall cell density (left). Tu-9648, MZ-54 and 4T1 cells are sensitive to rPP-C8 growth inhibition, NIH-3T3 and HUVEC cells are not.

Figure 7B) Purification of rPP-C8 and rPP-C8 cys in which the three cysteine residues present at positions 406, 418 and 456 have been replaced by serines. The proteins shown in lanes 1 and 4 are fused to a Flag-tag and a nuclear local- ization signal. The proteins shown in lanes 2, 3 and 5 comprise two His-tags as indicated in Fig. 4A. .

Figure 7Q rPP-C8, but not rPP-C8 cys effectively inhibits the growth of cancer cells.

4T1 and Tu-9648 cells were grown in the absence or presence of rPP-C8 and rPP-C8 cys in 6 well plates for 72 hours. After that time the cell density was visualized by crystal violet staining.

Figure 7D) rPP-C8 binds P-Stat3 more effectively in a co-immunoprecipitation assay than rPP-C8 cys. Tu-9648 cell lysates were mixed with purified rPP-C8 or rPP-C8 cys and P-Stat3 was immunoprecipitated with a specific antibody. The presence of rPP-C8 and rPP-C8 cys in the immune complexes was determined by western blotting with a His-tag specific antibody.

Figure 8: Transduction of rPP-C8 inhibits proliferation and causes the induction of apoptosis of breast and glioma tumor cells, but does not affect normal fibroblasts.

Figure 8A) depicts SK-BR-3, Tu-2449, Tu-9648, MZ-54 and NIH-3T3 cells, 2000 cells each, seeded into 96-well plates and grown for 3 days. 1 , 2 or 4 μΜ of rPP- C8 were added to the medium and the medium was changed every 24 hours. Addition of PBS was used as a control. The proliferation of the cells was determined after 72 hours by XTT proliferation assays. The assays were performed in duplicate and repeated three times (** = p<0.05).

Figure 8B) 5000 Tu-9648 cells were seeded in 1 6-well E-plates (Roche) and grown for

24 h at 37°C. Then, the cell index in all wells (determined in a cell analyzer, Xcelligence, Roche) was normalized and set to 0. Different concentrations of rPP-C8 were added to the medium and proliferation of the cells was recorded as a function of the rPP-C8 concentrations by growing the cells for an additional 24 h in the cell analyzer. The EC₅₀ for the two peptides was calculated using the Graphpad Prism program (p = 0.0038).

Figure 8C) depicts morphologic changes induced in Tu-2449 and Tu-9648 cells upon exposure to rPP-C8. Cells were treated for 72 hours with rPP-C8 and photographed at a 40x magnification. Early and late apoptotic cells were observed.

Figure 8D) depicts apoptosis induction by rPP-C8 in Tu-9648, Tu-2449 cells and NIH- 3T3 cells measured by the detection of histone-complexed DNA fragments (mono- and oligonucleosomes) in the cytoplasm of rPP-C8 treated cells. The enrichment factor indicates the increase in cytoplasmic nucleosomes in peptide treated Tu-9648 and Tu-2449 cells as compared to PBS treated cells and NIH-3T3 cells, the control cell value is considered 1 . Assays were performed with the Cell Death Detection ELISA^PLUS kit (Roche) according to the instructions of the manufacturer (* = p>0.05, ** = p<0.05).

Figure 9: Nucleic and amino acid sequence of rPP-C8

Figure 10: Nucleic and amino acid sequence of rPP-C5

Figure 1 1 : Nucleic and amino acid sequence of Flag-rPP-C8

Figure 12: Combined chemotherapeutic treatment of HCT1 16 cells Figure 12A) depicts a schematic presentation of a combined chemotherapeutic treatment of HCT1 1 6 cells with the rPP-C8 peptide and irinotecan. The effects of ir- initecan on the colon carcinoma cell line HCT1 16 were investigated in the absence and in the presence of rPP-C8. The formation of cell colonies (clonogenic assays) in vitro has been measured as a read-out. A small number of HCT1 1 6 cells (about 500 -1000) has been treated with irinotecan (2.5 ng/ml) for a period of 8 to 10 days. Upon cessation of treatment the number of cell colonies has been counted and thus the resistant cells could be quan- titated. In order to measure the effects of the Stat3 inhibitory peptide rPP-C8 on the formation of resistent cells clones, the cells were treated 3 times, during the 8 to 10 day exposure period to irinotecan, with the rPP-C8 peptide. Subsequently, colony formation was measured. The number of colonies was compared with those formed by control cells not treated with rPP-C8 or control cells treated with a neutral peptide (human thioredoxin). rPP-C8 and human thioredoxin have been applied at a concentration of 1 μΜ. The number of colonies formed was compared to the number formed by cells treated with irinotecan only. The data show that the Stat3-inhibitory peptide rPP-C8 enhances the cytotoxic action of irinotecan.

Figure 12B) depicts the colony formation of cells treated with irinotecan only, as de- scribed above for Figure 12A).

Figure 12C) depicts the colony formation of cells treated with irinotecan and a conrol peptide (human thioredoxin), as described above for Figure 12A). Figure 12D) depicts the colony formation of cells treated with irinotecan and the rPP-C8 peptide, as described above for Figure 12A).

Figure 13: Combined chemotherapeutic treatment of HT29 cells

Figure 13A) depicts a schematic presentation of a combined chemotherapeutic treatment of HT29 cells with the rPP-C8 peptide and irinotecan. The effects of ir- initecan on the colon carcinoma cell line HCT1 16 were investigated in the absence and in the presence of rPP-C8. The formation of cell colonies (clonogenic assays) in vitro has been measured as a read-out. A small number of HT29 cells (about 500 -1000) has been treated with irinotecan (2.5 ng/ml) for a period of 8 to 10 days. Upon cessation of treatment the number of cell colonies has been counted and thus the resistant cells could be quan- titated. In order to measure the effects of the Stat3 inhibitory peptide rPP-C8 on the formation of resistent cells clones, the cells were treated 3 times, during the 8 to 10 day exposure period to irinotecan, with the rPP-C8 peptide. Subsequently, colony formation was measured. The number of colonies was compared with those formed by control cells not treated with rPP-C8 or control cells treated with a neutral peptide (human thioredoxin). rPP-C8 and human thioredoxin have been applied at a concentration of 1 μΜ. The number of colonies formed was compared to the number formed by cells treated with irinotecan only. The data show that the Stat3-inhibitory peptide rPP-C8 enhances the cytotoxic action of irinotecan.

Figure 13B) depicts the colony formation of cells treated with irinotecan only, as described above for Figure 12 A).

Figure 13C) depicts the colony formation of cells treated with irinotecan and a conrol peptide (human thioredoxin), as described above for Figure 12A).

Figure 13D) depicts the colony formation of cells treated with irinotecan and the rPP-C8 peptide, as described above for Figure 12A).

Examples:

The following Examples are only illustrative to the present invention and shall describe particular embodiments of the present invention in further detail. However, these Examples are not intended to limit the subject matter of the present invention thereto.

General Materials and Methods

Reagents

Recombinant IL-6, mitomycin C, anti-Actin and -Tubulin were obtained from Sigma (Hamburg, Germany). P-Stat3, CyclinDI , His-tag and cMyc antibodies were purchased form CellSignaling (Frankfurt, Germany). Anti-Survivin and rabbit anti-Pias3 were from Acris Antibodies (Herford, Germany); mouse-derived anti-Pias3 and anti-Lamin B1 were obtained from Abeam (Cambridge, MA, USA). Stat3 and Stat5 antibodies were from Santa Cruz (Heidelberg, Germany). MG-132 was purchased from Calbiochem (Darmstadt, Germany). Cell lines

Human low grade (Hs-683), high grade (MZ-54, MZ-18, U-87 and U-373) and murine high- grade glioblastoma cell lines (Tu-9648, Tu-2449) were kindly provided by Donat Kogel and Jakob Weissenberger (Neuroscience Center, Frankfurt, Germany) [43]. These cell lines were not further tested, nor authenticated. Human breast cancer cell lines (SK-BR-3, MDA-MB- 468, MCF-7) as well as the murine melanoma cell line B1 6 were purchased from ATCC. The human breast cancer cells (MDA-MB-231 , MDA-MB-453) and human prostate cancer cells (PC3) were kindly provided by Cord Hartman and Gert Carra (Georg Speyer Haus, Institute for Biomedical Research, Frankfurt, Germany). The murine breast cancer cell line 4T1 was kindly provided by Nancy Hynes (Friedrich Miescher Insitute, Basel, Switzerland). The MDA-MB and 4T1 cells were verified by morphology and EGFR and ErbB2 receptor expression pattern. Primary human umbilical vein endothelial cells (HUVEC) were purchased from ATCC at passage 2 and kindly provided by Carmen Dobele (Institute of Cardiovascular Regeneration, Frankfurt, Germany). B16 and PC3 cells were grown in RPMI medium, normal mouse HC1 1 breast cells were cultivated in RPMI containing 10 ng/ml insulin and 5 g/ml EGF and all other cancer cell lines were grown in DMEM.

Preparation of tissue lysates

Mouse organs or tumors were dissected and dissociated in 5 ml standard RIPA buffer/g tissue. Samples were incubated on ice for 20 min and sonified. After centrifugation (1 5,000 rpm, 20 min, 4 °C) supernatants were stored at -20°C. For western blot analysis, 100 pg of each sample was loaded on a polyacrylamide gel. Western blots were performed according to standard procedures.

Plasmid construction

The hPias3L gene was amplified from cDNA. mPias3 was amplified from plasmids p513- flag-mPias3-WT, p513-flag-mPias3L-VVT (Duval, D., et al., 2003) kindly provided by Helene Boeuf (Universite Bordeaux, France) using specific primers. To generate the N- and C- terminal fragments primers 5 '-aaacatatggtgatgagtttccg-3 ' (SEQ ID NO: 15) and 5'- aaactcgagcatgagtgacacccggag-3 ' (SEQ ID NO: 1 6) or 5 '-aaacatatgtgcccgctagggaagatg-3 ' (SEQ ID NO: 1 7) and 5'-aaactcgaggtccagggaaatgatgtc-3' (SEQ ID NO: 18) were used, respectively. The products were cloned into vector pGAD-T7 (Clontech, Mountain View, CA, USA). Additionally, smaller fragments of hPias3 were amplified and cloned in pGAD-T7 as well as in pET-30a(+) (Merck, Darmstadt, Germany) for bacterial expression. To construct the bait plasmids, the different Stat genes were amplified from cDNA samples. The fragments were cloned into pGBK-T7 (Clontech, Mountain View, CA, USA). To prevent auto- activation of the bait constructs, the C-terminal transactivation domain of the Stats is not included in any of the bait constructs.

Co-lmmunoprecipitation experiments

Cell lysates were prepared with NP40 lysis buffer and 100 pg protein was mixed with 50 pg purified Pias3 peptides. The mixtures were rotated for 4 h at 4°C. Specific antibodies (1 -2 pg) bound to 5 μΙ protein A coated magnetic beads (Invitrogen, Karlsruhe, Germany) were added overnight. The beads were washed three times with phosphate buffer and the bound complexes were released by boiling. Immuno complexes were analyzed in a western blot according to standard procedures. mRNA analysis

Total RNA was isolated from cell lysates using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and the Superscript II Reverse Transcriptase kit (Invitrogen, Karlsruhe, Germany) was used for synthesis of cDNA. Q-PCR amplification of Pias3 and 1 8S transcripts was performed using forward primer 5'-gcctacatggacatgtcctgt-3' (SEQ ID NO: 19) and reverse primer 5'-tcccccctggactgggctgtact-3' (SEQ ID NO: 20) or primers 5'- gggaggtagtgacgaaaaataacaat-3' (SEQ ID NO: 21 ) and 5'-ttgccctccaatggatcct-3' (SEQ ID NO: 22), respectively. Q- PCR was performed using SYBR Green I (Abgene, Hamburg, Germany) for labeling. The reactions were performed in triplicate. To calculate the relative mRNA levels, the Ct value obtained by amplification of 18S mRNA was used for normalization. To amplify transcripts of Stat3 target genes in human (h) and murine (m) cell lysates, following primers were used: hCyclinDI 5'-tggccatgaactagctgg-3' (SEQ ID NO: 23) and 5'- ttggggtccatgttctgc-3' (SEQ ID NO: 24), mCyclinDI 5'-tggaacctggccgccatg-3' (SEQ ID NO: 25) and 5'-gtggccttggggtcgacg-3' (SEQ ID NO: 26), hBcl_XL 5'-acaggactgaggccccag-3' (SEQ ID NO: 27) and 5'-gccacagtcatgcccgtc-3' (SEQ ID NO: 28), mBclxL 5'-agtttggatgcgcgggag- 3' (SEQ ID NO: 29) and 5'-gccacagtcatgcccgtc-3' (SEQ ID NO: 30), hSurvivin 5'- ccgacgttgccccctgcc-3' (SEQ ID NO: 31 ) and 5'-ctcgatggcacggcgcac-3' (SEQ ID NO: 32), mSurvivin 5'-tggcagctgtacctcaag-3' (SEQ ID NO: 33) and 5'-tcaagaattcactgacgg-3' (SEQ ID NO: 34), hActin 5'- gagatggcgacggccgcc-3' (SEQ ID NO: 35) and 5'-tccacatctgctggaagg-3' (SEQ ID NO: 36), mActin 5'-atggccactgccgcatcc-3' (SEQ ID NO: 37) and 5'- tccacatctgctggaagg-3' (SEQ ID NO: 38). PCR products were generated at 58°C annealing temperature and 20 cycles for semi-quantitative analysis.

Cell proliferation assays

2000 cells were seeded in 96-well plates and the next day, medium was removed. 1 -5 μΜ of the peptides (or PBS as solvent control) was diluted in 100 μΙ medium and added to the cells every 24 h. Proliferation of the cells was measured by adding 50 μΙ XTT solution (Roche, Mannheim, Germany). After 4 h the proliferation rate was determined in a plate reader at 490 nm.

To determine the EC₅₀ of the peptides, 5000 cells were seeded in 16-well E-plates and grown over night in a cell analyzer (xCELLigence System, Roche, Mannheim, Germany). Cell indices were measured every 10 minutes over 24 h. They were normalized and peptides at increasing concentrations were added and growth of the cells was monitored for an additional 24 h. Dose response curves were generated by normalizing the cell index values and plotting them in percent of maximum response versus peptide concentration. The EC₅₀ values were calculated using Graphpad Prism.

Protein purification

BL21 CodonPlus (DE3)-RP competent cells (Stratagene, Lajolla, CA, USA) were freshly trans- formed with recombinant expression plasmids (pET-Pias3-N, pET-Pias3-C, pET-Pias3-C5 or pET-Pias3-C8). Cells were grown overnight in 500 ml standard TB medium containing the appropriate antibiotics and 4% glycerol. The next day cells were harvested and lysed in 30 ml urea buffer (8 M urea, 500 mM NaCI, 1 x PBS, pH7.5) and sonified three times for 1 min on ice. Cell lysates were filtered and purified onto a HiTrap chelating HP column (GE Healthcare, Freiburg, Germany) by FPLC (GE Healthcare). After elution, the samples were dialysed in 400 mM arginine, 250 mM NaCI, 10% glycerol and 1 x PBS (pH 7.5) overnight. The next day dialysis buffer was exchanged four times (2h intervals) with 1 x PBS/10% glycerol pH 7.5 The desalted proteins were centrifuged (14.000 rpm, 20 min, 4°C) and stored at -20 °C (45).

Migration assay

For the wound healing experiments, cells were seeded at high densities in 6-well plates and grown for two days in the presence of peptides. The cell monolayer was scratched with a pipette tip, which was photographed. Additionally, mitomycin c (10 g/ml) was added to prevent further proliferation of the cells. The cells were incubated for 16 h and the scratches were photographed again and the number of cells that moved into the scratch was determined. For the transwell assays, 1 x 10⁶ cells/ml pretreated for 2 days with peptides were seeded into a transwell insert (Greiner bio-one, Solingen, Germany) without serum. Transwell inserts were placed in a 12-well plate containing 1 ml DMEM (with 0.2% BSA) and plates were incubated overnight at 37 °C. The next day, the number of cells at the bottom of the transwell was determined by counting or staining with crystal violet. Example 1 : Pias3 expression

1 .1 Pias3 is differentially expressed in mouse tissues

The expression pattern of Stat3 in different tissues has been analyzed in multiple studies, but only limited data are available on the expression of its endogenous inhibitor Pias3. Northern blot analysis, reverse transcription PCR analysis and immuno histochemistry (IHC) analyses have been performed (Chung et al., 1997; Duval et al., 2003; Ma et al., 2009; Wang and Banerjee, 2004). In this respect these studies were extended and western blot analysis of freshly prepared mouse tissue protein extracts were carried out. These studies were complemented by the analysis of latent Stat3 and activated P-Stat3 levels. Previous studies showed that Pias3 mRNA can be detected in nearly all tissues, but the present experiments indicate that the Pias3 protein is absent in the kidney, liver, heart and brain (Fig. 1 A). High Pias3 expression in the muscles and in involuted breast tissue and low expression in the spleen and the lungs was found (Fig. 1 A). A band of 68 kDa, which corresponds to the expected size of Pias3 was detected. In addition, a band of higher molecular weight in spleen and lung extracts was found as well. This band represents the 85 kDa sumoylated form of Pias3 (Wang and Banerjee, 2004; Nakagawa and Yokosawa, 2002). Interestingly, the expression of Pias3 largely corresponds to the activation pattern Stat3 in the same tissues. These data could indicate a coordinate control of Pias3 expression and P-Stat3 activation in normal tissues.

High levels of Pias3 in cells of the involuting mammary gland were found. The studies were extended and the expression of Stat3, activated P-Stat3 and Pias3 during different stages of the developmental cycle of the mammary gland was analyzed (Fig. 1 B). Stat3 is rather uni- formly expressed during all stages of development, but it becomes activated mainly in developing glands during puberty and pregnancy. P-Stat3 levels decrease during lactation and increase again during involution. The involution phase, is induced by cessation of suckling, and is accompanied by massive cellular apoptosis and the activation of Stat3 (Baxter et al., 2007). Pias3 is expressed in glandular cells of virgin mice, these levels decrease during pregnancy and lactation, but again increase during involution (Fig. 1 B). The Pias3 protein levels thus correlate with the P-Stat3 levels. These data confirm the coordinate regulation of Stat3 activation and Pias3 expression in the mammary gland and suggest that Stat3 activity is dynamically counteracted by the up-regulation of Pias3-expression particularly at the end of the involution process.

1 .2 Pias3 protein expression is lost in cancer cell lines in vitro and in vivo

A correlation between Pias3 expression and P-Stat3 levels in normal tissues was found.

Since Stat3 is highly activated in e.g. breast cancers and glioblastomas, Pias3 expression in these cancer cells was investigated by western blotting. Five of seven breast cancer cell lines are both positive for P-Stat3 and negative for Pias3 expression (Fig. 1 C). In accordance with data reported earlier, all of the glioblastoma cell lines express Pias3, in contrast to most primary glioblastoma tissues (Brantley et al., 2008). Brantley et al. suggested that there is a difference in the in vitro and in vivo systems. However, an inverse correlation for P-Stat3 and Pias3 expression was found. Very high levels of P-Stat3 in Tu-2449 and Tu-9648 cells and relatively low levels in the low-grade glioma cell line HS-683 were obtained. The high- grade murine glioblastoma cells expressed very low levels of Pias3, whereas, the HS-683 cells showed the highest Pias3 expression (Fig. 1 D). Thus in both cancer types, the levels of Pias3 expression are inversely correlated with P-Stat3 levels.

It was further investigated, if the Pias3 expression levels are influenced by the in vivo growth conditions. PC3, B1 6, U-87, 4T1 and SK-BR-3 cancer cells, expressing constitutively activated Stat3, were injected subcutaneously in mice. The tumors were allowed to grow until they reached a volume of 100-200 mm³ before the mice were sacrificed. Protein ex- tracts were prepared and the P-Stat3 levels in the tumor tissues were compared to those found when the cells were grown on culture plates (Fig. I E). P-Stat3 was detectable in all tumor samples derived from these cell lines. A higher Stat3 activation was generally found in the cells derived from tumor tissue. The Pias3 levels in the tumor tissue derived cells were also investigated and it was found that they do not express Pias3 (Fig. 1 F). Only in PC3 cell derived tumors, a weak band of 85 kDa, corresponding to sumoylated Pias3, was detected. Thus, differently from HepG2 cells and prostate cancer cells (Gross et al., 2001 ), Pias3 is absent in most breast cancer and glioblastoma cell lines grown in cell culture or as xenografts in mice. The protection afforded by Pias3 to normal tissues against detrimental effects of P-Stat3 seems to be lost in these cancer cells.

1 .3 PIAS3 mRNA is constantly expressed in breast tissues and breast cancer cell lines

Pias3 protein expression seems largely absent in cancer cells. The molecular level at which the down-regulation of Pias3 gene expression occurs and measured the PIAS3 mRNA in cancer cells by quantitative real-time PCR (Q-PCR) was analyzed (Fig. 2A). The results show that Pias3 mRNA is present and that the levels of Pias3 mRNA do not vary greatly (0.9 - 1 .4 fold) when breast cancer cell lines expressing Pias3 (e.g. MDA-MB-453) and lines expressing no detectable Pias3 protein were compared. Similar results have previously been ob- tained in the analysis of glioblastoma cells (Brantley et al., 2008).

It was observed that the expression of Pias3 protein in the mammary gland depends on the differentiation stage of the epithelial cells and is dependent on the stage of the cycle of pregnancy and lactation (Fig. 1 B). Pias3 mRNA levels during these stages were also ana- lyzed and it was found that PIAS3 mRNA remains relatively constant (between 0.7 and 1 .0, Fig. 2B). These data suggest that Pias3 gene expression is post-transcriptionally regulated in normal cells as well as in cancer cells.

A possible mechanism for the absence of Pias3 protein in tumor cells, even in the presence of normal mRNA levels, could be found in an enhanced degradation rate of the protein. This option was investigated and, therefore, HepG2, 4T1 and Tu-9648 cells in the presence or absence of the proteasome inhibitor MG-132 were incubated (Fig. 2C). In Pias3- expressing HepG2 cells, the PIAS3 mRNA is translated into Pias3 protein and the cellular concentration is not influenced by MG-132. In 4T1 cells and in Tu-9648 cells only very little Pias3 is found in the absence of MG-132 (Fig. 2C). The addition of MG-132 resulted in a slight increase of Pias3 in 4T1 cells and an appreciable increase in Pias3 in Tu-9648 cells. These data indicate that increased proteasomal degradation constitutes at least one mechanism by which tumor cells downregulate the expression of Pias3. Example 2: Delimitation of the Pias3 and Stat3 interaction domains

The absence of Pias3 in most cancer cells suggests that the loss of Stat3 inhibition might be a crucial mechanism to maintain the tumor cells. This implies that the development of a Stat3 inhibiting peptide derived from Pias3 could be a promising approach. To define such an inhibitor, the molecular interactions of Stat3 and Pias3 with the help of the yeast two hybrid system was analyzed. Sequences of Stat3 (bp 1 -2004) or Stati (bp 1 -2085) were fused to the GAL4-DNA binding domain (GBD) and Stat3 and Stati bait constructs were derived. The sequences of human hPias3, murine mPiasL (variant 1 ) and its shorter variant 2 lacking the L-insert (mPias3) were fused to the GAL4-transactivation domain (GAD) and the hPias3, mPias3L and mPias3 prey constructs were obtained. Yeast cells were transformed with the bait and prey constructs and double transformants were grown on plates lacking leucine, tryptophane and histidine (-LTH), or on -LTH plates with 30 mM 3-AT. This selec- tive growth medium is used to select for protein interactions under stringent conditions. The growth of the yeast cells indicated strong interactions between Stat3 and hPias3 or mPias3L and a weaker interaction between Stat3 and mPias3 (Fig. 3A). hPias3 does not interact with the highly homologous Stati protein in which 52% of the amino acids are identical to those found in Stat3.

Similar to Stati and Stat3, Piasl and Pias3 exhibit a high sequence homology. It has been shown previously that Stati interacts with the C-terminal of hPiasl , comprising amino acid positions 392-541 . It was therefore reasonable to postulate that Pias3 and Stat3 might interact through similar domains. However, a previous report showed that an N-terminal frag- ment of mPias3, comprising amino acid positions 82-132, interacts with Stat3 in murine cell lines (Yagil et al., 2009; Sonnenblick et al., 2004). A yeast two hybrid analyses with the N- terminal, amino acid positions 1 -319, and the C-terminal, amino acid positions 320-619, fragments of hPias3 was perforemd. These fragments were integrated in prey constructs and Stat3 served in a bait construct (Fig. 3A-C). The yeast growth analysis indicated that Stat3 is not able to interact with the N-terminal fragment of hPias3 nor of mPias3. Instead, the results clearly show that Stat3 interacts with the C-terminal fragment of hPias3 (Fig. 3C). Therefore, it is concluded that a strong Stat3 -interacting motif is present in the C-terminal fragment of hPias3. Piasl and Pias3 are highly homologous in their N-terminal domains, but differ strongly in their C-terminal sequences. These differences might determine the specificities of their interactions with different Stats. The shortest fragment of Piasl , previously shown to interact with Statl , is located between amino acid positions 392-541 (Liao et al., 2000). In the corresponding region of Pias3, two short sequences of 15 amino acids with low homologies to Piasl (L1 : amino acid position 418-439 and L2: amino acid position 461 -494) flanking an acidic domain were found (Fig. 3B). This region of hPias3 was further investigated. A prey construct, comprising amino acids 400-523 and spanning the L1 , A and L2 domains (named C8) was derived. In a second prey construct, the GAD was fused to a fragment comprising the L2 domain, amino acids 474 to 543 (named C5). Fig. 3C and D show that the hPias3-C8 fragment and the overlapping hPias3-C5 construct both mediate strong interactions with Stat3. The domain within Stat3 which is being recognized by Pias3 was also defined. For this purpose four functional domains of Stat3 were fused to the GBD and used as bait constructs (Fig. 3D), the N-terminal domain (NID), the coiled-coil domain (CC), the DNA binding domain (DBD) and the dimerization domain (SH2). The Stat3 bait and the Pias3 prey constructs were introduced into yeast cells and analyzed in two hybrid experiments. All frag- ments derived from the C-terminal region of Pias3 (Pias3-C, -C5 and -C8), but not the fragments derived from the N-terminus, strongly interact with the coiled-coil domain of Stat3. There is no or only significantly less interaction with the other Stat3 domains (Fig. 3D). This result is not necessarily expected. It has previously been suggested that Pias3 might inhibit Stat3 through interference with its DNA-binding function (Chung et al., 1997). However, these results surprisingly show that Pias3 does not recognize the DNA binding domain of Stat3.

The experiments were verified by biochemical interaction studies with recombinant proteins. The N-terminal (amino acids 9-326) and C- (amino acids 329-628) terminal Pias3 fragments were fused to a His tag and cloned into bacterial expression vectors. The proteins were purified from bacterial lysates by FPLC (Fig. 4A). They were added to Tu-9648 cell lysates and co-immunoprecipitation experiments were performed with a Stat3 specific antibody. The C-terminal fragment, but not the N-terminal fragment of Pias3 is able to bind to Stat3 (Fig. 4A). These results confirm the yeast two hybrid analyses. Similar experiments were carried out with recombinantly expressed and purified Pias3-C5 and -C8 peptides, named rPP-C5 and rPP-C8. It was found that both rPP-C5 and -C8 are able to bind to Stat3, rPP-C8 slightly better than rPP-C5 (Fig. 4B). These differences in binding strengths were con- firmed in additional yeast two hybrid analyses. The ability of the yeast cells expressing the Pias3-C8 fragment to grow under more stringent selection conditions, i.e. on -LTA plates, shows that the Pias3-C8 fragment binds with higher affinity to Stat3 when compared to the Pias3-C5 fragment (Fig. 4C). Example 3: Expression, purification and cellular delivery of rPP-C8

The expression of the Pias3 gene in tumor cells was shown to cause the suppression of Stat3 function. These experiments were based on gene transfer methods. It was investigated, if the rPP-C8 peptide could be used to elicit a similar effect upon introduction into cancer cells. Since peptides do not readily enter cells, rPP-C8 was fused to a protein transduction domain. This domain is comprised of a homopolymeric stretch of 9 arginines and promotes the uptake of its cargo (Kotaja et al., 2002; Hetschko et al., 2008). Two His-tags were added for the affinity purification of the recombinant products (Fig. 5A). The purified peptides were found to be rather stable, when incubated for prolonged periods, up to 40 hours, in PBS, DMEM or cell lysates at 37° (data not shown). This indicates that the peptides might be suited for cellular transduction experiments.

Tumor cell lines were exposed to purified rPP-C8 for increasing periods of time and the uptake into cells was monitored. 4T1 cells were treated by addition of 1 μΜ rPP-C8 to the growth medium, cellular subfractions were prepared and the presence of rPP-C8 was analyzed in cytoplasmic and nuclear protein extracts by western blotting. The presence of rPP- C8 in the growth medium was also investigated. The peptides slowly decline in the medium over a period of about 4 hours and accumulate in the cytoplasm and the nucleus. They intracellular levels reach a maximum after about 2 hours and then decrease again (Fig. 5B). Similar results were obtained with Tu-9648, SK-BR-3 and NIH-3T3 cells. The uptake of rPP- C8 was also visualized by immunofluoresence microscopy. SK-BR-3 cells were treated for 4 hours with Flag-rPP-C8. This variant contains a Flag epitope and can thus be detected by immunoflourescence (Fig. 5A). An uptake of the peptide into the cytoplasm and nucleus of all cells was observed (Fig. 5C). Confocal laser scanning microscopy (CLSM) provided a higher resolution and it was found that the peptides accumulate in sub-nuclear dotted structures (Fig. 5D). This observation coincides with previous reports which showed that Pias3 accumulates in dot structures, also called nuclear granules (Kotaja et al., 2002; Rodel et al., 2000). The experiments show that the recombinantly expressed and purified rPP-C8 peptides quickly enter cells upon addition to the medium. Since the intracellular levels peaked at 2 to 4 hours, rPP-C8 was regularly replenished in experiments in which cells were treated for prolonged time periods. Example 4: Transduction of rPP-C8 inhibits StaO target gene expression

Stat3 regulates the expression of target genes involved in proliferation and survival, migration and angiogenesis. If the transduced peptides are able to bind to Stat3 and possibly sequester it to sub-nuclear sites, they also have the potential to inhibit Stat3 target gene ex- pression. Therefore, the immediate effects of the peptides on the expression level of two anti-apoptotic target genes of Stat3, Bcl_XL and Survivin and of two genes regulating proliferation, Cyclin D1 and c-Myc. MZ-54, Tu-9648 and 4T1 tumor cells were treated for 24 hours with rPP-C8 was analyzed. After this time period, fresh peptides were added to the medium. RNA was prepared from the cells at different time points after the second addition of rPP- C8. Transcript levels of the selected Stat3 target genes were determined by RT-PCR (Fig. 5E). The down-regulation of the Survivin, Bcl_XL and Cyclin D1 transcripts was observed within 4 hours in MZ-54 and Tu-9648 cells. The levels of protein expression of these targets as a function of rPP-C8 exposure were also monitored. 4T1 cells were treated for 48 hours with the peptides. After that time, the rPP-C8 was replenished and protein extracts were prepared at different times after addition (Fig. 5F). Treatment of 4T1 cells with rPP-C8 caused a strong reduction in the levels of Bcl_XL, Survivin and Cyclin D1 within 40 to 240 minutes. Similar decreases were found in Tu-9648 cells (not shown). Exposure of the cells to rPP-C8 did not cause a reduction in the level of c-Myc within the 4 hour observation period. The inhibition of Bcl_XL, Survivin and Cyclin D1 gene expression shows that the uptake of rPP-C8 regulates relevant proliferation and cell-cycle functions in cancer cells. It was investigated, if these effects are mediated through the regulation of Stat3 or P-Stat3 levels in the transduced cells. Increasing concentrations of rPP-C8 were added to the medium of Tu- 9648 cells and nuclear and cytoplasmic protein extracts were prepared. Fig. 5G shows that the levels of Stat3 and P-Stat3 remain rather stable and are not affected by the exposure of the cells to rPP-C8. It was concluded that the effects of rPP-C8 on target gene expression are caused by the functional inhibition of P-Stat3, possibly by subnuclear sequestration into dotted structures, and not by interference with tyrosine phosphorylation.

Example 5: Transduction of rPP-C8 inhibits migration of glioblastoma cells.

The phenotypic consequences for cancer cells, treated for two days with the Pias3-derived peptides, and initially focused on cell motility were investigated. One major characteristic of glioblastomas is their propensity to infiltrate surrounding normal tissues; this is a major obstacle for their surgical removal. The interference with the migratory potential of glioblastoma cells might therefore be of clinical significance. The Stat3 target genes MMP-2 and MMP-9 are suspected to be involved in the regulation of migration and invasion. Therefore the effects of rPP-C8 on glioblastoma cell migration were analyzed. Tu-2449, Tu-9648 and MZ-54 cells were grown for 2 days in the presence of the peptide. On the third day, the monolayer was scratched with a pipette tip and photographed. The extent to which the cells were able to migrate into the scratch was examined 16 hours later. The control cells were able to fill the scratch within this time period. 70% less cells were found in the gap area after rPP-C8 treatment (Fig. 6A). Additional migration assays were performed by seeding 5 x 10⁵ treated or non-treated Tu-2449, Tu9648 and MZ-54 cells in a transwell insert. MCF-7 cells, which are not able to migrate, were used as a negative control. After 16 hours the number of cells that migrated to the bottom surface of the membrane was counted (Fig. 6B). The rPP-C8 peptide clearly inhibited (2-4 fold) the migration of the three different glioblas- toma cell lines.

Example 6: Transduction of rPP-C8, but not rPP-C8 cys, affects cancer cell growth

The migration assays described above were carried out after treatment of the cells for 2 to 3 days. The exposure time was prolonged to 3 to 5 days and additional cellular phenotypes were observed. The Tu-9648, Tu-2449, MZ-54 and 4T1 cancer cell lines were seeded at low density and treated once a day with 2 μΜ of rPP-C8. After 5 days they were photographed to analyze their morphology and stained with crystal violet to visualize the overall cell density in the wells (Fig. 7 A). The PBS treated control cells were confluent after 5 days. Treatment of the glioblastoma and breast cancer cells with the peptides strongly reduced the cell density. The remaining cells displayed a rounded morphology or were detached from the plate. In contrast, the growth of normal primary cells (MEC; murine mammary epithelial cells and HUVEC; human endothelial cells) and of normal immortalized cells (NIH-3T3; fibroblasts and HC1 1 ; mammary epithelial cells) was largely unaffected (Fig. 7 A). These cells are not P-Stat3 -dependent. T47D cells which express Pias3 were also not affected. The differential sensitivity of the cell lines indicates that rPP-C8 is not generally cytotoxic, but selectively affects the growth and survival of cells which constitutively express activated Stat3 in the absence of endogenous Pias3.

It was reported earlier, that aggregation of recombinant proteins, attached to a protein transduction domain, strongly affects their cellular uptake and impedes the intracellular inhibition by peptide aptamers (Borghouts et al., 2008). Aggregation can be caused by the inter- molecular crosslinking and the formation of disulfide bridges. rPP-C8 contains three cysteine residues and partial aggregation of rPP-C8 during its purification was observed. It was investigated if its efficacy could be further improved by replacing the cysteine residues by serines through site directed mutagenesis. The mutated variant, rPP-C8 cys, was found to be highly soluble and could be purified in a monomeric form (Fig. 7B). However, when rPP- C8 cys was added to 4T1 cells or Tu-9648 cells, no inhibition of cell growth was observed (Fig. 7C). Although the peptides devoid of cysteines entered the cells more efficiently than the parental peptides, they were not able to affect growth of the cells. This indicates that at least one of the cysteines in rPP-C8 is required for the binding to Stat3. This conclusion was confirmed by co-immunoprecipitation experiments (Fig. 7D).

Example 7: Transduction of rPP-C8 inhibits proliferation and induces apoptosis in cancer cells. rPP-C8 suppresses the expression of Stat3 target genes involved in cellular growth (Fig. 5E-F) and consequently reduces cell density (Fig. 7A). The proliferation of the pepti de-treated cancer cells in XTT assays as a function of time was measured. SK-BR-3, 4T1 , Tu-2449, Tu- 9648, MZ-54 cancer cells and NIH-3T3 control cells were treated with 1 , 2 or 4 μΜ of rPP- C8 every 24 h for three days (Fig. 8A). The proliferation of the cancer cells was reduced by 70 to 80% upon addition of 4 μΜ rPP-C8 when compared to the PBS treated cells. 4T1 cell growth was reduced to 30% by treatment of the cells for four days with 2 μΜ rPP-C8 (not shown) when compared to control cells. The growth of NIH-3T3 cells remained largely unaffected by rPP-C8, even at the highest concentrations applied. This confirms the results shown in Fig. 7 A.

The effects of rPP-C8 exposure on Tu-9648 cell proliferation were measured in a cell analyzer (Xcelligence, Roche) in a concentration dependent manner. The cells were seeded in E-plates and grown for 24 hours. Then, the proliferation rates were normalized and increas- ing concentrations, between 0.1 μΜ to 10 μΜ, of the peptides were added for an additional 24 hours (Fig. 8B). The proliferation rates of the cells in different wells were dependent on the concentration of the peptides in the medium as shown by the sigmoid shape of the response curve. An EC₅₀ value of 2.7 μΜ for rPP-C8 in Tu-9648 glioblastoma cells was obtained.

Addition of rPP-C8 in the medium of Stat3-dependent cancer cells reduced the expression of the anti-apoptotic target genes Survivin and Bcl_xL. Treatment of the cells also affected their morphology and signs of apoptosis were observed (Fig. 8C). Therefore, the induction of apoptosis as a function of rPP-C8 exposure was quantitated. The apoptotic process is char- acterized by nuclear DNA fragmentation resulting in the release of nucleosomes into the cytoplasm. The presence of nucleosomes in the cytoplasm of Tu-9648, Tu-2449 and NIH- 3T3 cells after 24 or 48 hours of peptide treatment was determined by an ELISA assay. There is a clear increase (2 to 8 fold) in the number of apoptotic Tu-9648 and Tu-2449 cells when compared to PBS treated cells or the NIH-3T3 cells, used as controls (Fig. 8D).

These experiments show that rPP-C8, the fragment of Pias3 identified in the yeast two hybrid assay, can bind Stat3 specifically and efficiently in vitro. Fused to a PTD, rPP-C8 can be expressed as a recombinant protein. This protein is efficiently taken-up by cells, binds in- tracellularly to Stat3 and inhibits transactivation by P-Stat3. The peptide derived from the C- terminal acidic region of Pias3 acts as potent Stat3 inhibitor.

Example 8: Use of rPP-C8 in combination therapy with chemotherapeutic agents Treatment of colorectal carcinoma cells with the Stat3 inhibitor rPP-C8 and irinotecan.

The cytostatic drug irinotecan inhibits the activity of the enzyme topoisomerase I und thus induces DNA damage during the process of cell division. This causes death of rapidly divid- ing cells. The treatment of colon carcinoma cells with irinotecan has shown that the activation of Stat3 is able to counteract the effects of irinotecan and that the cells survive the treatment through a Stat3 regulated DNA repair process (Courapied S, Sellier H, de Carne Trecesson S, Vigneron A, Bernard AC, Gamelin E, Barre B, Coqueret O; J. Biol. Chem. 2010, 285:26765-78; "The cdk5 kinase regulates the STAT3 transcription factor to prevent DNA damage upon topoisomerase I inhibition").

The effects of irinitecan on colon carcinoma cell lines were investigated in the absence and in the presence of rPP-C8. The formation of cell colonies (clonogenic assays) in vitro has been measured as a read-out. A small number of HT29 (Figure 13) und HCT1 1 6 cells (Fig- ure 12) (about 500 -1000) has been treated with irinotecan (2.5 ng/ml) for a period of 8 to 10 days. Upon cessation of treatment the number of cell colonies has been counted and thus the resistant cells could be quantitated.

In order to measure the effects of the Stat3 inhibitory peptide rPP-C8 on the formation of resistent cells clones, the cells were treated 3 times, during the 8 to 10 day exposure period to irinotecan, with the rPP-C8 peptide. Subsequently, colony formation was measured. The number of colonies was compared with those formed by control cells not treated with rPP- C8 or control cells treated with a neutral peptide (human thioredoxin). rPP-C8 and human thioredoxin have been applied at a concentration of 1 μΜ. The number of colonies formed was compared to the number formed by cells treated with irinotecan only. The data show that the Stat3-inhibitory peptide rPP-C8 enhances the cytotoxic action of irinotecan.

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Items

The following items illustrate specific embodiments of the present invention:

1 . A (poly)peptide comprising a sequence of at least 10 contiguous amino acids selected from a basic amino acid sequence selected from

c) SEQ ID NO: 2, or

d) an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 2,

2. (Poly)peptide according to item 1 , comprising a sequence of about 10 to about 300 contiguous amino acids selected from a basic amino acid sequence selected from c) SEQ ID NO: 2, or

d) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 2,

wherein the entire (poly)peptide has a length of about 10 to about 500 amino acids. (Poly)peptide according to item 2, comprising an amino acid sequence selected from

c) SEQ ID NO: 3, or

d) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 3. (Poly)peptide according to item 2, comprising an amino acid sequence selected from

f) SEQ ID NO: 4,

g) SEQ ID NO: 5,

h) SEQ ID NO: 6, or

i) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. (Poly)peptide according to any of items 1 to 4, further comprising a transduction domain, a signaling domain, and/or a scaffold protein. (Poly)peptide according to any of items 1 to 5, comprising an amino acid sequence selected from

a) SEQ ID NO: 8,

b) SEQ ID NO: 9,

c) SEQ ID NO: 10, or

d) a sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequences according to SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 1 0.

7. (Poly)peptide according to any of items 1 to 6, wherein the (poly)peptide is capable of inhibiting Stat3 signalling, preferably by interacting with SEQ ID NO: 7, wherein the interaction preferably is characterized by an autonomous binding to an amino acid sequence of SEQ ID NO: 7, or to an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with SEQ ID NO: 7.

8. A nucleic acid comprising a nucleic acid sequence encoding a (poly)peptide according to any of items 1 to 7. 9. A nucleic acid hybridizing under stringent conditions with the nucleic acid according to item 8.

10. A recombinant vector comprising the nucleic acid according to item 8 or 9, wherein the recombinant vector is preferably a recombinant expression vector.

1 1 . A host cell comprising a nucleic acid or vector according to any of items 8 to 1 0.

1 2. A (poly)peptide according to any of items 1 to 7, a nucleic acid or vector according to any of items 8 to 1 0 for use in medicine.

1 3. A (poly)peptide according to any of items 1 to 7, a nucleic acid or vector according to any of items 8 to 1 0 for use in the treatment or prevention of a disease, of symptoms of a disease or causes for a disease connected to the transcription factor Stat3.

14. (Poly)peptide, nucleic acid or vector according to item 1 3, characterized in that the disease is cancer, an auto-immuno-disease, chronic inflammation, psoriasis, a liver disease, preferably cancer. A pharmaceutical composition comprising a (poly)peptide according to any of items 1 to 7, a nucleic acid or vector according to any of items 8 to 10 and optionally a pharmaceutically acceptable carrier, adjuvant, and/or vehicle. A targeted (poly)peptide consisting of a maximum of 700 contiguous amino acids comprising an amino acid sequence selected from

c) SEQ ID NO: 7 , or

d) an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with the sequence according to SEQ ID NO: 7. A method for measuring the binding affinity of a molecule to the targeted (poly)peptide according to item 1 6 comprising the steps of (a) bringing the molecule in contact with the targeted (poly)peptide according to item 1 6 and (b) measuring the affinity of the molecule to the targeted (poly)peptide according to item 1 6. A screening method for the identification of a molecule for the treatment of cancer, wherein the molecule is identified by its binding affinity to the targeted (poly)peptide according to item 16 comprising the steps of (a) bringing the molecule in contact with the targeted (poly)peptide according to item 1 6 and (b) measuring the affinity of the molecule to the targeted (poly)peptide according to item 1 6.

Claims

A (poly)peptide comprising:

• optionally, a sequence (I),

• a sequence (II) of at least 10 contiguous amino acids selected from an amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 2, and

• optionally, a sequence (III),

• with the proviso that the (poly)peptide does not comprise an amino acid sequence (I) comprising a sequence located N-terminally from sequence (II) and represented by SEQ ID NO: 76, or having identity of more than about 99%, or of more than about 95%, or of more than about 90%, or of more than about 80% with the amino acids of the sequence represented by SEQ ID NO: 76, in case sequence (II) comprises the amino acid sequence represented by SEQ ID NO: 77.

(Poly)peptide according to claim 1 , wherein the sequence (II) is at least 1 5, at least 20, at least 30, at least 40, or at least 50 contiguous amino acids in length.

(Poly)peptide according to any one of claims 1 or 2, wherein the sequence (II) of at least 10 contiguous amino acids is selected from the amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 5.

4. (Poly)peptide according to any one of claims 1 to 3, wherein the sequence (II) is represented by the amino acid sequence of SEQ ID NO: 5, or a sequence having identity of at least about 80%, preferably of at least about 90% even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequence represented by SEQ ID NO: 5.

5. (Poly)peptide according to any one of claims 1 to 3, wherein the sequence (II) is represented by the amino acid sequence of any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75, or a sequence having identity of at least about 80%, preferably of at least about 90% even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequences according to any one of SEQ ID NOs: 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74 or 75.

6. (Poly)peptide according to any one of claims 3 to 5, with the further proviso that the (poly)peptide does not comprise an amino acid sequence (III) comprising a sequence located C-terminally from sequence (II) and represented by SEQ ID NO: 78, or having identity of more than about 99%, or of more than about 95%, or of more than about 90%, or of more than 80% with the amino acids represented by SEQ ID NO: 78, in case sequence (II) comprises the amino acid sequence represented by SEQ ID NO: 55; and/or the (poly)peptide does not comprise an amino acid sequence (I) comprising a sequence located N-terminally from sequence (II) and represented by SEQ ID NO: 79, or having identity of more than about 99%, or of more than about 95%, or of more than about 90%, or of more than about 80% with the amino acids represented by SEQ ID NO: 79, in case (II) comprises the amino acid sequence represented by SEQ ID NO: 40.

7. Polypeptide according to any of claims 1 to 6, further comprising a transduction domain, a signaling domain, and/or a scaffold protein.

8. (Poly)peptide according to any of claims 1 to 7, wherein the (poly)peptide comprises the amino acid sequence of any one of SEQ ID NO: 8, SEQ ID NO: 10, or a sequence having identity of at least about 80%, preferably of at least about 90%, even more preferably of at least about 95%, and most preferably identity of at least about 99% with the sequences according to SEQ ID NO: 8, or SEQ ID NO: 10.

9. (Poly)peptide according to any of claims 1 to 8, wherein the (poly)peptide is capable of inhibiting Stat3 signalling, preferably by interacting with SEQ ID NO: 7, wherein the interaction preferably is characterized by an autonomous binding to an amino acid sequence of SEQ ID NO: 7, or to an amino acid sequence having an identity of at least about 60%, preferably of at least about 70% or about 80%, even more preferably of at least about 90% or about 95%, and most preferably an identity of at least about 99% with SEQ ID NO: 7.

10. A nucleic acid comprising a nucleic acid sequence encoding a (poly)peptide according to any of claims 1 to 9.

1 1 . A nucleic acid hybridizing under stringent conditions with the nucleic acid according to claim 10.

12. A recombinant vector comprising the nucleic acid according to claim 10 or 1 1 , wherein the recombinant vector is preferably a recombinant expression vector.

13. A host cell comprising a nucleic acid or vector according to any of claims 10 to 12.

14. A (poly)peptide according to any of claims 1 to 9, a nucleic acid or vector according to any of claims 10 to 12 for use in medicine.

15. A (poly)peptide according to any of claims 1 to 9, a nucleic acid or vector according to any of claims 10 to 12 for use in the treatment or prevention of a disease, of symptoms of a disease or causes for a disease connected to the transcription factor Stat3.

16. (Poly)peptide, nucleic acid or vector for use according to claim 15, characterized in that the disease is cancer, an auto-immuno-disease, chronic inflammation, psoriasis, a liver disease, preferably cancer.

1 7. (Poly)peptide, nucleic acid or vector for use according to any one of claims 15 or

16, wherein the (poly)peptide, nucleic acid or vector is used together with a che- motherapeutic agent.

18. (Poly) peptide, nucleic acid or vector for use according to any one of claims 15 to

1 7, wherein the (poly)peptide, nucleic acid or vector is used as a sensitizer.

19. A pharmaceutical composition comprising a (poly)peptide according to any of claims 1 to 9, a nucleic acid or vector according to any of claims 10 to 12 and optionally a pharmaceutically acceptable carrier, adjuvant, and/or vehicle.

20. Pharmaceutical composition according to claim 19, additionally comp

chemotherapeutic agent.