WO2004064710A2 - Applications of substances binding to gstm for the diagnosis and treatment of cystic carcinomas - Google Patents

Applications of substances binding to gstm for the diagnosis and treatment of cystic carcinomas Download PDF

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WO2004064710A2
WO2004064710A2 PCT/DE2004/000092 DE2004000092W WO2004064710A2 WO 2004064710 A2 WO2004064710 A2 WO 2004064710A2 DE 2004000092 W DE2004000092 W DE 2004000092W WO 2004064710 A2 WO2004064710 A2 WO 2004064710A2
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gstm
substance
protein
tumor
peptide
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PCT/DE2004/000092
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German (de)
French (fr)
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WO2004064710A3 (en
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Bernd Hinzmann
Edgar Dahl
Thomas Specht
Christian Pilarsky
Alexander Herr
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Bernd Hinzmann
Edgar Dahl
Thomas Specht
Christian Pilarsky
Alexander Herr
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Publication of WO2004064710A2 publication Critical patent/WO2004064710A2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to new uses of GSTM or sequences derived therefrom for screening for substances binding to it, and the use of substances binding to GSTM for the diagnosis and / or treatment of bladder carcinoma.
  • GSTM stands for glutathione-S-transferase class ⁇ member 4.
  • GSTM belongs to a multi-gene family of enzymes which, by binding glutathione to cell-toxic substances, initiate their detoxification in the cell. Furthermore, they bind and mediate lipophilic substances
  • GSTM genes mainly GSTM1
  • GSTM1 GSTM1
  • bladder tumors but some, but not all, low-grade tumors show high expression values of this molecule on.
  • high-grade, invasive forms on the other hand, no GSTM could be detected at all in small amounts (Celis et al., Cencer Res 15, 56 (20): 4782-4790 (1996)).
  • the expression pattern shows a very heterogeneous picture, which is why it cannot be predicted whether or in which tumors overexpression will take place and what such overexpression ultimately means.
  • Bladder cancer is the second most common urological tumor. It occurs in men with a frequency of 245 to 100,000 and in women from 65 to 100,000. Among cancer deaths, bladder cancer ranks fourth in men and sixth in women. Treatment is usually through cystectomy, i.e. by partially or completely removing the bladder. This often leads to further complications for the sick person.
  • Bladder cancer develops through the degeneration of individual urothelial cells.
  • the urothelium is the layer of cells that shields the inside of the body from the urine that is formed. It lines the lumen of the renal pelvis, ureter, bladder and urethra.
  • Bladder cancer develops almost completely (> 93%) as adenocarcinoma and is characterized by the strong tendency to recurrence, the regular recurrence even after treatment.
  • the development of bladder cancer is mainly due to the influence of chemical noxae, such as aromatic amines, but mainly to smoking as a cause recycled. It can be diagnosed relatively early and take two forms.
  • the more common variant that of papillary or superficial pTa-Zumoren, has a good prognosis because it is not invasive and can be removed surgically.
  • the invasive forms grow into the tissue and, if left untreated, lead to the most severe symptoms, such as lymph node involvement and metastases. The deaths arise almost exclusively from this second group. It is characteristic of the course that the papillary tumors very often remain stable and therefore show no progress towards the dangerous invasive forms. In 10-20% of cases, however, there is a progression to aggressive, cancer-invasive tumors in the course of 5 years.
  • a number of genes have so far been examined for their suitability as a prognostic marker in bladder cancer. It is of primary interest whether a patient with non-invasive, papillary tumors remains stable at this stage or whether he will develop invasive forms.
  • the markers examined so far mainly include p53, p21 and the retinoblastoma gene Rb. However, none of the markers mentioned enables a prognosis for an individual patient with sufficient certainty (Marberger et al., Eur Urol, 40/5, Curric Urol 1-9, (2001)). More recent, commercially available tests (BTA-STAT, NMP22) focus more on the detection of urothelium than on the prognosis of the course.
  • the invention is therefore based on the technical problem of specifying pharmaceutical compositions for diagnosis, in particular for prognosis for progression or progression, and / or for treating bladder carcinoma and means for identifying them.
  • a nucleic acid coding for GSTM and / or a GSTM peptide or protein for the detection of bladder carcinoma or for the detection of a risk of the disease of such a carcinoma or for the detection of a risk of a progression of papillary urinary carcinoma to one teaches invasive carcinoma, a urothelial cell tissue sample, in particular a urinary bladder urothelial cell tissue sample, being examined for transcription or over-transcription of GSTM RNA or for expression or over-expression of a GSTM protein.
  • a nucleic acid coding for GSTM or a detector substance binding to GSTM protein or peptide, preferably containing a reporter group, can be used, whereby binding of said nucleic acid and / or said protein or peptide to the detector substance is detected semi-quantitatively or quantitatively.
  • the invention further teaches a test system for (in vitro) detection of a carcinoma mentioned above or a risk of the disease thereof or prognosis progression, comprising means for quantitative measurement of the expression of GSTM in tissue samples, these means being, for example, means for amplification and specific Detection of GSTM RNA and / or a detector substance, in particular specific for GSTM protein, can be.
  • the invention further teaches the use of a GSTM RNA or a GSTM protein or peptide for screening for substances which bind to it, in particular prospective active ingredients for modulating, in particular inhibiting, said RNA or said protein or peptide, or pro-prospective detector substances, a prospective substance or a mixture of such prospective substances is contacted with said RNA or said protein or peptide, binding events being ascertained using a binding assay, and a binding prospective substance being selected, if appropriate after deconvolution.
  • the invention further teaches a screening system for determining active substances suitable for the treatment of the above tumor diseases, comprising a GSTM nucleic acid or a GSTM protein or peptide, means for determining (in vitro) binding events to the GSTM nucleic acid or to the GSTM protein or peptide, and / or means for determining the (in vitro) activity of GSTM protein.
  • GSTM can be present in a cell-free or a cell-based system, the latter in particular having urothelial cells of the urogenital tract, in particular the urinary bladder, or a cell line developed therefrom.
  • Means for determining binding events can, for example, naturally include substances or association partners which bind naturally in normal cells or in tumor cells, for example to GSTM protein, with their (free) concentration or change in concentration when prospective active substances and / or detector substances are added to allow a binding binding of a binding active substance. or detector substance is determined.
  • Such means can also include physical or physico-chemical methods, such as X-ray structure analysis and / or NMR, in particular two-dimensional 1H / 1H or 15N / 1H or 14C / 1H correlation spectroscopy.
  • spectra are compared with each other before and after the addition of a prospective active substance or detector substance, and in the event of changes, a binding event is determined.
  • Spectra or the like of either GSTM or the prospective substance or a combination of the two can be used.
  • all other standard methods of determining binding events and / or protein activities can also be used.
  • a prospective substance or several substances, spatially separated from one another
  • GSTM is applied.
  • a binding event is then determined after application and subsequent rinsing by detection, possibly locally resolved, of the label-bound FABP4s.
  • GSTM can be immobilized and a labeled prospective substance or a mixture thereof is applied. Binding events are determined analogously to the variant above.
  • the invention teaches the use of a substance which inhibits or binds to GSTM for the production of a pharmaceutical composition for the treatment and / or diagnosis of bladder carcinoma or prognosis of progression in bladder carcinoma diseases.
  • the substance can be an antibody which is obtained by immunizing a non-human mammal with a GSTM peptide or protein with cDNA coding therefor transfected cells, with tumor cells expressing such a peptide or protein endogenously, or with recombinantly produced GSTM peptides or proteins, or a phage display antibody.
  • the substance can also be a mimicry compound of an antibody against a GSTM peptide or protein.
  • the substance can be an aptamer, an antisense RNA, a ribozyme or an siRNA against GSTM nucleic acids.
  • the substance can additionally carry a cytotoxic and / or immune-stimulating component.
  • the above substances binding to GSTM protein specifically bind to the GSTM protein when used for therapeutic purposes and modulate its biological activity. This is not necessary in the case of fusion or connection of the substance with a cytotoxic component. This is also not necessary if the substance is used to obtain an anti-idiotypic antibody which is recognized by a patient's immune system as foreign to the body due to its non-humanized form and which otherwise presents a GSTM antigen to the immune system.
  • the pharmaceutical composition can be used for any application, for example i.v. or i.p. Injection, be prepared. Preparation for local application in tissue containing tumor cells is recommended if a cytotoxic component is used.
  • the invention can be used in the context of a method for the diagnosis or prognosis of progression of a tumor disease of the bladder carcinoma, whereby one Detector substance is applied in one embodiment with a reporter group in the tissue to be examined, if appropriate in vitro after tissue removal, the tissue to be examined then being subjected to a detection method stage which is sensitive to the reporter group, and in the case of the detection of a defined one minimum value of the reporter group in the tissue, the tissue is classified as containing tumor cells or classified as prdgressionsgefährdet or not hrdet progressionsgef, and a method for treatment of a urinary bladder tumor disease, wherein 'a Inventions contemporary pharmaceutical composition is administered in a physiologically effective dose to a patient.
  • a tissue sample can additionally or alternatively be examined for GSTM expression using a test system according to the invention.
  • the invention is based in particular on the knowledge that GSTM is expressed differently in different papillary tumors of bladder carcinoma, ie in said tumor tissues the expression is higher in comparison with normal cells of the same tissue in the case of such papillary carcinomas which later become invasive, and in the case of papillary carcinomas which remain stable, on the other hand, low, and the technical teaching which can be derived therefrom that GSTM can be used as a target molecule in diagnosis, in particular prognosis of progression, and therapy or prophylaxis, in particular of invasive tumor diseases. GSTM can thus serve as a specific marker for identifying tumor cells in said tumor tissues which have a risk of forming invasive tumor tissues.
  • the inhibition of GSTM offers the opportunity to intervene in the tumor-specific GSTM associations with other processes in the tumor cells and thus ultimately to disrupt the tumor cell-specific metabolism and to die or at least inhibit the growth of the tumor cells, but especially to inhibit the progression invasive tumors.
  • a sample from a tissue which is identified as tumor tissue by other methods in advance of treatment with a pharmaceutical composition according to the invention and to examine the tissue sample for expression or overexpression of GSTM.
  • a detector substance according to the invention can be used to test in vivo for GSTM dependency. If an expression or overexpression of GSTM with respect to normal tissue of the same type is found, the use of the pharmaceutical composition according to the invention is indicated.
  • the substance binding to GSTM is additionally a cytotoxic and / or immunostimulating component. This ultimately leads to the fact that almost exclusively tumor cells are killed, either by cytotoxicity or by attack by the stimulated immune system, while normal cells are practically completely preserved in the tissue.
  • the binding substance itself does not have to have an inhibitory effect on GSTM, since the binding substance then only has to function as a marker which carries the components to target tumor cells. If a cytotoxic component is used, it will be particularly recommended if the pharmaceutical composition is prepared for local application in tissue containing tumor cells, for example for injection.
  • GSTM is used for all human isoforms, known or new, based on nucleic acids or amino acids. These terms also include the short sequences disclosed in the context of this description, which originate from the isoforms, for example immunization sequences. Also included are homologs, the homology being at least 80%, preferably more than 90%, most preferably more than 95%, calculated with the MEGALIGN (DNASTAR LASERGENE) program at the time the present application current version. In the case of the nucleic acid sequences, complementary or allelic variants are also included.
  • sequences for example, an exon or more exons, or complementary sequences thereto, with the proviso that these partial sequences in the case of nucleic acids, one for hybridization with a ER- fin ' nucleic acid of the invention sufficient length, at least
  • nucleic acids hybridizing with nucleic acids according to the invention are included, namely those which are stringent
  • the invention also includes expression cassettes, ie one or more of the nucleic acid sequences according to the invention with at least one operatively linked control or regulatory sequence.
  • Such an expression cassette can also comprise a sequence for a known protein, a fusion protein being formed in the course of the translation from a known protein and a protein or peptide according to the invention. Antisense sequences to the above nucleic acid sequences are also included.
  • GSTM as well as correlating DNA and vice versa are included, as well as genomic DNA as well as correlated cDNA and vice versa.
  • GSTM homo- or heterodimers can also be used within the scope of the invention. In this respect, the term GSTM also includes such homo- or heterodimers.
  • GSTM include nucleic acids or protein or peptides in addition to the full lengths of the disclosed sequences (see also the preceding paragraph) and also partial sequences thereof, with a minimum length of 12 to 30 nucleotides, preferably 30 to 90 nucleotides , in the case of nucleic acids and a minimum length of 4 to 10 amino acids, preferably 10 to 30 amino acids, in the case of peptides or proteins.
  • These partial sequences can be incorporated into nucleic acid or protein or peptide sequences that are otherwise different from GSTM.
  • treatment also includes prophylaxis, especially prophylaxis of progression to invasive tumors.
  • An inhibitor is a compound or substance which either inhibits the formation of GSTM protein or reduces the activity of GSTM protein formed, based on the GSTM activity in the absence of the inhibitor.
  • an inhibitor can be a substance that interferes with the GSTM cascade.
  • an inhibitor can be a substance that binds with the GSTM formed, in such a way that further physiological interactions with endogenous substances are at least reduced.
  • Mimicry molecules are compounds that simulate the variable region, in particular the binding region of an antibody, and bind to a target molecule in the same place as the underlying antibody.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, non-human, human and humanized antibodies, as well as phage display antibodies, but also chimeric antibodies and specific fragments of the light and / or heavy chain of the variable
  • bispecific antibodies combine a defined immune cell activity with a specific tumor cell recognition, whereby tumor cells are killed.
  • a bispecific antibody binds on the one hand to a trigger molecule of the immune effector cell (e.g. CD3, CD16, CD64) and on the other hand to antigens of the tumor target cell.
  • Counter ions for ionic compounds are, for example, Na + , K + , Li + or cyclohexylammonium.
  • Suitable solid or liquid galenical forms of preparation are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions (iV, ip, i...) And Preparations with protracted release of active ingredients, which are common in their manufacture Aids such as carriers, disintegrants, binders, coating agents, swelling agents, lubricants or lubricants, flavoring agents, sweeteners and dissolving agents are used.
  • a pharmaceutical composition according to the invention can be produced by mixing at least one inhibitor used according to the invention in a defined dose with a pharmaceutically suitable and physiologically compatible carrier and, if appropriate, other suitable active ingredients, additives or auxiliaries with a defined inhibitor dose and preparing the desired dosage form.
  • Tumor cells express GSTM differentially if normal cells of the same tissue type (of the same or different volunteers) do not express it. Tumor cells overexpress GSTM specifically or differentially if GSTM is expressed at least twice as much as normal cells of the same tissue type.
  • Cytotoxic components or groups are compounds which directly or indirectly induce apoptosis or lead to necrosis or at least inhibit growth.
  • radioisotopes for example 188Re, 213Bi, 99mTc, 90Y, 131J, 177Lu
  • groups or compounds can in particular be cytostatics which are used in tumor therapy. Examples include: alkylating agents (e.g. mechlorethamine, ifosfamide, chlorambucil, Cyclophosphamide, melphalan, alkylsulfonates, busulphan, nitrosoureas, carmustine, lomustine, semustine, triazenes, dacarbazine), antimetabolites (e.g.
  • folic acid antagonists methotrexate, pyrimidine analogs, fluorouracil, fluorodeoxyuridine, cytarabine, gemitone purine, gemitone purine, gemitone purine, gemitone purine) , Mitosis inhibitors (e.g. vinca alkaloids, voncristin, vinblastine, paclitaxal, docetaxel, protaxel), epipodophyllotoxins (e.g. etoposide, teniposide), antibiotics (e.g. dactinomycin, daunorubicin, idarubicin, anthracycline, blearaginase), L-Asparaginase, L-Asparaginase
  • Platinum complex compounds e.g. cisplatin
  • hormones and related compounds e.g. adrenal cortex steroids, aminogluthetimide, progestogens, estrogens, androgens, antioestrogens, tamoxifen, steroid analogues, flutamide.
  • the coupling takes place in such a way that the affinity for GSTM is reduced by no more than 90%, preferably 50%, based on the substance without a cytostatic group, and the cytostatic activity of the group is not more than 90%, preferably 50%, based on the compound without substance, is reduced.
  • An immunostimulating component is usually a protein or an effective component thereof, which stimulates cells of the immune system.
  • cytokines such as M-CSF, GM-CSF, G-CSF, interferons such as IFN-alpha, beta, gamma, interleukins such as IL-1 to -16 (except -8), human LIF, chemokines such as Rantes, MCAF, MIP-1-alpha, -beta, NAP-1 and IL-8.
  • a reporter group is an atom, molecule or a compound which, in conjunction with an assay placed thereon, is used to detect the reporter group and thus compound or substance associated with the reporter group.
  • reporter groups and associated detection methods are: 32P labeling and intensity measurement using phosphoimager. Many other examples are known to the person skilled in the art and do not need to be listed in detail.
  • a substance that binds to GSTM can be a substance that binds to a GSTM protein or a GSTM RNA.
  • Definitions expanded in the context of the above definition in relation to the narrow sense of the word also include the specific terms in the narrow sense of the word.
  • Example 1 Examined tissue samples 46 tissues from 17 patients with non-invasive papillary pTA tumors at the time of collection were removed. The further course of the disease of all patients was monitored in the following three years and assigned to the tissue samples. Twenty-one of the tissues came from patients who had progressed to an invasive form of bladder cancer within three years. The remaining 24 tissues belonged to patients whose bladder tumor remained stable in the pTa stage during the same period.
  • Example 1 The samples from Example 1 were subjected to an expression analysis on GSTM using the GeneChip technology (Affimetrix). The results are shown in FIG. 3. The median expression values of different stages and subtypes of bladder carcinoma are shown. The values for the pTA tumors with and without subsequent progression are highlighted in dark. It can be seen that the median of the progressive tumors is 4.8 times higher than that of the non-progressive tumors, i.e. Expression of the GSTM gene is found almost exclusively in the tumor epithelium of patients in whom the tumor later took an invasive course. Specifically, GSTM was severely overexpressed in 17 of 21 tissues that showed progression to invasive tumors. In contrast, increased expression was only found in 8 of 24 tissues that showed a stable course.

Abstract

The invention relates to applications of GSTM for the diagnosis and treatment of urogenital tumours, in particular, cystic carcinomas and for screening for substances for such purposes.

Description

Verwendungen von an GSTM bindenden Substanzen zur Diagnose und Behandlung des Harnblasenkarzinoms . Uses of substances binding to GSTM for the diagnosis and treatment of bladder cancer.
Gebiet der ErfindungField of the Invention
Die Erfindung betrifft neue Verwendungen von GSTM oder daraus abgeleiteten Sequenzen zum Screenen nach daran bindenden Substanzen, sowie die Verwendung von an GSTM bin- denden Substanzen zur Diagnose und/oder Behandlung des Harnblasenkarzinoms .The invention relates to new uses of GSTM or sequences derived therefrom for screening for substances binding to it, and the use of substances binding to GSTM for the diagnosis and / or treatment of bladder carcinoma.
Hintergrund der Erfindung und Stand der TechnikBackground of the Invention and Prior Art
GSTM steht für Glutathion-S-Transferase Klasse μ member 4. GSTM gehört zu einer Multigenfamilie von Enzymen, die durch die Bindung von Glutathion an zelltoxische Substanzen deren Entgiftung in der Zelle einleiten. Weiterhin binden sie lipophile Substanzen und vermitteln derenGSTM stands for glutathione-S-transferase class μ member 4. GSTM belongs to a multi-gene family of enzymes which, by binding glutathione to cell-toxic substances, initiate their detoxification in the cell. Furthermore, they bind and mediate lipophilic substances
Löslichkeit. GST's einer Klasse bilden untereinander Ho o- oder Heterodimere, die verschiedene elektrophile Substanzen, so eine Reihe bekannter Karzinogene, prozessieren. Der stöchiometrische Anteil der M4-Variante ist dabei sehr gering (Rowe et al . , Bioche . J., 15, 325 (Pt2) : 481-486Solubility. GSTs of one class form ho or heterodimers with one another, which process various electrophilic substances, such as a number of known carcinogens. The stoichiometric proportion of the M4 variant is very low (Rowe et al., Bioche. J., 15, 325 (Pt2): 481-486
(1997)), so dass ihr Nachweise oft mit der Tumorentstehung assoziiert werden kann.(1997)), so that their evidence can often be associated with tumor development.
Die Expression von GSTM-Genen (hauptsächlich GSTM1) in Normal- und Tumorgeweben des Urogenitaltraktes ist sehr unterschiedlich. Man findet in Blasentumoren geringe Mengen an GSTM, einige, jedoch nicht alle niedriggradige Tu- more weisen dagegen hohe Expressionswerte dieses Moleküls auf. In hochgradigen, invasiven Formen konnte dagegen überhaupt kein GSTM in allenfalls geringen Mengen detek- tiert werden (Celis et al . , Cencer Res 15, 56 (20) : 4782-4790 (1996) ) . Insgesamt ergibt sich für die Expressionsmuster ein sehr heterogenes Bild, weshalb es nicht prognostizierbar ist, ob oder in welchen Tumoren Überexpression stattfindet und was eine solche Überexpression letzlich bedeutet .The expression of GSTM genes (mainly GSTM1) in normal and tumor tissues of the urogenital tract is very different. Small amounts of GSTM are found in bladder tumors, but some, but not all, low-grade tumors show high expression values of this molecule on. In high-grade, invasive forms, on the other hand, no GSTM could be detected at all in small amounts (Celis et al., Cencer Res 15, 56 (20): 4782-4790 (1996)). Overall, the expression pattern shows a very heterogeneous picture, which is why it cannot be predicted whether or in which tumors overexpression will take place and what such overexpression ultimately means.
Das Harnblasenkarzinom ist der zweithäufigste urologische Tumor. Es tritt bei Männern mit einer Häufigkeit von 245 zu 100000 und bei Frauen von 65 zu 100000 auf. Unter den durch Krebs verursachten Todesfällen nimmt das Blasenk- arzinom bei Männern die vierte und bei Frauen die sechste Position ein. Die Behandlung erfolgt zumeist durch Cystek- to ie, d.h. durch teilweise oder vollständige Entfernung der Harnblase. Dadurch ergeben sich nicht selten weitere Komplikationen für die erkrankte Person.Bladder cancer is the second most common urological tumor. It occurs in men with a frequency of 245 to 100,000 and in women from 65 to 100,000. Among cancer deaths, bladder cancer ranks fourth in men and sixth in women. Treatment is usually through cystectomy, i.e. by partially or completely removing the bladder. This often leads to further complications for the sick person.
Das Harnblasenkarzinom entwickelt sich durch Entartung einzelner Zellen des Urothels. Das Urothel ist die Zellschicht, die das Körperinnere gegen den gebildeten Urin abschirmt. Es kleidet das Lumen des Nierenbeckens, der Harnleiter, der Harnblase und der Harnröhre aus.Bladder cancer develops through the degeneration of individual urothelial cells. The urothelium is the layer of cells that shields the inside of the body from the urine that is formed. It lines the lumen of the renal pelvis, ureter, bladder and urethra.
Blasenkrebs entwickelt sich fast vollständig (>93%) als Adenokarzinom und ist gekennzeichnet durch die starke Tendenz zur Rezidivität, dem regele äßigen Wiederauftreten auch nach erfolgter Behandlung. In den Industrieländern wird in die Entsteheung von Blasenkrebs vor allem auf den Einfluss von chemischen Noxen, wie aromatischen Aminen, hauptsächlich aber auf das Rauchen als Ursache zurückgeführt. Er kann relativ früh diagnostiziert werden und zwei Verlaufsformen annehmen.Bladder cancer develops almost completely (> 93%) as adenocarcinoma and is characterized by the strong tendency to recurrence, the regular recurrence even after treatment. In industrialized countries, the development of bladder cancer is mainly due to the influence of chemical noxae, such as aromatic amines, but mainly to smoking as a cause recycled. It can be diagnosed relatively early and take two forms.
Die häufigere Variante, die der papillären oder oberfläch- liehen pTa-Zumoren, hat eine gute Prognose, da sie nicht invasiv ist und sich gut operativ entfernen läßt. Die in- vasiven Formen wachsen dagegen in das gewebe hinein und führen unbehandelt zu schwerster Symptomatik, wie Lymphknotenbefall und Metastasen. Die Todesfälle entsprin- gen fast ausschließlich dieser zweiten Gruppe. Kennzeichnend für den Verlauf ist, dass die papillären Tumoren sehr häufig stabil bleiben und somit keinen Progress zu den gefährlichen invasiven Formen zeigen. In 10-20% der Fälle kommt es aber im Verlauf von 5 Jahren zu einem Progress zu den aggresiven uskelinvasiven Tumoren.The more common variant, that of papillary or superficial pTa-Zumoren, has a good prognosis because it is not invasive and can be removed surgically. In contrast, the invasive forms grow into the tissue and, if left untreated, lead to the most severe symptoms, such as lymph node involvement and metastases. The deaths arise almost exclusively from this second group. It is characteristic of the course that the papillary tumors very often remain stable and therefore show no progress towards the dangerous invasive forms. In 10-20% of cases, however, there is a progression to aggressive, cancer-invasive tumors in the course of 5 years.
Eine Reihe von Genen wurde bisher auf die Eignung als prognostischer Marker im Harnblasenkarzinom untersucht. Hierbei ist vorrangig von Interesse, ob ein Patient mit nichtinvasiven, papillären Tumoren stabil in diesem Stadium verbleibt, oder ob er invasive Formen entwickeln wird. Zu den bisher untersuchten Markern gehören vor allem p53, p21 und das Retinoblastomgen Rb . Keiner der genannten Marker ermöglicht jedoch eine Prognose für einen individu- eilen Patienten mit ausreichender Sicherheit (Marberger et al., Eur Urol, 40/5, Curric Urol 1-9, (2001)). Neuere, kommerziell erhältliche Tests (BTA-STAT, NMP22) fokussieren sich eher auf den Nachweis eines Urotheliums als auf die Prognose des Verlaufes.A number of genes have so far been examined for their suitability as a prognostic marker in bladder cancer. It is of primary interest whether a patient with non-invasive, papillary tumors remains stable at this stage or whether he will develop invasive forms. The markers examined so far mainly include p53, p21 and the retinoblastoma gene Rb. However, none of the markers mentioned enables a prognosis for an individual patient with sufficient certainty (Marberger et al., Eur Urol, 40/5, Curric Urol 1-9, (2001)). More recent, commercially available tests (BTA-STAT, NMP22) focus more on the detection of urothelium than on the prognosis of the course.
Technisches Problem der Erfindung Der Erfindung liegt daher das technische Problem zugrunde, pharmazeutische Zusammensetzungen zur Diagnose, insbesondere zur Verlaufs- bzw. Progressionsprognose, und/oder zur Behandlung des Harnblasenkarzinoms anzugeben sowie Mittel zu deren Identifizierung.Technical problem of the invention The invention is therefore based on the technical problem of specifying pharmaceutical compositions for diagnosis, in particular for prognosis for progression or progression, and / or for treating bladder carcinoma and means for identifying them.
Grundzüge der Erfindung und bevorzugte Ausführungsformen.Basics of the invention and preferred embodiments.
Zur Lösung dieses technischen Problems lehrt die Verwendung einer für GSTM codierenden Nukleinsäure und/oder eines GSTM Peptids oder Proteins zur Detektion des Harnblasenkarzinoms oder zur Detektion eines Risikos der Erkrankung an einem solchen Karzinom oder zur Detektion eines Risikos einer Progression eines papillären Harn- balsenkarzinoms zu einem invasiven Karzinom, wobei eine Urothelzellen-Gewebeprobe, insbesondere eine Harnblasen- Urothelzellen-Gewebeprobe, auf Transkription oder Übertranskription von GSTM RNA oder auf Expression oder Über- expression eines GSTM Proteins untersucht wird. Eine an für GSTM codierende Nukleinsäure oder eine an GSTM Protein oder Peptid bindende Detektorsubstanz, vorzugsweise enthaltend eine Reportergruppe, kann verwendet werden, wobei Bindung besagter Nukleinsäure und/oder besagten Proteins oder Peptids an die Detektorsubstanz halbquantitativ oder quantitativ detektiert wird. In diesem Zusammenhang lehrt die Erfindung weiterhin ein Testsystem zur (in vitro) Detektion eines vorstehend genannten Karzinoms oder eines Risikos der Erkrankung hieran oder der Progressionsprog- nose, enthaltend Mittel zur quantitativen Messung der Expression von GSTM in Gewebeproben, wobei diese Mittel beispielsweise Mittel zur Amplifikation und spezifischen Detektion von GSTM RNA und/oder eine Detektorsubstanz, insbesondere spezifisch für GSTM Protein, sein können.To solve this technical problem, the use of a nucleic acid coding for GSTM and / or a GSTM peptide or protein for the detection of bladder carcinoma or for the detection of a risk of the disease of such a carcinoma or for the detection of a risk of a progression of papillary urinary carcinoma to one teaches invasive carcinoma, a urothelial cell tissue sample, in particular a urinary bladder urothelial cell tissue sample, being examined for transcription or over-transcription of GSTM RNA or for expression or over-expression of a GSTM protein. A nucleic acid coding for GSTM or a detector substance binding to GSTM protein or peptide, preferably containing a reporter group, can be used, whereby binding of said nucleic acid and / or said protein or peptide to the detector substance is detected semi-quantitatively or quantitatively. In this context, the invention further teaches a test system for (in vitro) detection of a carcinoma mentioned above or a risk of the disease thereof or prognosis progression, comprising means for quantitative measurement of the expression of GSTM in tissue samples, these means being, for example, means for amplification and specific Detection of GSTM RNA and / or a detector substance, in particular specific for GSTM protein, can be.
Die Erfindung lehrt weiterhin die Verwendung einer GSTM RNA oder eines GSTM Proteins oder Peptids zum Screenen nach daran bindenden Substanzen, insbesondere prospektiven Wirkstoffen zur Modulierung, insbesondere Inhibierung, von besagter RNA oder besagtem Protein oder Peptid, oder pro'spektiven Detektorsubstanzen, wobei eine prospektive Substanz oder eine Mischung solcher prospektiver Substanzen mit besagter RNA oder besagtem Protein oder Peptid kontaktiert wird, wobei mit einem Bindungsassay Bindungsereignisse festgestellt werden, und wobei eine bindende prospektive Substanz, ggf. nach Dekonvolutierung, selek- tiert wird. In diesen Zusammenhängen lehrt die Erfindung weiterhin ein Screeningsystem zur Ermittlung von für die Behandlung von vorstehenden Tumorerkrankungen geeigneten Wirksubstanzen enthaltend eine GSTM Nukleinsäure oder ein GSTM Protein bzw. Peptid, Mittel zur Bestimmung von (in vitro) Bindungsereignissen an die GSTM Nukleinsäure oder an das GSTM Protein bzw. Peptid, und/oder Mittel zur Bestimmung der (in vitro) Aktivität von GSTM Protein. Hierbei kann GSTM in einem zellfreien oder einem zellbasierten System, letzteres insbesondere aufweisend Urothelzellen des Urogenitaltraktes, insbesondere der Harnblase, bzw. eine hieraus entwickelte Zelllinie, vorliegen. Mittel zur Bestimmung von Bindungsereignissen können beispielsweise natürlicherweise in normalen oder in Tumorzellen z.B. an GSTM Protein bindende Substanzen bzw. Assoziationspartner umfassen, wobei über deren (freie) Konzentration bzw. Konzentrationsänderung bei Zugabe prospektiver Wirksubstanzen und/oder Detektorsubstanzen eine ko petitive Bindung einer bindenden Wirk- oder Detektorsubstanz bestimmt wird. Solche Mittel können aber auch physikalische bzw. physikalisch-chemische Methoden umfassen, wie beispielsweise Röntgenstrukturanalyse und/oder NMR, insbesondere zweidimensionale 1H/1H oder 15N/1H oder 14C/1H Korrelationsspektroskopie. Hierbei werden Spektren vor und nach der Zugabe einer prospektiven Wirk- oder Detektorsubstanz miteinander verglichen und im Falle von Änderungen ist ein Bindungsereignis festgestellt. Es kann mit Spektren oder dergleichen entweder von GSTM oder der prospektiven Substanz oder mit einer Kombination aus bei- dem gearbeitet werden. Selbstverständlich sind auch alle anderen fachüblichen Methoden der Bestimmung von Bindung- sereignissen und/oder Proteinaktivitäten einsetzbar. Beispielsweise kann eine prospektive Substanz (oder me- hrere Substanzen, räumlich voneinander getrennt) immobilisiert sein, wobei dann markiertes GSTM aufgetragen wird. Ein Bindungsereignis wird dann nach Auftrag und folgender Spülung durch Detektion, ggf. ortlich aufgelöst, der Markierung gebundenen FABP4s festgestellt. Umgekehrt kann GSTM immobilisiert sein und es wird eine markierte prospektive Substanz oder eine Mischung hieraus aufgetragen. Bindungsereignisse werden analog der vorstehenden Variante festgestellt .The invention further teaches the use of a GSTM RNA or a GSTM protein or peptide for screening for substances which bind to it, in particular prospective active ingredients for modulating, in particular inhibiting, said RNA or said protein or peptide, or pro-prospective detector substances, a prospective substance or a mixture of such prospective substances is contacted with said RNA or said protein or peptide, binding events being ascertained using a binding assay, and a binding prospective substance being selected, if appropriate after deconvolution. In these contexts, the invention further teaches a screening system for determining active substances suitable for the treatment of the above tumor diseases, comprising a GSTM nucleic acid or a GSTM protein or peptide, means for determining (in vitro) binding events to the GSTM nucleic acid or to the GSTM protein or peptide, and / or means for determining the (in vitro) activity of GSTM protein. Here, GSTM can be present in a cell-free or a cell-based system, the latter in particular having urothelial cells of the urogenital tract, in particular the urinary bladder, or a cell line developed therefrom. Means for determining binding events can, for example, naturally include substances or association partners which bind naturally in normal cells or in tumor cells, for example to GSTM protein, with their (free) concentration or change in concentration when prospective active substances and / or detector substances are added to allow a binding binding of a binding active substance. or detector substance is determined. Such means can also include physical or physico-chemical methods, such as X-ray structure analysis and / or NMR, in particular two-dimensional 1H / 1H or 15N / 1H or 14C / 1H correlation spectroscopy. Here, spectra are compared with each other before and after the addition of a prospective active substance or detector substance, and in the event of changes, a binding event is determined. Spectra or the like of either GSTM or the prospective substance or a combination of the two can be used. Of course, all other standard methods of determining binding events and / or protein activities can also be used. For example, a prospective substance (or several substances, spatially separated from one another) can be immobilized, in which case marked GSTM is applied. A binding event is then determined after application and subsequent rinsing by detection, possibly locally resolved, of the label-bound FABP4s. Conversely, GSTM can be immobilized and a labeled prospective substance or a mixture thereof is applied. Binding events are determined analogously to the variant above.
Die Erfindung lehrt schließlich die Verwendung einer GSTM inhibierenden oder daran bindenden Substanz zur Herstellung einer pharmazeutischen Zusammensetzung zur Behandlung und/oder Diagnose des Harnblasenkarzinoms bzw. der Progressionsprognose bei Harnblasenkarzinom-Erkrankungen.Finally, the invention teaches the use of a substance which inhibits or binds to GSTM for the production of a pharmaceutical composition for the treatment and / or diagnosis of bladder carcinoma or prognosis of progression in bladder carcinoma diseases.
Die Substanz kann ein Antikörper sein, welcher durch Immunisierung eines nicht-menschlichen Säugetiers mit einem GSTM Peptid oder Protein, mit hierfür codierender cDNA transfizierte Zellen, mit endogen ein solches Peptid oder Protein exprimierenden Tumorzellen, oder mit rekombinant hergestellten GSTM Peptiden oder Proteinen, erhältlich ist, oder ein Phage-Display-Antikörper sein. Die Substanz kann aber auch eine Mimikryverbindung eines Antikörpers gegen ein GSTM Peptid oder Protein sein. Die Substanz kann schließlich ein Aptamer, eine antisense RNA, ein Ribozym oder eine siRNA gegen GSTM Nukleinsäuren sein. Die Substanz kann zusätzlich eine zytotoxische und/oder immun- stimulierende Komponente tragen.The substance can be an antibody which is obtained by immunizing a non-human mammal with a GSTM peptide or protein with cDNA coding therefor transfected cells, with tumor cells expressing such a peptide or protein endogenously, or with recombinantly produced GSTM peptides or proteins, or a phage display antibody. However, the substance can also be a mimicry compound of an antibody against a GSTM peptide or protein. Finally, the substance can be an aptamer, an antisense RNA, a ribozyme or an siRNA against GSTM nucleic acids. The substance can additionally carry a cytotoxic and / or immune-stimulating component.
Bevorzugt ist es, wenn die vorstehenden, an GSTM Protein bindenden Substanzen in der Verwendung zu therapeutischen Zwecken spezifisch an das GSTM Protein binden und es in seiner biologischen Aktivität modulieren. Dies ist nicht erforderlich im Falle der Fusion bzw. Verbindung der Substanz mit einer zytotoxischen Komponente. Dies ist weiterhin nicht erforderlich, wenn die Substanz der Gewinnung eines anti-idiotypischen Antikörpers dient, welcher vom Immunsystem eines Patienten aufgrund seiner nicht- humanisierten Form als körperfremd erkannt wird und dem Immunsystem ansonsten ein GSTM-Antigen präsentiert.It is preferred if the above substances binding to GSTM protein specifically bind to the GSTM protein when used for therapeutic purposes and modulate its biological activity. This is not necessary in the case of fusion or connection of the substance with a cytotoxic component. This is also not necessary if the substance is used to obtain an anti-idiotypic antibody which is recognized by a patient's immune system as foreign to the body due to its non-humanized form and which otherwise presents a GSTM antigen to the immune system.
Die pharmazeutische Zusammensetzung kann zur beliebigen Applikation, beispielsweise i.v. oder i.p. Injektion, hergerichtet sein. Eine Herrichtung zur lokalen Applikation in Tumorzellen enthaltendem Gewebe wird sich empfehlen im Falle des Einsatzes einer zytotoxischen Komponente.The pharmaceutical composition can be used for any application, for example i.v. or i.p. Injection, be prepared. Preparation for local application in tissue containing tumor cells is recommended if a cytotoxic component is used.
Die Erfindung läßt sich im Rahmen eines Verfahrens zur Diagnose bzw. Progressionsprognose einer Tumorerkrankung des Harnblasenkarzinoms verwenden, wobei eine Detektorsubstanz in einer Ausführungsform mit einer Reportergruppe in zu untersuchendes Gewebe, ggf. in vitro nach Gewebeentnahme, appliziert wird, wobei das zu untersuchende Gewebe dann einer Detektionsverfahrenstufe unter- worfen wird, welche sensitiv für die Reportergruppe ist, und wobei im Fall der Detektion eines definierten Mindestwertes der Reportergruppe im Gewebe das Gewebe als Tumorzellen enthaltend qualifiziert bzw. als prdgressionsgefährdet oder nicht progressionsgef hrdet eingestuft wird, sowie eines Verfahrens zur Behandlung einer Harnblasentumor-Erkrankung, wobei ' eine erfindungs- gemäße pharmazeutische Zusammensetzung in einer physiologisch wirksamen Dosis einem Patienten dargereicht wird. Im Falle der Diagnose bzw. Progressionsprognose kann zusätzlich oder alternativ eine Gewebeprobe mit einem erfindungsgemäßen Testsystem auf GSTM Expression untersucht werden .The invention can be used in the context of a method for the diagnosis or prognosis of progression of a tumor disease of the bladder carcinoma, whereby one Detector substance is applied in one embodiment with a reporter group in the tissue to be examined, if appropriate in vitro after tissue removal, the tissue to be examined then being subjected to a detection method stage which is sensitive to the reporter group, and in the case of the detection of a defined one minimum value of the reporter group in the tissue, the tissue is classified as containing tumor cells or classified as prdgressionsgefährdet or not hrdet progressionsgef, and a method for treatment of a urinary bladder tumor disease, wherein 'a Inventions contemporary pharmaceutical composition is administered in a physiologically effective dose to a patient. In the case of diagnosis or prognosis, a tissue sample can additionally or alternatively be examined for GSTM expression using a test system according to the invention.
Die Erfindung beruht insbesondere auf der Erkenntnis, daß GSTM in verschiedenen papillären Tumoren des Harnblasenkarzinoms unterschiedlich exprimiert wird, i.e. in besagten Tumorgeweben ist die Expression im Falle solcher papillären Karzinome, die in späteren Verlauf invasiv werden, höher, verglichen mit normalen Zellen gleichen Gewebes, und im Falle solcher papillärer Karzinome, die stabil bleiben, dagegen niedrig, und der daraus herleitbaren technische Lehre, daß GSTM als Zielmolekül bei der Diagnostik, insbesondere Progressionsprognose, und Therapie bzw. Prophylaxe insbesondere der invasiven Tumorer- krankungen eingesetzt werden kann. GSTM kann also als spezifischer Marker zur Identifizierung von Tumorzellen in den besagten Tumorgeweben dienen, welche ein Risiko aufweisen, invasive Tumorgewebe zu bilden. Auf der anderen Seite bietet die Inhibierung von GSTM die Möglichkeit, in die Tumor-spezifischen GSTM Assoziationen mit anderen Prozessen in den Tumorzellen einzugreifen und somit letztendlich den tumorzellenspezifisch veränderten Stoffwechsel zu stören und zu einem Absterben oder zumindest einer Wachstumshemmung der Tumorzellen, insbsondere aber einer Hemmung der Progression zu invasiven Tumoren, beizutragen.The invention is based in particular on the knowledge that GSTM is expressed differently in different papillary tumors of bladder carcinoma, ie in said tumor tissues the expression is higher in comparison with normal cells of the same tissue in the case of such papillary carcinomas which later become invasive, and in the case of papillary carcinomas which remain stable, on the other hand, low, and the technical teaching which can be derived therefrom that GSTM can be used as a target molecule in diagnosis, in particular prognosis of progression, and therapy or prophylaxis, in particular of invasive tumor diseases. GSTM can thus serve as a specific marker for identifying tumor cells in said tumor tissues which have a risk of forming invasive tumor tissues. On the other On the one hand, the inhibition of GSTM offers the opportunity to intervene in the tumor-specific GSTM associations with other processes in the tumor cells and thus ultimately to disrupt the tumor cell-specific metabolism and to die or at least inhibit the growth of the tumor cells, but especially to inhibit the progression invasive tumors.
Im Rahmen der Efindung kann es sich empfehlen, im Vorfeld einer Behandlung mit einer erfindungsgemäßen pharmazeutischen Zusammensetzung eine Probe aus einem Gewebe, welches als Tumorgewebe mit anderen Methoden identifiziert ist, zu entnehmen und die Gewebeprobe auf Expression bzw. Überexpression von GSTM zu untersuchen. Alternativ kann mit einer erfindungsgemäßen Detektorsubstanz zur Diagnose in vivo auf GSTM Abhängigkeit getestet werden. Wird eine Expression bzw. Überexpression von GSTM gegenüber Normalgewebe gleichen Typs festgestellt, so ist die Anwendung der erfindungsgemäßen pharmazeutischen Zusammensetzung indiziert .Within the scope of the invention, it may be advisable to take a sample from a tissue which is identified as tumor tissue by other methods in advance of treatment with a pharmaceutical composition according to the invention and to examine the tissue sample for expression or overexpression of GSTM. Alternatively, a detector substance according to the invention can be used to test in vivo for GSTM dependency. If an expression or overexpression of GSTM with respect to normal tissue of the same type is found, the use of the pharmaceutical composition according to the invention is indicated.
Generell ist es im Rahmen der Erfindung möglich, patientenspezifisch auf differentielle Expression zu unter- suchen, wobei Normalgewebeprobe und Tumorgewebeproben bzw. tumorverdächtige Gewebeproben dem (gleichen) Patienten entnommen und vergleichend auf Werte der GSTM Expression untersucht werden.In general, it is possible within the scope of the invention to examine patient-specifically for differential expression, normal tissue sample and tumor tissue samples or suspected tumor samples being taken from the (same) patient and being compared for values of the GSTM expression.
Handelt es sich bei dem Tumor um einem Typus, bei welchem Tumorzellen GSTM exprimieren, Normalzellen gleichen Gewebetyps jedoch nicht, so ist es besonders bevorzugt, wenn die an GSTM bindende Substanz zusätzlich eine zytotoxische und/oder immunstimulierende Komponente trägt. Dies führt dann letztendlich dazu, dass praktisch ausschließlich Tumorzellen getötet werden, sei es durch die Zytotoxizität, sei es durch Angriff durch das stimulierte Immunsystem, während Normalzellen in dem Gewebe praktisch vollständig erhalten bleiben. In dieser Ausführungsform braucht die bindende Substanz selbst nicht inhibierend auf GSTM zu wirken, da die bindende Substanz dann lediglich als Marker funktionieren muß, welcher die Komponenten zu Ziel- Tumorzellen trägt. Im Falle des Einsatzes einer zytotoxischen Komponente wird es sich besonders empfehlen, wenn die pharmazeutische Zusammensetzung zur lokalen Applikation in Tumorzellen enthaltendem Gewebe hergerichtet ist, beispielsweise zur Injektion.If the tumor is a type in which tumor cells express GSTM, but normal cells of the same tissue type are not, it is particularly preferred if the substance binding to GSTM is additionally a cytotoxic and / or immunostimulating component. This ultimately leads to the fact that almost exclusively tumor cells are killed, either by cytotoxicity or by attack by the stimulated immune system, while normal cells are practically completely preserved in the tissue. In this embodiment, the binding substance itself does not have to have an inhibitory effect on GSTM, since the binding substance then only has to function as a marker which carries the components to target tumor cells. If a cytotoxic component is used, it will be particularly recommended if the pharmaceutical composition is prepared for local application in tissue containing tumor cells, for example for injection.
Definitionen und weitere Ausführungsformen der Erfindung.Definitions and further embodiments of the invention.
Die Nukleinsäure- sowie Proteinsequenzen von zwei GSTM 1 Varianten, einer GSTM2, 3 GSTM4 Varianten und einer GSTM5 sind in den Figuren la-g (RNA) und 2a-g (Protein) dargestellt (Seq.-ID Nos. 1 bis 14) .The nucleic acid and protein sequences of two GSTM 1 variants, a GSTM2, 3 GSTM4 variants and a GSTM5 are shown in Figures la-g (RNA) and 2a-g (protein) (SEQ ID Nos. 1 to 14).
Im Rahmen dieser Beschreibung wird die Bezeichnung GSTM für alle humanen Isoformen, bekannt oder neu, auf Nukleinsäuren- oder Aminosäurenbasis, verwendet. Mit diesen Begriffen mit umfaßt sind auch die im Rahmen dieser Beschreibung offenbarten kurzen Sequenzen, welche aus den Isoformen stammen, beispielsweise Immunisierungssequenzen. Weiterhin mit umfaßt sind auch Homologe, wobei die Homologie zumindest 80%, vorzugsweise mehr als 90%, höchstvor- zugsweise mehr als 95%, beträgt, berechnet mit dem Programm MEGALIGN (DNASTAR LASERGENE) in der zum Zeitpunkt der vorliegenden Anmeldung aktuellen Fassung. Im Falle der Nukleinsäuresequenzen sind auch komplementäre oder al- lelische Varianten mit umfaßt. Weiterhin sind Sequenzen umfaßt, welche lediglich Teilsequenzen der explizit offen- 5 barten Sequenzen, beispielsweise ein Exon oder mehrere Exons, oder komplementärer Sequenzen hierzu darstellen, mit der Maßgabe, daß diese Teilsequenzen im Falle der Nukleinsäuren eine für eine Hybridisierung mit einer er- fin'dungsgemäßen Nukleinsäure hinreichende Länge, zumindestIn the context of this description, the term GSTM is used for all human isoforms, known or new, based on nucleic acids or amino acids. These terms also include the short sequences disclosed in the context of this description, which originate from the isoforms, for example immunization sequences. Also included are homologs, the homology being at least 80%, preferably more than 90%, most preferably more than 95%, calculated with the MEGALIGN (DNASTAR LASERGENE) program at the time the present application current version. In the case of the nucleic acid sequences, complementary or allelic variants are also included. Furthermore, comprising sequences that only partial sequences of the explicit disclosure, 5 are disclosed sequences, for example, an exon or more exons, or complementary sequences thereto, with the proviso that these partial sequences in the case of nucleic acids, one for hybridization with a ER- fin ' nucleic acid of the invention sufficient length, at least
10 50 Basen, aufweisen und im Falle der Proteine bzw. Peptide mit zumindest gleicher Affinität an ein protein- oder pep- tidspezifisches Zielmolekül binden. Weiterhin sind alle mit erfindungsgemäßen Nukleinsäuren hybridisierende Nukleinsäuren umfaßt, nämlich solche, die unter stringenten10 50 bases, and, in the case of proteins or peptides, bind to a protein- or peptide-specific target molecule with at least the same affinity. Furthermore, all nucleic acids hybridizing with nucleic acids according to the invention are included, namely those which are stringent
15 Bedingungen (5°C bis 25°C unterhalb der AufSchmelztemperatur; siehe ergänzend J.M. Sambrook et al . , A laboratory manual, Cold Spring Harbor Laboratory Press (1989) und E.M. Southern, J Mol Biol, 98:503ff (1975)) hybridisieren. Es versteht sich, daß die Erfindung auch Expressionskas- 0 setten umfaßt, i.e. eine oder mehrere der erfindungsgemäßen Nukleinsäuresequenzen mit mindestens einer operativ verbundenen Kontroll- oder regulatorischen Sequenz. Eine solche Expressionskassette kann auch eine Sequenz für ein bekanntes Protein umfassen, wobei im Zuge 5 der Translation ein Fusionsprotein aus einem bekannten Protein und einem erfindungsgemäßen Protein oder Peptid entsteht. Ebenso sind auch antisense Sequenzen zu den vorstehenden Nukleinsäuresequenzen umfaßt. Schließlich sind RNA sowie damit korrelierende DNA und umgekehrt umfaßt, 0 ebenso wie genomische DNA als auch korrelierte cDNA und umgekehrt. Im Rahmen der Erfindung können auch GSTM Homo- oder Het- erodimere verwendet werden. Insofern umfaßt der Begriff GSTM auch solche Homo- oder Heterodimere .15 conditions (5 ° C to 25 ° C below the melting temperature; see in addition JM Sambrook et al., A laboratory manual, Cold Spring Harbor Laboratory Press (1989) and EM Southern, J Mol Biol, 98: 503ff (1975)) hybridize , It goes without saying that the invention also includes expression cassettes, ie one or more of the nucleic acid sequences according to the invention with at least one operatively linked control or regulatory sequence. Such an expression cassette can also comprise a sequence for a known protein, a fusion protein being formed in the course of the translation from a known protein and a protein or peptide according to the invention. Antisense sequences to the above nucleic acid sequences are also included. Finally, RNA as well as correlating DNA and vice versa are included, as well as genomic DNA as well as correlated cDNA and vice versa. GSTM homo- or heterodimers can also be used within the scope of the invention. In this respect, the term GSTM also includes such homo- or heterodimers.
Im Zusammenhang mit erfindungsgemäßen Verwendungen umfassen die Begriffe der GSTM Nukleinsäuren oder Protein bzw. Peptide neben den Volllängen der offenbarten Sequenzen (siehe auch vorstehender Absatz) auch Teilsequenzen hieraus, und zwar mit einer Mindestlänge von 12 bis 30 Nuk- leotiden, vorzugsweise 30 bis 90 Nukleotiden, im Falle der Nukleinsäuren und einer Mindestlänge von 4 bis 10 Aminosäuren, vorzugsweise 10 bis 30 Aminosäuren, im Falle der Peptide oder Proteine. Diese Teilsequenzen können in ansonsten von GSTM verschiedene Nukleinsäuren- oder Protein- bzw. Peptidsequenzen eingebaut sein.In connection with uses according to the invention, the terms GSTM include nucleic acids or protein or peptides in addition to the full lengths of the disclosed sequences (see also the preceding paragraph) and also partial sequences thereof, with a minimum length of 12 to 30 nucleotides, preferably 30 to 90 nucleotides , in the case of nucleic acids and a minimum length of 4 to 10 amino acids, preferably 10 to 30 amino acids, in the case of peptides or proteins. These partial sequences can be incorporated into nucleic acid or protein or peptide sequences that are otherwise different from GSTM.
Der Begriff der Behandlung umfaßt auch die Prophylaxe, insbesondere die Prophylaxe der Progression zu invasiven Tumoren.The term treatment also includes prophylaxis, especially prophylaxis of progression to invasive tumors.
Als Inhibitor ist eine Verbindung oder Substanz bezeichnet, welche entweder die Bildung von GSTM Protein inhibiert oder gebildetes GSTM Protein in der Aktivität reduziert, bezogen auf die GSTM Aktivität in Abwesenheit des Inhibitors. Insofern kann ein Inhibitor einerseits eine Substanz sein, welche in der Entstehungskaskade von GSTM inhibierend eingreift. Auf der anderen Seite kann ein Inhibitor eine Substanz sein, welche mit gebildetem GSTM eine Bindung eingeht, und zwar dergestalt, dass weitere physiologische Wechselwirkungen mit endogenen Substanzen zumindest reduziert sind. Mimikry-Moleküle sind Verbindungen, die den variablen Bereich, insbesondere den Bindungsbereich eines Antikörpers, nachbilden und an gleicher Stelle eines Zielmoleküls binden, wie der zu Grunde liegende Antikörper.An inhibitor is a compound or substance which either inhibits the formation of GSTM protein or reduces the activity of GSTM protein formed, based on the GSTM activity in the absence of the inhibitor. In this respect, an inhibitor can be a substance that interferes with the GSTM cascade. On the other hand, an inhibitor can be a substance that binds with the GSTM formed, in such a way that further physiological interactions with endogenous substances are at least reduced. Mimicry molecules are compounds that simulate the variable region, in particular the binding region of an antibody, and bind to a target molecule in the same place as the underlying antibody.
Der Begriff der Antikörper umfaßt polyklonale Antikörper, monoklonale Antikörper, nicht-humane, humane und humanisierte Antikörper, sowie Phage-Display-Antikörper, aber auch chimäre Antikörper sowie spezifische Fragmente der leichten und/oder der schweren Kette des variablenThe term antibodies includes polyclonal antibodies, monoclonal antibodies, non-human, human and humanized antibodies, as well as phage display antibodies, but also chimeric antibodies and specific fragments of the light and / or heavy chain of the variable
Bereiches zu Grunde liegender Antikörper vorstehender Art sowie anti-idiotypische Antikörper. Die Herstellung bzw. Gewinnung solcher Antikörper mit vorgegebenen Immunogenen ist dem Durchschnittsfachmann wohl vertraut und braucht nicht näher erläutert zu werden. Weiterhin umfaßt der Begriff der Antikörper bispezifische Antikörper. Bispezifische Antikörper kombinieren eine definierte Immunzellaktivität mit einer spezifischen Tumorzellerkennung, wodurch Tumorzellen getötet werden. Ein bispezi- fischer Antikörper bindet einerseits an ein Auslösemolekül der Immun-Effektorzelle (z.B. CD3, CD16, CD64) und andererseits an Antigene der Tumorzielzelle.Range of underlying antibodies of the above type and anti-idiotypic antibodies. The production or production of such antibodies with predetermined immunogens is well known to the person skilled in the art and need not be explained in more detail. The term antibody also includes bispecific antibodies. Bispecific antibodies combine a defined immune cell activity with a specific tumor cell recognition, whereby tumor cells are killed. A bispecific antibody binds on the one hand to a trigger molecule of the immune effector cell (e.g. CD3, CD16, CD64) and on the other hand to antigens of the tumor target cell.
Die galenische Herrichtung einer erfindungsgemäßen phar- mazeutischen Zusammensetzung kann in fachüblicher Weise erfolgen. Als Gegenionen für ionische Verbindungen kommen beispielsweise Na+, K+, Li+ oder Cyclohexylammonium infrage. Geeigente feste oder flüssige galenische Zubereitungsfor- men sind beispielsweise Granulate, Pulver, Dragees, Ta- bletten, (Mikro-) Kapseln, Suppositorien, Sirupe, Säfte, Suspensionen, Emulsionen, Tropfen oder injizierbare Lösungen (i.V., i.p., i. .) sowie Präparate mit protrahierter Wirkstoff-Freigabe, bei deren Herstellung übliche Hilfsmittel wie Trägerstoffe, Spreng-, Binde-, Überzugs-, Quellungs-, Gleit- oder Schmiermittel, Geschmacksstoffe, Süßungs ittel und Lösungsverrαittler, Verwendung finden. Als Hilfsstoffe sei Magnesiumcarbonat, Titandioxyd, Lac- tose, Mannit und andere Zucker, Talcum, Milcheiweiß, Gelatine, Stärke, Zellulose und ihre Derivate, tierische und pflanzliche Öle wie Lebertran, Sonnenblumen-, Erdnuss- oder Sesamöl, Polyethylenglycole und Lösungsmittel, wie etwa steriles Wasser und ein- oder mehrwertige Alkohole, beispielsweise Glycerin, genannt. Eine erfindungsgemäße pharmazeutische Zusammensetzung ist dadurch herstellbar, dass mindestens ein erfindungsgemäß verwendeter Inhibitor in definierter Dosis mit einem pharmazeutisch geeigneten und physiologisch verträglichen Träger und ggf. weiteren geeigneten Wirk-, Zusatz- oder Hilfsstoffen mit definierter Inhibitordosis gemischt und zu der gewünschten Darreichungsform hergerichtet ist.The pharmaceutical preparation of a pharmaceutical composition according to the invention can be carried out in a customary manner. Counter ions for ionic compounds are, for example, Na + , K + , Li + or cyclohexylammonium. Suitable solid or liquid galenical forms of preparation are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions (iV, ip, i...) And Preparations with protracted release of active ingredients, which are common in their manufacture Aids such as carriers, disintegrants, binders, coating agents, swelling agents, lubricants or lubricants, flavoring agents, sweeteners and dissolving agents are used. Magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talcum, milk protein, gelatin, starch, cellulose and their derivatives, animal and vegetable oils such as cod liver oil, sunflower, peanut or sesame oil, polyethylene glycols and solvents, such as, for example, may be used as auxiliary substances sterile water and monohydric or polyhydric alcohols, for example glycerol. A pharmaceutical composition according to the invention can be produced by mixing at least one inhibitor used according to the invention in a defined dose with a pharmaceutically suitable and physiologically compatible carrier and, if appropriate, other suitable active ingredients, additives or auxiliaries with a defined inhibitor dose and preparing the desired dosage form.
Tumorzellen exprimieren GSTM differenziell, wenn Normal- zellen des gleichen Gewebetyps (des gleichen oder verschiedener Probanden) dieses nicht exprimieren. Tumorzellen überexprimieren GSTM spezifisch bzw. differen- ziell, wenn GSTM im Vergleich zu Normalzellen des gleichen Gewebetyps zumindest in doppelter Menge exprimiert wird.Tumor cells express GSTM differentially if normal cells of the same tissue type (of the same or different volunteers) do not express it. Tumor cells overexpress GSTM specifically or differentially if GSTM is expressed at least twice as much as normal cells of the same tissue type.
Zytotoxische Komponenten bzw. Gruppen sind Verbindungen, welche direkt oder indirekt Apoptose einleiten bzw. zu Nekrose führen oder zumindest wachstumshemmend wirken. Solche Gruppen bzw. Verbindungen können neben Radioiso- topen (z.B. 188Re, 213Bi, 99mTc, 90Y, 131J, 177Lu) insbesondere Zytostatika sein, welche in der Tumortherapie eingesetzt werden. Beispiele hierfür sind: Alkylantien (z.B. Mechlorethamin, Ifosfamid, Chlorambucil, Cyclophosphamid, Melphalan, Alkylsulfonate, Busulphan, Nitrosoharnstoffe, Carmustin, Lomustin, Semustin, Tri- azene, Dacarbazin) , Antimetaboliten (z.B. Folsäure- Antagonisten, Methotrexat, Pyrimidin-Analoga, Fluoruracil, Fluordesoxyuridin, Cytarabin, Gemcitabin, Purin-Analoga, Mercaptopurin) , Mitosehemmer (z.B. Vincaalkaloide, Von- cristin, Vinblastin, Paclitaxal, Docetaxel, Protaxel) , Epipodophyllotoxine (z.B. Etoposid, Teniposid) , Antibiotika (z.B. Dactinomycin, Daunorubicin, Idarubicin, An- thracycline, Bleomycin, L-Asparaginase) ,Cytotoxic components or groups are compounds which directly or indirectly induce apoptosis or lead to necrosis or at least inhibit growth. In addition to radioisotopes (for example 188Re, 213Bi, 99mTc, 90Y, 131J, 177Lu), such groups or compounds can in particular be cytostatics which are used in tumor therapy. Examples include: alkylating agents (e.g. mechlorethamine, ifosfamide, chlorambucil, Cyclophosphamide, melphalan, alkylsulfonates, busulphan, nitrosoureas, carmustine, lomustine, semustine, triazenes, dacarbazine), antimetabolites (e.g. folic acid antagonists, methotrexate, pyrimidine analogs, fluorouracil, fluorodeoxyuridine, cytarabine, gemitone purine, gemitone purine, gemitone purine, gemitone purine) , Mitosis inhibitors (e.g. vinca alkaloids, voncristin, vinblastine, paclitaxal, docetaxel, protaxel), epipodophyllotoxins (e.g. etoposide, teniposide), antibiotics (e.g. dactinomycin, daunorubicin, idarubicin, anthracycline, blearaginase), L-Asparaginase, L-Asparaginase
Platinkomplexverbindungen (z.B. Cisplatin) , Hormone und verwandte Verbindungen (z.B. Nebennierenrindensteroide, Aminogluthetimid, Gestagene, Östrogene, Androgene, An- tiöstrogene, Tamoxifen, Steriodanaloga, Flutamid) . Bei Bindung einer solchen Verbindung mit einer an GSTM bindenden Substanz erfolgt die Kopplung dergestalt, daß die Affinität zu GSTM um nicht mehr als 90%, vorzugsweise 50%, bezogen auf die Substanz ohne zytostatische Gruppe, reduziert ist und die zytostatische Wirkung der Gruppe um nicht mehr als 90%, vorzugsweise 50%, bezogen auf die Verbindung ohne Substanz, reduziert ist.Platinum complex compounds (e.g. cisplatin), hormones and related compounds (e.g. adrenal cortex steroids, aminogluthetimide, progestogens, estrogens, androgens, antioestrogens, tamoxifen, steroid analogues, flutamide). When such a compound is bound to a substance which binds to GSTM, the coupling takes place in such a way that the affinity for GSTM is reduced by no more than 90%, preferably 50%, based on the substance without a cytostatic group, and the cytostatic activity of the group is not more than 90%, preferably 50%, based on the compound without substance, is reduced.
Eine immunstimulierende Komponente ist meist ein Protein oder ein wirksamer Bestandteil hiervon, welches Zellen des Immunsystems stimuliert. Beispiele hierfür sind: Zytokine, wie M-CSF, GM-CSF, G-CSF, Interferone, wie IFN-alpha, -beta, -gamma, Interleukine wie IL-1 bis -16 (außer -8) , human LIF, Chemokine wie Rantes, MCAF, MIP-1-alpha, -beta, NAP-1 und IL-8.An immunostimulating component is usually a protein or an effective component thereof, which stimulates cells of the immune system. Examples include: cytokines such as M-CSF, GM-CSF, G-CSF, interferons such as IFN-alpha, beta, gamma, interleukins such as IL-1 to -16 (except -8), human LIF, chemokines such as Rantes, MCAF, MIP-1-alpha, -beta, NAP-1 and IL-8.
Eine Reportergruppe ist ein Atom, Molekül oder eine Verbindung, welche in Verbindung mit einem hierauf abgestellten Assay den Nachweis der Reportergruppe und der somit mit der Reportergruppe verbundenen Verbindung oder Substanz ermöglicht. Beispiele für Reportergruppen und hiermit assoziierte Detektionsmethoden sind: 32P-Labeling und Intensitätsmessung mittels Phosphoimager . Viele weitere Beispiele sind dem Durchschnittsfachmann bekannt und bedürfen nicht der detaillierten Aufzählung.A reporter group is an atom, molecule or a compound which, in conjunction with an assay placed thereon, is used to detect the reporter group and thus compound or substance associated with the reporter group. Examples of reporter groups and associated detection methods are: 32P labeling and intensity measurement using phosphoimager. Many other examples are known to the person skilled in the art and do not need to be listed in detail.
Eine an GSTM bindende Substanz kann eine Substanz sein, welche an ein GSTM Protein oder eine GSTM RNA bindet.A substance that binds to GSTM can be a substance that binds to a GSTM protein or a GSTM RNA.
Im Rahmen der vorstehenden Definition gegenüber dem engen Wortsinn erweiterte Begriffsbestimmungen umfassen auch die bestimmten Begriffe im engen Wortsinn.Definitions expanded in the context of the above definition in relation to the narrow sense of the word also include the specific terms in the narrow sense of the word.
Beispiele .Examples.
Im Folgenden wird die Erfindung anhand von lediglich bevorzugte Ausführungsformen darstellenden Beispielen und Figuren näher erläutert. Es zeigen:The invention is explained in more detail below on the basis of examples and figures which merely illustrate preferred embodiments. Show it:
Fig. 1: Nukleinsäuresequenzen von GSTMFigure 1: GSTM nucleic acid sequences
Fig. 2: Aminosäurensequenzen von GSTMFigure 2: Amino acid sequences from GSTM
Fig. 3: Expressionsanalyse von GSTM, insbesondere GSTM4, in verschiedenen Stadien und Subtypen des Harnblasenkarzinoms,3: Expression analysis of GSTM, in particular GSTM4, in different stages and subtypes of bladder carcinoma,
Beispiel 1: Untersuchte Gewebeproben Es wurden 46 Gewebe von 17 Patienten mit zum Zeitpunkt der Erhebung nichtinvasiven, papillären pTA-Tumoren entnommen. Der weitere Krankheitsverlauf aller Patienten wurde in den folgenden drei Jahren überwacht und den Gewebeproben zugeordnet. 21 der Gewebe stammten dabei von Patienten, die innerhalb der drei Jahre Progress zu einer invasiven Form des Harnblasenkarzinoms zeigten. Die verbliebenen 24 Gewebe gehörten zu Patienten, deren Blasentumor in gleichen Zeitraum stabil im pTa-Stadium verblieb.Example 1: Examined tissue samples 46 tissues from 17 patients with non-invasive papillary pTA tumors at the time of collection were removed. The further course of the disease of all patients was monitored in the following three years and assigned to the tissue samples. Twenty-one of the tissues came from patients who had progressed to an invasive form of bladder cancer within three years. The remaining 24 tissues belonged to patients whose bladder tumor remained stable in the pTa stage during the same period.
Beispiel 2: Expressionsprofile der untersuchten GewebeExample 2: Expression profiles of the examined tissues
Die Proben aus Beispiel 1 wurden einer Expressionsanalyse auf GSTM mittels der GeneChip-Technologie (Affimetrix) unterworfen. Die Ergebnisse sind in der Figur 3 dargestellt. Dargestellt sind die medianen Expression- swerte verschiedener Stadien und Subtypen des Harnblasenkarzinoms. Die Werte für die pTA Tumoren mit und ohne späterer Progradienz sind dunkel hervorgehoben. Man erkennt, dass der Mediän der progradienten Tumoren 4,8x höher als der der nicht-progradienten Tumoren ist, i.e. Expression des Gens GSTM fast ausschließlich im Tumorepithel von Patienten gefunden wird, bei denen der Tumor zu einem späteren Zeitpunkt einen invasiven Verlauf nahm. Im Einzelnen wurde in 17 von 21 Geweben, die Progression zu invasiven Tumoren zeigten, GSTM stark überexprimiert . Demgegenüber wurde erhöhte Expression nur in 8 von 24 Geweben gefunden, die stabilen Verlauf zeigten. The samples from Example 1 were subjected to an expression analysis on GSTM using the GeneChip technology (Affimetrix). The results are shown in FIG. 3. The median expression values of different stages and subtypes of bladder carcinoma are shown. The values for the pTA tumors with and without subsequent progression are highlighted in dark. It can be seen that the median of the progressive tumors is 4.8 times higher than that of the non-progressive tumors, i.e. Expression of the GSTM gene is found almost exclusively in the tumor epithelium of patients in whom the tumor later took an invasive course. Specifically, GSTM was severely overexpressed in 17 of 21 tissues that showed progression to invasive tumors. In contrast, increased expression was only found in 8 of 24 tissues that showed a stable course.

Claims

Patentansprüche : Claims:
1. Verwendung einer für GSTM, insbesondere GSTM4, codier- enden Nukleinsäure und/oder eines GSTM, insbesondere1. Use of a nucleic acid coding for GSTM, in particular GSTM4, and / or a GSTM, in particular
GSTM4, Peptids oder Proteins zur Detektion von Tumoren des Urogenitaltraktes, insbesondere des Harnblasenkarzinoms, oder zur Detektion eines Risikos der Erkrankung an -einem solchen Tumor oder zur Detektion eines Risikos einer Progression eines papillären Urogenitaltumors zu einem invasiven Karzinom, wobei eine Urothelzellen- Gewebeprobe, insbesondere eine Harnblasen-Urothelzellen- Gewebeprobe, auf Transkription oder Übertranskription von GSTM RNA oder auf Expression oder Überexpression eines GSTM Proteins untersucht wird.GSTM4, peptide or protein for the detection of tumors of the genitourinary tract, in particular of the bladder carcinoma, or for the detection of a risk of the disease of such a tumor or for the detection of a risk of a progression of a papillary urogenital tumor to an invasive carcinoma, wherein a urothelial cell tissue sample, in particular a urinary bladder urothelial cell tissue sample is examined for transcription or over-transcription of GSTM RNA or for expression or overexpression of a GSTM protein.
2. Verwendung nach Anspruch 1, wobei eine an für GSTM codierende Nukleinsäure oder eine an GSTM Protein oder Pep- tid bindende Detektorsubstanz, vorzugsweise enthaltend eine Reportergruppe, verwendet wird, wobei Bindung besagter Nukleinsäure und/oder besagten Proteins oder Peptids an die Detektorsubstanz halbquantitativ oder quantitativ detektiert wird.2. Use according to claim 1, wherein a nucleic acid coding for GSTM or a detector substance binding to GSTM protein or peptide, preferably containing a reporter group, is used, binding of said nucleic acid and / or said protein or peptide to the detector substance semi-quantitative or is detected quantitatively.
3. Verwendung einer GSTM, insbesondere GSTM4, RNA oder eines GSTM, insbesondere GSTM4, Proteins oder Peptids zum Screenen nach daran bindenden Substanzen, insbeson- dere nach prospektiven Wirkstoffen zur Inhibierung von besagter RNA oder besagtem Protein oder Peptid oder nach prospektiven Detektorsubstanzen, wobei eine prospektive Substanz oder eine Mischung solcher prospektiver Substanzen mit besagter RNA oder besagtem Protein oder Peptid kontaktiert wird, wobei mit einem Bindungsassay Bindungsereignisse festgestellt werden, und wobei eine bindende prospektive Substanz, ggf. nach Dekonvolu- tierung, selektiert wird.3. Use of a GSTM, in particular GSTM4, RNA or a GSTM, in particular GSTM4, protein or peptide for screening for substances which bind to it, in particular for prospective active substances for inhibiting said RNA or said protein or peptide or for prospective detector substances, one prospective substance or a mixture of such prospective Substances are contacted with said RNA or said protein or peptide, binding events being ascertained using a binding assay, and a binding prospective substance being selected, if appropriate after deconvoluting.
4. Verwendung einer GSTM, insbesondere GSTM4, inhibier- -enden oder daran bindenden Substanz zur Herstellung - einer pharmazeutischen Zusammensetzung zur Behandlung von Urogenitaltumoren, insbesondere des Harnblasenkarzinoms oder zur Herstellung einer pharmazeutischen Zusammensetzung zur Diagnose eines solchen Tumors oder zur Diagnose eines Progressionsrisikos eines nicht invasiven solchen Tumors zu einer invasiven Form.4. Use of a GSTM, in particular GSTM4, inhibiting or binding substance for the production of a pharmaceutical composition for the treatment of urogenital tumors, in particular urinary bladder cancer or for the production of a pharmaceutical composition for the diagnosis of such a tumor or for the diagnosis of a progression risk of a non-invasive such tumor into an invasive form.
5. Verwendung nach Anspruch 4, wobei die Substanz ein Antikörper ist, welcher beispielsweise durch Immunisierung eines nicht-menschlichen Säugetiers mit einem GSTM Peptid oder Protein, mit GSTM transfizierten Zellen, oder einer hierfür für codierenden cDNA, erhältlich ist, oder ein Phage-Display Antikörper ist.5. Use according to claim 4, wherein the substance is an antibody which can be obtained, for example, by immunizing a non-human mammal with a GSTM peptide or protein, cells transfected with GSTM, or a cDNA coding therefor, or a phage display Is antibody.
6. Verwendung nach Anspruch 4, wobei die Substanz eine Mimikriverbindung eines Antikörpers gegen ein GSTM Peptid oder Protein ist.6. Use according to claim 4, wherein the substance is a facial expression compound of an antibody against a GSTM peptide or protein.
7. Verwendung nach Anspruch 4, wobei die Substanz, ein Aptamer, eine antisense RNA, eine siRNA, oder ein Ri- bozym ist. 7. Use according to claim 4, wherein the substance is an aptamer, an antisense RNA, an siRNA, or a ribozyme.
Verwendung nach einem der Ansprüche 4 bis 7, • wobei die Substanz zusätzlich eine zytotoxische und/oder immunstimulierende Komponente trägt. Use according to one of claims 4 to 7, • wherein the substance additionally carries a cytotoxic and / or immunostimulating component.
9. Verwendung nach einem der Ansprüche 4 bis 8, wobei die pharmazeutische Zusammensetzung zur lokalen Applikation -in Tumorzellen enthaltendem Gewebe hergerichtet ist.9. Use according to one of claims 4 to 8, wherein the pharmaceutical composition is prepared for local application in tissue containing tumor cells.
10. Verfahren zur Diagnose einer Urogenitaltumorerkrankung, insbesondere eines Harnblasenkarzinoms, oder des Risikos der Progression eines solchen Tumors zu einer invasiven Form wobei eine an GSTM, insbesondere GSTM4, bindende Detektorsubstanz in einer Ausführungsform mit einer Reportergruppe in zu untersuchendes Gewebe appliziert wird, wobei das zu untersuchende Gewebe dann einer Detektionsverfahren- stufe unterworfen wird, welche sensitiv für die Reportergruppe ist, und wobei im Fall der Detektion eines definierten Mindestwertes der Reportergruppe im Gewebe das Gewebe als Tumorzellen enthaltend qualifiziert oder als progressionsgefährdet wird.10. A method for diagnosing a urogenital tumor disease, in particular a bladder carcinoma, or the risk of such a tumor progressing to an invasive form, wherein a detector substance which binds to GSTM, in particular GSTM4, is applied in one embodiment with a reporter group to the tissue to be examined, with the addition of examining tissue is then subjected to a detection method step which is sensitive to the reporter group, and in the case of detection of a defined minimum value of the reporter group in the tissue the tissue is qualified as containing tumor cells or is at risk of progression.
11. Verfahren zur Behandlung einer Urogenitaltumorerkrankung, insbesondere eines Harnblasenkarzinoms, wobei eine pharmazeutische Zusammensetzung nach einem der Ansprüche 4 bis 9 in einer physiologisch wirksamen Dosis und galenisch für die anzuwendende Darreichungsform hergerichtet einem Patienten dargereicht wird. 11. A method for treating a urogenital tumor disease, in particular a bladder carcinoma, wherein a pharmaceutical composition according to any one of claims 4 to 9 in a physiologically effective dose and galenically prepared for the dosage form to be administered is given to a patient.
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