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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
  • C12Y207/04006—Nucleoside-diphosphate kinase (2.7.4.6)
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  • C12Y207/10—Protein-tyrosine kinases (2.7.10)
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  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
  • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/11—Antisense
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• the present invention relates to novel target structures and modulators thereof for the prevention, diagnosis, prognosis and treatment of hyperproliferative and/or metastatic disorders, in particular cancer diseases.
• the novel targets are useful for the identification, characterization and development of novel effective anti-tumor drugs.
• the present invention relates to compositions comprising agents suitable for anticancer therapy and to novel approaches for combating hyperproliferative and/or metastatic diseases.
• Protein kinases e.g. tyrosine kinases
• tyrosine kinases are part of intracellular signal cascades which regulate cell division, differentiation and/or apoptosis and thus are involved directly or indirectly in the control of cell proliferation.
• tyrosine kinases are involved in the transmission of growth signals from receptors on the cell surface to the nucleus, where the cell division is started.
• These signal cascades are often disturbed in hyperproliferating cells such as cancer cells, for example due to mutations in the sequence of tyrosine kinase encoding genes involved in the cell cycle regulation.
• genes encoding tyrosine kinases which are involved in the intracellular transmission of growth signals and the expression of which is altered in hyperproliferating cells belong to the group of proto-oncogenes.
• ROS1 V-ros UR2 sarcoma virus oncogene homolog 1
• ROS1 belongs to the "Sevenless" subfamily of tyrosine kinase insulin receptor genes.
• the protein encoded by this gene is a type 1 integral membrane protein with tyrosine kinase activity. Studies have shown that ROS1 protein may function as a growth or differentiation factor receptor.
• ROS1 was also identified (among numerous other kinases) as a cancer-related protein kinase (WO 2008/066489).
• the roles of the receptor tyrosine kinase ROS in development and cancer is described byAcquaviva et al. (Biochem. Biophys. Act. 1795 (2009), 37-52).
• Evidence for increased expression of the ROS oncogene in primary human gliomas was found by Watkins et al. (Cancer Genet. Cytogenet. 72 (1994), 130-136).
• Sunitinib also known as SU11248, which is the active ingredient of the therapeutic agent Sutent ® is an orally administered, small molecule, multi- targeted receptor tyrosine kinase inhibitor for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumor (GIST).
• RRC renal cell carcinoma
• GIST imatinib-resistant gastrointestinal stromal tumor
• Sunitinib was the first cancer drug simultaneously approved for two different indications and has become a standard of care for both of these cancers.
• Administration of SU11248 inhibits intracellular signal transmission of multiple receptor tyrosine kinases leading to both reduced tumor vascularization and cancer cell death, and ultimately tumor shrinkage. In some cases, however, administration of SU11248 has been less effective.
• the present inventors have identified specific polypeptides as novel targets of SU11248. Binding of SU11248 to the these polypeptides in a cell modulates, e.g. inhibits cell division, invasion and migration or reduces the proliferation rate of a number of cancer cell lines and tumors. Additionally, an interaction between SU11248 and said polypeptides leads to an inhibition of the activity of said polypeptides which induces apoptosis. Further, it was shown for the first time that these novel targets of SU11248 are expressed in different cell lines in a rate which is characteristic for a specific cell species or tissue and in particular characteristic for a specific cancer cell type. Therefore, polypeptides with a binding affinity to SU11248 or genes encoding said polypeptides are excellent targets for
• the present invention provides target structures, which may be modulated for therapy and/or diagnosis of a hyperproliferative disorder.
• the target structures according to the present invention may be selected from
• hyperproliferating cells e.g. in an amount which is different from that of the corresponding non- proliferating (wild-type; wt) or normal cells, and
• polypeptides may be identified by affinity chromatography of cell extracts using an SU11248 molecule which is coupled to a solid phase, e.g. a Sepharose material.
• the coupling is preferably effected by a linker.
• Polypeptides identified by this procedure may be targets for therapeutic and/or diagnostic methods in conditions associated with dysfunctional cell proliferation.
• the target polypeptide may be a protein involved in the control of the cell cycle and/or apoptosis.
• the target polypeptide may be a protein with enzymatic activity, e.g. a kinase, as for example a tyrosine kinase.
• a kinase as for example a tyrosine kinase.
• the target polypeptide is a human protein.
• the target polypeptide is a human protein with a tyrosine kinase activity.
• the target polypeptide is selected from ROS1 as defined in SEQ ID NO:1 , BMP2k as defined in SEQ ID NO:2 or 3, NEK9 as defined in SEQ ID NO:4, NME4 as defined in SEQ ID NO:5 or TBK1 as defined in SEQ ID NO:6 or allelic variants and homologs thereof having at least 70-80%, preferably at least 80-90%, more preferably at least 90-95%, and most preferably at least 95- 99.9% or more identity with the sequences of SEQ ID NO:1 , 2, 3, 4, 5 or 6 over the entire length, provided that said allelic variants and homologs exhibit the characteristics of a target polypeptide as defined above.
• the target polypeptide is ROS1 as defined in SEQ ID NO:1.
• the target structures according to the invention may be further selected from nucleic acids encoding at least one target polypeptide as described above.
• the target nucleic acid of the invention may be an endogenous gene or a transcript resulting from said gene.
• the target nucleic acid may be in the form of DNA, RNA or DNA RNA hybrid and comprises an open reading frame encoding the primary structure of at least one target polypeptide as described above.
• the target nucleic acid encodes a target polypeptide selected from ROS1 (SEQ ID NO:1 ), BMP2k (SEQ ID NO:2 or 3), NEK9 (SEQ ID NO:4), NME4 (SEQ ID NO:5) or TBK1 (SEQ ID NO:6) or allelic variants and homologs thereof as defined above.
• a target polypeptide selected from ROS1 (SEQ ID NO:1 ), BMP2k (SEQ ID NO:2 or 3), NEK9 (SEQ ID NO:4), NME4 (SEQ ID NO:5) or TBK1 (SEQ ID NO:6) or allelic variants and homologs thereof as defined above.
• the target nucleic acid is a ROS1 nucleic acid having the sequence of SEQ ID NO:7, a BMP2k nucleic acid having the sequence of SEQ ID NO:8 or 9, or a NEK9 nucleic acid having the sequence of SEQ ID NO:10, a NME4 nucleic acid having the sequence of SEQ ID NO:11 or a TBK1 nucleic acid having the sequence of SEQ ID NO:12.
• the target polypeptides or nucleic acids of the invention may be expressed in hyperproliferating (e.g. tumor) cell types.
• they may be over- or under-expressed in a variety of hyperproliferating (e.g. tumor) cell types as compared to the corresponding non-hyperproliferating wild-type cells.
• over- or under-expression means a degree of polypeptide synthesis leading to an amount of said polypeptide within or at the surface of the hyperproliferating cell, which differs from that of the wild-type or normal cell by at least factor 1.5, preferably at least factor 2, more preferably by at least factor 5.
• the expression profile of the target structures according to the invention may be cell- and/or cancer-type specific. Many of the investigated cancer cell lines exhibit a high expression of a target structure as defined above. For example, ROS1 is highly expressed in NSCLC cell lines, glioblastoma, pancreas cancer and metastatic renal cancer carcinoma (mRCC), whereas ROS1 is rarely expressed in certain cancer cells of breast and colon tissue.
• mRCC metastatic renal cancer carcinoma
• the activity of the target polypeptide according to the invention may be increased or decreased in a variety of hyperproliferating (e.g. tumor) cell types as compared to that of the corresponding non-hyperproliferating wildtype cells.
• an increased or decreased enzymatic activity means a change in the activity by at least factor 1.5, preferably by at least factor 2, more preferably by at least factor 5.
• Modulation of a target structure according to the invention e.g.
• hyperproliferating cell lines or individuals having hyperproliferating cells leads to a dramatic alteration, e.g. inhibition of the proliferation rate of said hyperproliferating cells, e.g. cancer cells derived from glioblastoma or lung as shown in the examples below.
• Further modulating a target structure according to the invention may reduce migration, invasiveness and/or metastatic potential and may induce apoptosis of hyperproliferating cells.
• the targets as defined above can be used for the identification, characterization and/or for designing of compounds having an antiproliferative and/or pro-apoptotic activity profile.
• Such compounds or agents may have the ability to specifically modulate or modulate at least one target as described herein, e.g. due to binding to the at least one target, and can therefore be used for detecting, preventing, treating and/or monitoring conditions associated with, accompanied by and/or caused by the occurrence of hyperproliferating cells.
• the present invention relates to molecules or agents which modulate a target structure as defined above, e.g. its expression and/or activity, and to compositions comprising at least one of said molecules or agents for therapy, prognosis and/or diagnosis of a hyperproliferative disorder, e.g. cancer disease.
• a target structure as defined above, e.g. its expression and/or activity
• compositions comprising at least one of said molecules or agents for therapy, prognosis and/or diagnosis of a hyperproliferative disorder, e.g. cancer disease.
• modulate or “modulating” within the context of the present invention relates to the ability of a molecule or a composition comprising at least one of such a molecule (so-called modulator) to influence the synthesis, activity and/or biological function of a target as described herein and thus to change the proliferation behaviour of the cell.
• a modulator can be a molecule which interacts directly or indirectly with the target polypeptide, wherein, as a consequence of the interaction, the activity or biological function is inhibited, activated or enhanced, preferably inhibited.
• a modulator directed against ROS1 may be for example a molecule which inhibits, activates or enhances, preferably inhibits the tyrosine kinase activity of ROS1 polypeptide and/or its interaction with its components of the signal transduction cascade, e.g. with its substrates, co-factors or chaperones.
• a modulator can be a molecule interacting directly or indirectly with the target nucleic acid, wherein as a consequence of the interaction, the expression, i.e. transcription and/or translation, of said target nucleic acid is inhibited, activated or enhanced, preferably inhibited.
• a modulator directed against a ROS1 nucleic acid may be, for example, a molecule which inhibits, activates or enhances, preferably inhibits the expression of a ROS1 polypeptide on the transcription and/or translation level.
• a modulator according to the invention is useful for the treatment, detection and monitoring of a hyperproliferative disease, e.g. cancer.
• a modulator according to the invention is one which inhibits the expression, activity and/or biological function of a target structure, e.g. a kinase activity as defined above.
• Such inhibitors may be selected from:
• nucleic acids or nucleic acid analogues directed against target structures in particular small interfering RNA (siRNA) molecules, micro RNA (miRNA) molecules or precursors thereof, oligonucleotide aptamers, anti-sense oligonucleotides, or ribozymes;
• siRNA small interfering RNA
• miRNA micro RNA
• oligonucleotide aptamers anti-sense oligonucleotides
• ribozymes oligonucleotide aptamers
• polypeptides binding to target structures in particular antibodies or peptidic aptamers
• organic molecules e.g. organic molecules having a molecular weight up to 1000 Da or up to 2000 Da;
• the inhibitor is directed against a target polypeptide, i.e. an inhibitor acting on the protein level.
• the inhibitor is a polypeptide, e.g. an antibody or an antibody fragment having at least one binding site for the target polypeptide.
• the term "antibody” covers monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, polyclonal antibodies, and recombinant antibodies, such as multispecific antibodies (e.g. bispecific antibodies) or single-chain antibodies or fragments thereof having at least one antigen binding site.
• the antibody may be an IgM, IgG, e.g. lgG1 , lgG2, lgG3 or lgG4.
• antibody fragments include Fab, Fab", F (ab') 2 and Fv fragments, diabodies and multispecific antibody fragments.
• Fab fragments
• Fab fragments
• Fab fragments
• F (ab') 2 fragments
• Fv fragments diabodies
• multispecific antibody fragments diabodies
• the administration of chimeric antibodies, humanized antibodies or human antibodies is especially preferred.
• a monoclonal antibody may be obtained by the hybridoma method as described by Kohler et al. (Nature 256 (1975), 495) or by recombinant DNA methods (cf. e.g. US patent 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques which are known to the person skilled in the art. Chimeric and humanized antibodies may be obtained from monoclonal antibodies according to known methods. Human antibodies may be obtained from animals having a xenogenic human immune system, e.g. mice.
• the antibodies according to the invention may be coupled to a labelling group, particularly for diagnostic applications.
• suitable labelling groups such as radioactive groups, fluorescent groups or other labelling groups are known in the art.
• the antibody may be coupled to an effector group, which may be effective in the treatment of hyperproliferative diseases, e.g. a cytotoxic group such as a radioactive group, a toxin or another effector group as known in the art.
• the inhibitor directed against a target polypeptide on the protein level may also be an aptamer, i.e. a nucleic acid or a peptidic molecule that specifically binds to the target polypeptide.
• a nucleic acid aptamer may have a sequence length of between about 20-100 nucleotides, preferably about 25-75, more preferably about 30-50 nucleotides.
• a peptide aptamer generally consists of a variable peptide loop attached at both ends to a protein scaffold. The variable loop length is between 5-50, preferably about 10-30, and more preferably about 10-20 amino acids. Aptamers may be prepared by chemical synthesis and selected by a series of evolution cycles as known by the person skilled in the art.
• An aptamer according to the invention may be coupled to a labelling group as described above. Further, particularly for therapeutic applications, the aptamer may be coupled to an effector group as described above.
• the modulator is an inhibitory antibody, an antigen-binding fragment thereof or an aptamer directed against a ROS1 , NEK9, BMP2k, NME4 or TBK1 polypeptide, i.e. the modulator inhibits the biological activity of the target polypeptide. More preferred is an antibody, an antigen-binding fragment thereof or an aptamer which is directed against the ROS1 polypeptide as defined in SEQ ID NO:1 and in particular against the active center of the ROS1 kinase.
• the modulator according to the invention is directed against a target nucleic acid, i.e. a modulator acting on the nucleic acid level.
• the modulator is a nucleic acid compound, e.g. a RNAi inducing molecule like siRNA, a miRNA, an anti-sense oligonucleotide, a ribozyme, or a precursor thereof or a DNA template molecule encoding the nucleic acid compound or the precursor.
• An RNAi-inducing molecule may refer to a nucleic acid molecule, wherein at least one polynucleotide strand of said nucleic acid molecule has a sequence which is sufficiently complementary to a target RNA, preferably to a target mRNA, in order to effect its processing, i.e. its degradation by RNA interference mechanisms.
• the complementarity is at least 80 %, preferably at least 90 % and more preferably at least 99 %.
• the RNAi-inducing molecule may be a siRNA molecule, for example, a double-stranded of RNA and/or nucleic acid analogue, preferably with a 3' overhang on at least one strand, more preferably on both strands.
• each RNA strand of the double strand is 19 to 30 nucleotides long, more preferably 20 to 28 nucleotides and most preferably 21 to 23 nucleotides.
• the 3' overhang on the end of a RNA strand is preferably 2 nucleotides long.
• the siRNA double- strand consists of two 21 nucleotides long RNA strands each having a 2 nucleotides long 3' overhang.
• the siRNA molecule may also be single- stranded RNA-molecule having a length of 19-30 nucleotides, preferably 20- 28 nucleotides and particularly having a length of 21-23 nucleotides, whereby the single-stranded RNA molecule is for at least 80%, preferably for at least 90% and more preferably for more than 99% complementary to a sequence of a target RNA, in particular of a target mRNA, and a binding of siRNA to the target RNA effects a sequence specifc decrease.
• Examples of siRNA molecules are described in WO 02/44321 and WO 2004/007718, the contents of which are herein incorporated by reference.
• a miRNA molecule may be a single- or double-stranded RNA molecule of 19- 30, preferably 20-28, and more preferably 21-23 nucleotides in length, which can regulate gene expression, or a precursor thereof, which is then processed to the major form having a sequence which is at least partially complementary to messenger RNA of a target molecule according to the invention.
• miRNA molecules are described in WO 03/029459, the content of which is herein incorporated by reference.
• An antisense oligonucleotide may be a single, double, or triple-stranded nucleic acid or nucleic acid analogue, e.g. a DNA, RNA, PNA (peptide nucleic acid) or a combination thereof (e.g. hybrids of DNA and RNA strands) having a length of between about 10-100, preferably 20-50, and more preferably 20-30 nucleotides in length, which can interfere with mRNA targets by hybrid formation and therefore inhibit translation of said mRNA.
• nucleic acid analogue e.g. a DNA, RNA, PNA (peptide nucleic acid) or a combination thereof (e.g. hybrids of DNA and RNA strands) having a length of between about 10-100, preferably 20-50, and more preferably 20-30 nucleotides in length, which can interfere with mRNA targets by hybrid formation and therefore inhibit translation of said mRNA.
• Ribozymes may be selected from RNA molecules and nucleic acid analogs known in the art.
• ROS1 tyrosine kinase inhibitor a pyrazol compound
• Park et al. Bioorganic and Med. Chem. Lett. 19 (2009), 4720-4723
• This compound exemplifies low molecular weight ROS1 inhibitors.
• a modulator molecule acting on the nucleic acid level may be a nucleic acid such as DNA or RNA or a nucleic acid analogue, comprising at least one modified nucleotide within its sequence.
• nucleotide analogs may, for example, increase the stability of the molecule.
• Nucleotide analogs are well known to the person skilled in the art and are modified compared to the original RNA molecules by base modification, sugar modification, e.g. modification of the 2'-OH group of ribose, e.g. by H, alkyl, alkenyl, alkynyl, halo, CN etc., and/or phosphate backbone modifications.
• Precursors of nucleic acid molecules as described above are nucleic acid molecules which are processed to the desired modulators, e.g. by cellular enzymes.
• RNA precursor molecules such as long double-stranded RNA (dsRNA) or short hairpin RNA-molecules (shRNA)
• dsRNA long double-stranded RNA
• shRNA short hairpin RNA-molecules
• the invention encompasses DNA constructs encoding nucleic acid effector molecules or precursors thereof, operatively linked to regulatory sequences allowing an expression in the target cell. Examples for such regulatory sequences are promoters such as polymerase II promoters or polymerase III promoters.
• the modulator is a nucleic acid selected from siRNA, miRNA, ribozymes or antisense oligonucleotides, directed against an mRNA encoding a ROS1 , NEK9, BMP2k, NME4 or TBK1 polypeptide as defined above, more preferably directed against a ROS1.
• Nucleic acid molecules may be coupled to a labelling group, e.g. for diagnostic purposes or may be coupled to an effector molecule as described above.
• the polypeptides as described above may be administered to a target cell or organism by known techniques.
• the nucleic acid molecules may be administered to a target cell or organism by any technique which is known to a person skilled in the art, such as by administration of the nucleic acid molecule as such or on an appropriate vector, e.g. a viral or a non-viral vector.
• the modulator/effector is an inhibitor of the expression of a target nucleic acid, which preferably acts by down-regulation or knock-down of the target.
• Down-regulation or knockdown of the target results preferably in a reduction of the steady state level of the target mRNA or polypeptide by at least factor 2, 5, 10 or more, or in a disappearance of the target mRNA from the cell.
• ROS1 , NEK9, BMP2k, NME4 and/or TBK1 may lead to an inhibition of cell proliferation, migration, invasion and/or metastasis formation or to an induction of apoptosis.
• the modulator/effector may also be an inhibitor of the activity, e.g. the catalytic activity, of a target polypeptide selected from ROS1 , NEK9, BMP2k, NME4 and/or TBK1.
• the modulator may directly interact with the target, e.g. by binding the target.
• a preferred modulator inhibits the enzymatic tyrosine kinase activity of a target polypeptide which may lead to an inhibition of cell proliferation, migration, invasion and/or metastatis formation.
• the modulator of the target polypeptide and/or a nucleic acid may be used for the treatment of conditions associated with, accompanied by or caused by the occurrence of cellular hyperproliferation, particularly associated with invasiveness, migration and/or metastasis formation. More particularly, the condition is cancer associated with metastasis formation. Further, the condition may be characterized in that it exhibits an overexpression of at least one of said target molecules. Furthermore, the cancer may be an invasive, migratory and/or metastatic cancer or non-metastatic cancer. Particularly, the cancer is metastatic.
• the cancer may be a bone, brain, breast, cervix, colon, gastric, kidney, liver, lung, ovary, pancreas, prostate and skin cancer, and preferably glioblastoma, non-small cell lung cancer (NSCLC), metastatic renal carcinoma (mRCC) or gastro-intestinal stromal tumor (GIST).
• NSCLC non-small cell lung cancer
• mRCC metastatic renal carcinoma
• GIST gastro-intestinal stromal tumor
• the modulator may be an activator of a target molecule.
• Such an activator may inhibit proliferation, migration and/or invasion of cancer cells characterized which exhibit a reduced or impaired expression of at least one of said target molecules.
• Examples for hyperproliferating cells showing a reduced or no expression of a target molecule, e.g. a reduced or no ROS1 expression, are certain types of breast cancer cells.
• the modulator may be the target polypeptide or the nucleic acid molecule coding therefor, e.g. a nucleic acid molecule operatively linked to an appropriate expression control sequence.
• the administration of such polypeptides and nucleic acid molecules may be performed according to known methods.
• the cell is a hyperproliferating cell.
• the cell is a hyperproliferating cell, e.g. a cancer cell, which over- or underexpresses at least one of a target molecule selected from ROS1 , BMP2k, NEK9, NME4 and TBK1 , and the modulator is an inhibitor or an activator of a ROS1 , NEK9, BMP2k, NME4 and/or TBK1 polypeptide or nucleic acid as defined above.
• a modulator according to the invention may be used in medicine, particularly in human medicine, e.g. for the treatment or prevention of hyperproliferative diseases, i.e. conditions associated with, accompanied by and/or caused by the occurrence of hyperproliferation, e.g. cancer, particularly metastatic cancer as described above.
• Administration of the modulator may lead to reduction or loss of hyperproliferation, invasiveness and/or metastatic potential and/or to an induction of apoptosis.
• the modulator may be formulated as a pharmaceutical composition with pharmaceutically acceptable diluents, excipients, adjuvants and/or carriers known in the art.
• the composition may contain further active ingredients, e.g further anti-hyperproliferative agents.
• additional agents may be selected from cytostatic, cytotoxic, chemotherapeutic and/or immunotherapeutic agents, e.g. kinase inhibitors such as Sunitinib, Lapatinib, Gefitinib, Eriotinib, and/or anti-tumour antibodies, such as Herceptin or Erbitux, and combinations thereof.
• a further aspect to the invention refers to a method which comprises administering to a subject in need thereof, e.g. a human subject, particularly a human cancer patient, a therapeutically effective amount of a pharmaceutical composition comprising a modulator of (i) a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 polypeptide and/or (ii) a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 nucleic acid.
• the modulator may be selected from compounds as described above.
• Inhibitors of target polypeptides may be administered to a subject suffering from a disorder characterized by expression or overexpression of target polypeptides.
• Activators of target polypeptides may be administered to a subject suffering from a disorder characterized by underexpression of target polypeptides.
• the method may additionally comprise determining the amount, activity and/or localization of the target molecule (target polypeptide and/or target nucleic acid) before the treatment and/or during the treatment.
• target molecule target polypeptide and/or target nucleic acid
• a medicament comprising the modulator as described above can be formulated to be suitable as any known dosage form, e.g. as a solution (e.g. an aqueous solution), syrup, suspension, emulsion, dispersion, tablet, powder, capsule, aerosol, suppository, creme, lotion, ointment, gel or patch for topical, transdermal, enteral (e.g. orally, rectally or by feeding tube) or parenteral (e.g. by intravenous, intraarterial, intramuscular, subcutaneous, intratecal, intraperitoneal injection or infusion) application.
• a solution e.g. an aqueous solution
• syrup e.g. an aqueous solution
• emulsion dispersion
• tablet e.g. an aqueous solution
• powder e.g. orally, rectally or by feeding tube
• parenteral e.g. by intravenous, intraarterial, intramuscular, subcutaneous, intratecal,
• the composition may be formulated as a single dose or as multiple doses for appropriate administration.
• the administration may involve the use of a targeted drug delivery system, for example, in a liposome presenting a tumour-specific targeting molecule, e.g. an antibody.
• the liposomes will be targeted to and taken up selectively by the hyperproliferating cells of a target tissue.
• the effective amount of the active agent, i.e. the modulator, in the composition may be determined by the skilled person without undue burden depending on the kind of active agent and the kind of conditions to be treated.
• about 1 pg/kg to 15 mg/kg of a modulator may be administered to a human patient, e.g. by one or more separate administrations or by continuous infusion.
• a typical daily dosage may range from about 1 pg/kg to about 100 mg/kg or more, depending on the factors such as age, gender, weight, tumour-size and condition of the person to be treated etc.
• the administration may be continued over a longer time period of several days, weeks, or months, depending on the condition to be treated.
• a pharmaceutical composition as defined in the present invention may be further administered as a part of a combination therapy.
• “combination therapy” refers to the simultaneous coadministration of at least two active agents during a treatment period, wherein at least one of these agents is a modulator of a target molecule according to the present invention.
• the co-administration may involve the administration of a single pharmaceutical composition or the simultaneous or non-simultaneous administration of at least two pharmaceutical compositions.
• the term “combination therapy” also includes the administration of the modulator of the invention together with radiation therapy and/or surgery.
• a preferred embodiment of the invention relates to a pharmaceutical composition for the treatment of patients overexpressing a target molecule, e.g. ROS1 , NEK9, BMP2k, NME4 and/or TBK1.
• the present invention relates to a diagnostic composition for (i) a polypeptide selected from a ROS1 , BMP2k, NEK9, NME4 or TBKI or (ii) a nucleic acid selected from a ROS1 , BMP2k, NEK9, NME4 or TBK1 nucleic acid for the diagnosis and/or monitoring of a hyperproliferative disorder.
• the detection reagent comprises an agent selected from
• an antibody directed against a target polypeptide e.g. an anti-ROS1 antibody, or an antigen-binding fragment thereof, or
• a hybridization probe or amplification primer specific for a target nucleic acid such as a ROS1 nucleic acid.
• the agent may be labelled as described above. It may be used in methods for detecting target structures by known methods.
• Target polypeptides may be detected e.g. by immunological methods.
• Target nucleic acids may be detected e.g. by nucleic acid hybridization and/or amplification protocols.
• Another aspect of the present invention relates to a method for screening, identifying, designing and/or characterizing a test compound as a candidate agent for the treatment of a hyperproliferative disease, e.g. cancer.
• the method may comprise the step of determining if a test compound acts as a modulator, e.g. inhibitor or activator of a target polypeptide or nucleic acid as described herein.
• the test compound is preferably tested if it binds to a target polypeptide and/or inhibits the activity, e.g. the enzymatic activity, of a target polypeptide.
• the compound is tested for being a kinase inhibitor which is preferably suitable for inhibiting tyrosine kinase activity of ROS1 , NEK9, BMP2k, NME4 and/or TBK1.
• the test compound may e.g. be a low molecular weight organic compound, e.g. a compound having a molecular weight of up to 2000 Da or up to 1000 Da.
• the screening method may comprise the steps of:
• the test compound may be contacted with the target polypeptide in a cell, e.g. in a cancer cell or in a recombinant cell over- or underexpressing the target polypeptide.
• the test compound may be contacted with the target polypeptide in vitro, wherein the target polypeptide may be present in an at least partially isolated or purified form.
• the determination of the modulating activity of a test compound in a cellular system may comprise the steps of:
• test sample and control sample comprise a cell expressing the at least one target polypeptide, and wherein the test sample is contacted with the test compound and wherein the control sample is not contacted with the compound to be tested,
• test compound determining if the test compound exhibits a modulating activity.
• the modulating activity may be determined in a single sample before and after contacting the sample with the test compound.
• the determination of the modulating activity of a test compound in vitro may comprise the steps of:
• test and the control sample comprise the at least one target polypeptide in an extract or in an at least partially purified or isolated form, and wherein the test sample is contacted with the test compound and wherein the control sample is not contacted with the test compound,
• test compound determining if the test compound exhibits a modulating activity.
• the modulating activity may be determined in a single sample before and after contacting the sample with the test compound.
• the determination of the modulating activity of a test compound may involve determination of the binding of the test compound to the target polypeptide.
• the amount or concentration of the target polypeptide in a cellular sample may be determined by immunological methods. Therefore, the cell sample may be contacted with a detection reagent directed to said target.
• the detection reagent may comprise an antibody, an antibody fragment or an aptamer as defined above. Quantitative or semiquantitative immunoassays which can be used herein are known in the art.
• the amount of a target polypeptide may also be determined by measuring the expression rate on nucleic acid level. Therefore, the cell sample may be contacted with a detection reagent directed to the target nucleic acid, e.g. a hybridization probe and optionally an amplification primer.
• a detection reagent directed to the target nucleic acid, e.g. a hybridization probe and optionally an amplification primer.
• the reagent may comprise a hybridization probe directed to a ROS1 , NEK9, BMP2k, NME4 or TBK1 nucleic acid as described above.
• Hybridization assays which can be used herein are known in the art.
• the present invention further relates to a transgenic cell, a non-human transgenic organism and to a tumor cell showing an altered, e.g. an increased or decreased, expression of a target molecule, e.g. a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 polypeptide, compared to a wild-type cell for use in a screening method as described above.
• a target molecule e.g. a ROS1 , BMP2k, NEK9, NME4 and/or TBK1 polypeptide
• the invention further relates to a method for the identification and/or characterization of target molecules for the therapy and/or diagnosis of a hyperproliferative disease.
• the method comprises
• step (iv) determining, if modulating the molecule obtained in step (iii) has antiproliferative effects.
• the anti-proliferative and/or pro-apoptotic agent may be a low molecular weight organic compound, e.g. an indolin-2-one compound as described above.
• the anti-proliferatively and/or pro-apoptotically effective agent is a tyrosine kinase inhibitor as, for example, SU11248.
• the agent is immobilized to a suitable solid phase, e.g. Sepharose, preferably via a linker.
• the linker has a chain length of 2-20 atoms.
• a preferred example of an immobilized SU11248 molecule is shown in Fig. 1.
• Polypeptides bound to the solid phase may be qualitatively and/or quantitatively determined e.g. by gel electrophoresis and subsequent chromatographic and/or mass- spectroscopic analysis.
• kits for modulating a target molecule as described herein and thus for therapy and/or diagnosis of a hyperproliferative disorder.
• the kit comprises a modulator, e.g. in a pharmaceutical or a diagnostic composition as described above.
• Figure 1 (A) is a schematic depiction of a method for the identification of target structures molecules in a cell.
• the compound SU11248 was coupled to Sepharose and contacted with a total cell lysate under in vitro association conditions. Cellular components bound to SU11248 were identified.
• Figure 1(B) shows the identification of ROS1 as a target polypeptide for SU11248.
• Figure 2 shows the results of an ROS1 -expression profiling within different cancer cell lines.
• Figure 3 shows the effect of siRNA molecules directed against ROS1 , BMP2k, NEK9, NME4 and TBK1 mRNA on the viability (A), caspase activity (B1 ), apoptosis (B2), cell proliferation (C) and cell morphology (D) of different cancer cell lines.
• Figure 4 shows the effect of siRNA molecules directed against ROS-1 , BMP2k, NME4 and TBK1 mRNA on SU11248 treatment efficacy in different cancer cell lines.
• Figure 5 shows the nucleotide (A) and amino acid (B) sequences of ROS1 , BMP2k, NEK9, NME4 and TBK1 mRNAs or polypeptides.
• Inhibition Rate (%) [1-(ODtreated- ODtreated (day0)/ODcontrol- ODcontrol (day0))]*100%.
• the IC 5 o i.e. the drug concentration that reduced the absorbance observed in untreated cells by 50% was calculated by using Hill three parameter log fit in SIGMA Plot 10.
• Inhibition Rate (%) [1- (ODtreated- ODtreated (day0)/ODcontrol- ODcontrol (day0))]*100%.
• the IC 5 o i.e. the drug concentration that reduced the absorbance observed in untreated cells by 50% was calculated by using Hill threeparameter log fit in SIGMA Plot 10.0.
• the assay was performed as described in the manufacturer ' s protocol (Roche). Briefly, in a 96- well flat-bottomed plate, 1000- 2000 cells/100 ⁇ cell suspension was seeded. After 24 h, cells were exposed to different concentrations of compound. Each treatment was tested in triplicate wells. At the end of exposure (24 h, 48 h and 72 h) cells were incubated with BrdU for 6- 18 h at 37°C. The labeled cells were fixed with ethanol and prior to incubation with a monoclonal antibody to BrdU, the DNA was partially digested with nucleases to allow the antibody to access BrdU.
• the anti- BrdU antibody labeled with peroxidase (POD)
• POD peroxidase
• Transfected or compound- treated cells were trypsinized after 72h of siRNA transfection or drug application and collected by centrifugation. For fixation, cells were washed once with PBS, resuspended in 1 ml cold 70% ethanol and stored overnight at 4°C. Cells were then collected by centrifugation, washed once with PBS and incubated with 0.01 % Triton, 0.1 % sodium citrate, 0.02mM propidium iodide (Sigma) in the dark for 2h at 4°C. Cells were analyzed by flow cytometry (FACS Calibur, BD Bioscience). Using the CellQuestPro software, each of the three peaks (representing cells in Gi, S, and G 2 /M phases, respectively) obtained in the flow cytometry profile of fluorescence plotted against cell number was gated and quantified.
• Caspase 3/7 activity of siRNA transfected or compound- treated cells was measured using the Caspase 3/7 Glo- Assay from Promega according to manufacturer ' s instruction.
• Sunitinib maleate (SU11248) was purchased from ACC Corporation (San Diego, USA).
• SU11248 was chemically modified and kindly provided by VICHEM CHEMIE Ltd., Budapest, Hungary.
• Drained epoxy-activated Sepharose 6B was resuspended in 2 vol of 0.1 , 0.3, 1 or 3 mM Sunitinib dissolved in 50% DMSO/50% 0.05M Na 2 C0 3 (pH11 ). After adding of 10 mM NaOH, coupling was performed overnight at 30°C in the dark. After three washes with 50% DMSO/50% 0.05M Na 2 CO 3 (pH11 ), remaining reactive groups were blocked with 1 M ethanolamine. Subsequent washing steps were performed according to the manufacturer's instructions. To generate the control matrix, epoxy-activated Sepharose 6B was incubated with 1 M ethanol-amine and equally treated as described above. The beads were stored at 4°C in the dark as a suspension in 20% ethanol.
• Cells (80 mg protein) were lysed in 30 ml of buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCI, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT plus additives (10 mM sodium fluoride/1 mM orthovanadate/10 pg/ml aprotinin/10 pg/ml leupeptin/1 mM phenylmethylsulfonylfluoride/10% glycerol), cleared by centrifugation, and adjusted to 1 M NaCI.
• the filtrated lysate was loaded with a flow rate of 100 ⁇ /min on an HR 5/2 chromatography column (Amersham Biosciences) containing 600 pi of SU11248 matrix equilibrated to lysis buffer without additives containing 1 M NaCI.
• the column was washed with 15 column volumes and equilibrated to lysis buffer without additives containing 150 mM NaCI, and bound proteins were eluted in the same buffer containing 1 mM SU11248, 10 mM ATP, and 20 mM MgCI 2 with a flow rate of 50 ⁇ /min.
• the volume of protein-containing elution fractions was reduced to 1/10 in a Centrivap concentrator (Labonco, Kansas City, MO) before precipitation according to Wessel and Fluegge ((Wessel and Flugge, 1984)). Protein pellets were desolved in 1.5x SDS- sample buffer, pooled and subjected to 1 D- SDS- Gel electrophoresis.
• Protein pellets after Wessel-Fluegge precipitation were boiled in 1.5 x LDS- sample buffer with 0.5 mM DTT for 10 min at 70°C and subsequently separated by one-dimensional SDS-PAGE using NuPage Novex Bis- Tris gels and NuPage MOPS SDS running buffer (Invitrogen) according to the manufacturer's instruction.
• the gel was stained with Coomassie Blue using the colloidal blue staining kit (Invitrogen).
• Gel bands were cut into 1 mm 3 cubes and washed with 50 mM ammonium bicarbonate, 50 % ethanol.
• gel pieces were incubated with 10 mM DTT in 50 mM ammonium bicarbonate for 1 h at 56°C.
• Alkylation of cysteines was performed by incubating the samples with 55 mM iodoacetamide in 50 mM ammonium bicarbonate for 45 min at 25°C in the dark.
• Gel pieces were washed two times with 50 mM ammonium bicarbonate and incubated with 12.5 ng/ ⁇ Trypsin in 50 mM ammonium bicarbonate for 16h at 37°C for protein digestion.
• Supernatants were transferred to fresh tubes and remaining peptides were extracted by incubating the gel pieces two times in 30% acetonitrile in 3% TFA followed by dehydration with 100% acetonitrile.
• the extracts were combined and used for direct peptide identification by mass spectrometry after desalting the samples using RP-C18 stage tip columns.
• the peptide mixture was injected onto the column with a flow of 0.5 ⁇ /min and subsequently eluted with a 5-40 % acetonitrile gradient in 0.5 % acetic acid with a flow of 0.25 ⁇ /min.
• the gradient was 140 min.
• Mass spectra were acquired in the positive ion mode applying a data-dependent automatic switch between survey scan and tandem mass spectra (MS/MS) acquisition.
• the hybrid linear ion trap/orbitrap instrument was used with the lock mass option in both MS and MS/MS mode.
• the polydimethylcyclosiloxane (PCM) ions generated in the electrospray process from ambient air were used for internal mass recalibration in real time.
• Mass spectrometric data were analyzed with the in-house developed software MaxQuant (version 1.0.12.0 for the proteome and version 1.0.11.5 for the phosphoproteome analysis). MS/MS spectra were searched by Mascot (version 2.2.2, Matrix Science) against the Human IPIBase (version 3.37) combined with common contaminants and concatenated with the reversed versions of all sequences. The following parameters were set for the Mascot searches. Trypsin allowing for cleavage N-terminal to proline and cleavage between aspartic acid and proline was chosen as enzyme specificity.
• Cysteine carbamidomethylation eas selected as a fixed modification, while protein N-terminal acetylation, methionine oxidation and serine, threonine and tyrosine phosphorylation were selected as variable modifications. Maximally three missed cleavages and up to three labeled amino acids according to SILAC or non- SILAC study were allowed. Initial mass deviation of precursor ion and fragment ions were up to 5 ppm and 0.5 Da, respectively. MaxQuant automatically identified and quantified SILAC peptides and proteins. SILAC protein ratios were calculated as the median of all peptide ratios assigned to the protein.
• a false discovery rate (FDR) of 0.01 was required for proteins and peptides with a minimum length of 6 amino acids. Furthermore, a posterior error probability (PEP) for each MS/MS spectrum below or equal to 0.01 was required. In case the identified peptides of two proteins were the same or the identified peptides of one protein included all peptides of another protein, these proteins (e.g. isoforms and homologs) were combined by MaxQuant and reported as one protein group.
• FDR false discovery rate
• PEP posterior error probability
• A498 kidney cancer cells were seeded at a density of 150.000 cells/well in 6- well flat-bottom cell culture dishes. 24h prior to SU11248 treatment, cells were starved for 24h in medium containing 0% FCS. Drug incubation was performed for 2h, followed by pervanadate stimulation for 5min at 37°C. Cells were lysed and subjected to immunoprecipitation with an antiROSI antibody over night.
• RNAi transfection 15.000 cells/ml were seeded into 6-well cell culture plates. At 30% confluency cells were transfected with 30 pmol of validated or pre-designed siRNA from Ambion using RNAiMax (Invitrogen) according to the manufacturer ' s instruction. GI2 siRNA was taken as control. 5d after transfection cells were used for cell cycle analysis by flow cytometry, MTT-, SRB-, BrdU-assay and western blotting. The knock-down efficiency was monitored by RT-PCR and Western Blotting.
• Membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated antimouse, antirabbit or antigoat secondary antibody in TBST + 4% nonfat dry milk for 1 h at room temperature. Membranes were washed three times with TBST and visualized by ECL (Western Lightning, Perkin Elmer) on X-ray films.
• Binding partners of the indolin-2-one compound sunitinib were identified using affinity chromatography and mass spectrometry.
• SU11248 Binding partners of the indolin-2-one compound sunitinib
• For FPLC- affinity chromatography an SU11248 matrix by coupling SU11248 to ECH/epoxy-Sepharose was prepared. Whole cell lysate was incubated with the SU11248 Sepharose matrix under in vitro association conditions. Bound polypeptides were eluted and subjected to LC/MS analysis after in-gel trypsin digestion of PAGE-separated protein bands (Fig. 1A). By this method, the polypeptides ROS1 , BMP2k, NEK9, NME4 and TBK1 could be identified as binding partners of SU 11248 (cf. Fig. 1 B for ROS1 ).
• Fig. 3A MTT colorimetric assay
• Fig. 3B1 caspase assay
• Fig. 3B2 FACS-PI assay
• Fig. 3C The effect of siRNA molecules specific for ROS1 , BMP2k and NEK9 mRNA on the proliferation of different cancer cell lines is shown in Fig. 3C (BrdU- Assay). In several cancer cell lines a significant decrease of cell proliferation was observed.
• siRNA specific for ROS1 mRNA, BMP2k mRNA, or NEK9 mRNA versus control (ctrl) siRNA is shown in Fig. 3D (microscopic picture).
• Administration of siRNA leads to morphological changes and a reduction in cell growth.
• Target depletion was achieved by transient RNAi experiments and the SU11248 efficacy was measured using a standard MTT colorimetric method. Cancer cells were either transfected with control or target-specific siRNA, grown for two days under serum conditions and treated with increasing SU11248 concentrations for 72h. SU11248 efficacy is shown at 5 ⁇ and expressed in percent relative to mock-transfected, SU11248 treated cells. Results were averaged over three to five independent experiments (error bars: SEM). In Fig. 4, it is shown that depletion of ROS1 , BMP2k, NME4 and TBK1 by siRNA leads to a dramatic decrease in SU11248 treatment efficacy.

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