WO2003102173A2

WO2003102173A2 - Protein or peptide (mimpd), nucleic acid that codes therefor, and uses of these substances

Info

Publication number: WO2003102173A2
Application number: PCT/DE2003/001824
Authority: WO
Inventors: Bernd Hinzmann; Edgar Dahl; Christian Pilarsky; Thomas Specht; Andr� ROSENTHAL; Rosemarie Lichtner; Irina Klamann; Katrin Gottlob; Karin Blechschmidt; Anke Stein; Georg Beckmann; Armin Schmidt
Original assignee: Metagen Pharmaceuticals Gmbh; Edgar Dahl
Priority date: 2002-05-31
Filing date: 2003-05-30
Publication date: 2003-12-11
Also published as: WO2003102173A8; AU2003243913A1; DE10225180A1

Abstract

.

Description

Protein or peptide (MIMPD), nucleic acid coding therefor and uses of these substances.

Field of the Invention

The invention relates to a new protein, hereinafter referred to as MIMPD, partial sequences thereof, nucleic acids coding for MIMPD or for partial sequences thereof, new uses of MIMPD or sequences derived therefrom for screening for substances binding to it, and the use of substances binding to MIMPD for diagnosis and / or or treatment of tumor diseases.

Background of the Invention and Prior Art

From the references DW Waggoner et al. , Biochem Biophys Acta 1439: 299-316 (1999) and H. Kanoh et al. , Biochem Biophys Acta 1348: 56-62 (1997) a lipid phosphate phosphatase family is known, which are also referred to as PAP (phosphatic acid phosphatases). PAPs catalyze the conversion of phosphatidic acid to diacylglycerols and inorganic phosphate. Diacylglycerols play an important role in cell signaling and should therefore be of importance in signal transduction. Other lipid phosphates such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (SIP) are also PAP substrates. The cleavage of LPA and SIP by PAP prevents their binding to specific receptors in the cell membrane and thus inhibits the receptor-mediated signal transduction. From the bibliography of biochemistry and molecular biology, Herder, 1992, 3rd volume, it is known that acidic serum phosphatases are suitable as indications for prostate cancer.

A sequence is known from reference US Pat. No. 6, 225, 054, there sequence 275, which has approximately 90% sequence identity with a partial sequence of the new protein MIMPD described below in the context of the invention. Furthermore, the differential expression of said sequence in breast tumor and breast normal tissue is known from this literature reference.

Cancer is a disease with a considerable incidence as it ages. So far, cancer has been diagnosed essentially pathologically and mostly treated by removing the malignant tissue. Removal of organs usually has various adverse effects on a patient. Improved diagnosis and treatment of cancer, particularly without the need to remove an organ or parts thereof, is therefore highly desirable.

Technical problem of the invention

The invention is based on the technical problem of specifying pharmaceutical compositions for diagnosing and / or treating cancer and means for identifying them. Fundamentals of the invention and preferred exemplary embodiments.

The invention first teaches a nucleic acid containing at least one partial sequence with a length of at least 12 to 21 nucleotides from SEQ ID 1 or 16. Of particular interest is a partial sequence or a partial partial sequence thereof up to the stop codon TGA starting at nucleotide 1166. In contrast, the sequence 275 known from the literature US Pat. No. 6,225,054 lies beyond the stop codon, ie in the untranslated 3 'region (3'-UTR) of the overall sequence SEQ ID 1 according to the invention.

The nucleic acid sequences according to the invention are cDNA or genomic DNA which codes for a previously unknown PAP, hereby called MIMPD. The sequence of the new PAP is given in SEQ ID 2. The invention therefore also relates to a peptide or protein containing at least one partial sequence with a length of at least 4 to 7 amino acids from SEQ-ID 2.

The lower limits of the lengths of the partial sequences from the sequences given in the sequence listing can also be 12 to 30 (nucleic acids; NS) or 4 to 10 (amino acids; AS). Lower limits of 60 or 90 (NS) or 20 or 30 (AS) are also possible. It is understood that lower limits higher than 21 (NS) or 7 (AS) can only be used if the sequence used from the sequence listing has the required number of NS or AS.

MIMPD is an integral membrane protein with probably six transmembrane regions, with three loops being formed are which are on the outside of the membrane. The invention therefore also relates to proteins or peptides containing at least one partial sequence with a length of at least 7 amino acids from one or more of the sequences SEQ.ID 3-9 (membrane-external regions) and / SEQ.ID 10-15 (conserved PAP Partial sequences), or consisting of one or more such partial sequences.

The partial sequences can preferably have a length of at least 15 or 30 amino acids or 45 or 90 nucleotides. In the extreme case of the sequence length, however, they can also be formed as a whole by the specified sequences. In the embodiments in which the nucleic acids or protein or peptides contain the said sequences or partial sequences, any connecting sequences can be added to one or both ends of the sequences. A wide variety of non-natural molecules or chemical groups can also be connected or connected to the connection sequences.

In the context of the invention it was found that MIMPD is expressed differentially in a wide variety of tumor tissues. An essential area of application of the invention is therefore to be seen in the diagnosis and / or treatment of cancer, as well as a target or tool for finding active substances for the diagnosis and / or treatment of cancer.

Specifically, the invention therefore also teaches the use of a nucleic acid coding for MIMPD and / or a MIMPD peptide or protein for the detection of cancer or for the detection of a risk of the disease of cancer, a tissue sample for over-transcription of MIMPD RNA or for overexpression of a MIMPD protein or peptide is examined. A nucleic acid coding for MIMPD or a detector substance binding to MIMPD protein or peptide, preferably containing a reporter group, can be used, binding of said nucleic acid and / or said protein or peptide to the detector substance being detected semi-quantitatively or quantitatively.

The invention further teaches the use of a MIMPD RNA or a MIMPD 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, a prospectively binding substance or a mixture thereof Substances are contacted with said RNA or said protein or peptide, binding events being determined using a binding assay, and a binding prospective substance, optionally after deconvolution, being selected as a substance suitable for detecting or treating cancerous diseases.

The invention further teaches the use of a MIMPD inhibiting or binding substance for producing a pharmaceutical composition for the detection and / or treatment of cancer. The substance can be an antibody which is obtained by immunizing a non-human mammal with a MIMPD peptide or protein, or with cells transiently or stably transfected with cDNA of MIMPD (eg tumor cell lines, NIH3T3 cells), or with endogenously expressing MIMPD or with in MIMPD produced by insect cells or a phage display is antibody. MIMPD cDNA in non-human mammals can also be used for immunization.

However, the substance can also be a mimicry compound of an antibody against a MIMPD peptide or protein. Alternatively, the substance can be an aptamer, an antisense RNA, an inhibitory RNA, or a ribozyme against MIMPD. The substance can additionally carry a cytotoxic, radioactive and / or immunostimulating component. The pharmaceutical composition can be prepared for local or systemic application in tissue containing tumor cells.

The invention further relates to a method for diagnosing cancer, wherein an inventive detector substance in one embodiment with a reporter group is applied to the tissue to be examined, in vitro or in vivo, the tissue to be examined then being subjected to a detection method step which is sensitive to the Is 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.

Finally, the invention relates to a method for treating a cancer, wherein a pharmaceutical composition according to the invention is administered to a patient in a physiologically effective dose.

The invention can be used in particular for the following cancers: breast cancer, uterine cancer, Colon cancer, stomach cancer, ovarian cancer, lung cancer and / or rectal cancer.

The invention is based on the knowledge that MIMPD is overexpressed or differentially expressed in the tumor tissues mentioned, i.e. In said tumor tissues, the expression is higher or can be observed at all, compared to normal cells of the same tissue, and the technical teaching that can be derived therefrom that MIMPD can be used as a target molecule in the diagnosis and therapy of these diseases. MIMPD can thus serve as a marker for the identification of tumor cells in said tumor tissues. On the other hand, the inhibition of MIMPD, especially in the case of local application, offers the possibility to intervene in tumor-specific MIMPD associations with other processes in the tumor cells and thus ultimately to disrupt the metabolism which is changed for the tumor cell and to die or at least inhibit growth Contribute to tumor cells. The inhibition of MIMPD can also be used in combination with chemotherapy or radiotherapy.

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 and to examine the tissue sample for expression or overexpression of MIMPD prior to treatment with a pharmaceutical composition according to the invention. Alternatively, a detector substance according to the invention can be used to test in vivo for MIMPD dependency. Is an expression or overexpression of MIMPD compared to normal tissues of the same type or an expression at all determined, the use of the pharmaceutical composition according to the invention is indicated.

If the tumor is a type in which tumor cells express MIMPD, but normal cells of the same tissue type do not, it is particularly preferred if the substance binding to MIMPD additionally carries a cytotoxic, radioactive 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 MIMPD, since the binding substance then only has to function as a marker which carries the components to target tumor cells. If a cytotoxic, radioactive and / or immunostimulating 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.

Definitions.

In the context of this description, the term MIMPD is used as a generic term for all human isoforms, splice variants and regulatory RNA, based on nucleic acids or amino acids. These terms also include the short sequences disclosed in the context of this description. Also included are homologs, the homology being at least 80%, preferably more than 90%, most preferably more than 95%, but not sequence 275 from US 6,225,054. In the case of the nucleic acid sequences, complementary or allelic variants are also included. Furthermore, sequences are included which are only partial sequences, for example one or more exons, of the explicitly disclosed sequences or complementary sequences, with the proviso that, in the case of the nucleic acids, these partial sequences are of sufficient length for hybridization with a nucleic acid according to the invention, have at least 50 bases and, in the case of the proteins or peptides, optionally coded by a nucleic acid, bind to a protein- or peptide-specific target molecule with at least the same affinity. It is understood that the invention also includes expression cassettes, ie one or more of the nucleic acid sequences according to the invention with at least one 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 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. Also included are expression vectors containing nucleic acids according to the invention and host cells transformed therewith, for example

Mammalian cells, e.Coli or vertebrate cells. RNA and correlating DNA and vice versa are also included, as are genomic DNA and correlated cDNA and vice versa. Finally, based on amino acid (part), sequences include the nucleic acids coding for it. An inhibitor is a compound or substance which either inhibits the formation of MIMPD or reduces the activity of MIMPD formed, based on the MIMPD in vitro or in vivo activity in the absence of the inhibitor. In this respect, an inhibitor can be a substance that interferes with the cascade of MIMPD. On the other hand, an inhibitor can be a substance that binds with the MIMPD 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.

The term antibodies encompasses human, humanized and non-humanized, polyclonal or monoclonal antibodies and phage display antibodies, but also anti-idiotypic or chimeric antibodies and specific fragments of the light and / or heavy chain of the variable region lying antibody of the above type. The production or production of such antibodies with given immunogens is well known to the average person skilled in the art and need not be explained in more detail. Also included are bispecific antibodies which on the one hand bind to a trigger molecule of an immune effector cell (eg CD3, CD16, CD64) and on the other hand to an antigen according to the invention, for example a loop on the outside of the tumor target cell. In the event of binding, this ultimately causes the tumor cell to be killed. 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 pharmaceutical preparation forms are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions (IV, IP, IM) as well as preparations with a protracted active ingredient release , usual in their manufacture

Auxiliaries such as carriers, disintegrants, binders, coating agents, swelling agents, lubricants or lubricants, flavoring agents, sweeteners and solubilizers can be 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 mono- or polyhydric alcohols, such as glycerin. A pharmaceutical composition according to the invention can be produced by mixing at least one MIMPD inhibitor 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 MIMPD differentially if normal cells of the same tissue type do not express it. Tumor cells overexpress MIMPD specifically or differentially if MIMPD compared to normal cells of the same tissue is expressed in higher, for example at least double, amount.

Cytotoxic components or groups are compounds which directly or indirectly induce apoptosis or at least inhibit growth. In addition to radioisotopes (eg 188Re, 213Bi, 99mTc, 90Y, 131J, 177Lu), such groups or compounds can in particular be cytostatics, including so-called prodrugs, which are used in tumor therapy. Examples of these are: alkylating agents (for example mechlorethamine, ifosfamide, chlorambucil, cyclophosphamide, melphalan, alkylsulfonates, busulphan, nitrosoureas, carmustine, lomustine, semustin, triazines, dacarbazine), antimetabolites (for example folic acid antagonists, methotacrexilators, pyrimuridine analogs, pyrurine , Fluorodeoxyuridine, cytarabine, gemcitabine, purine analogues, mercaptopurine), mitosis inhibitors (e.g. vinca alkaloids, vincristine, vinblastine, paclitaxal, docetaxel, protaxel), epipodophyllotoxins (e.g. etoposide, teniposide), antibiotics, eg antibiotic, eg antibiotic, eg antibiotics, eg antibiotics, , Antracycline, bleomycin, L-asparaginase), platinum complex compounds (eg cisplatin), hormones and related compounds (eg adrenal gland steroids, aminogluthetimide, progestogens, estrogens, androgens, antioestrogens, tamoxifen, steroid analogues, flutamide). When such a compound is bound to a substance that binds to MIMPD, the coupling takes place in such a way that the affinity for MIMPD is not reduced by 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. Examples of these are: cytokines such as M-CSF, GM-CSF, G-CSF, interferons such as IFN-alpha, beta, gam a, 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, enables the detection of the reporter group and the compound or substance thus connected to 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 average person skilled in the art and do not require a detailed list.

A substance that binds to MIMPD can be a substance that binds a MIMPD protein or MIMPD RNA.

The term external to the membrane denotes parts of a protein or peptide located in or on a membrane which do not span the membrane or are not built into the membrane. The term external membrane is not limited to one side of the membrane; rather, a part outside the membrane can be arranged on any side of the membrane.

Detailed description of the invention as well as examples. The invention is explained in more detail below on the basis of examples and figures which merely illustrate preferred embodiments. Show it:

1: MIMPD cDNA (a) and MIMPD amino acid sequence (b),

2: genomic organization of MIMPD,

3: topology model of MIMPD (a) and sequence comparison MIMPD with conserved phosphatase motifs (b),

4: Chip-based expression profile for breast cancer,

5: cancer tissue profiling,

FIG. 6: Quantifications from FIG. 5 for breast cancer (a), colon cancer (b), lung cancer (c), ovarian cancer (d), endometrial cancer (e), stomach cancer (f), rectal cancer

(g) •

7: in situ hybridization in uterus (7a) and breast (7b) samples,

Fig. 8-14: see example 8

15: a hammerhead ribozyme which cuts MIMPD mRNA,

Fig. 16: antisense sequences.

FIG. 1 a shows the MIMPD cDNA sequence according to the invention and FIG. 1 b encodes it Amino acid sequence shown. These sequences correspond to SEQ ID 1 and SEQ ID 2, respectively.

The genomic organization of the MIMPD gene is shown in FIG. It is 133 kb in size and contains seven exons. The 5 'and 3' non-translated areas (UTR) are of similar size. The locus of the gene is 10q26.13.

FIG. 3a shows a topology model that was created with TMHMM Vers. 2.0 (A. Krogh et al., J. Mol. Biol. 305 (3): 567-580 (2001). A total of six transmebrane areas can be seen 7 membrane-outside areas are formed. In particular, the membrane-outside areas are suitable as targets for diagnostics and / or therapeutics. Figure 3b compares areas of MIMPD with conserved phosphatase areas according to the literature reference J. Stukey et al., Protein Sei. 6: 469- 472 (1997).

Example 1: Chip-based expression profile for breast cancer.

There were microdissections from breast tumor tissue and

Breast normal tissue, if possible from the same patient, performed and the RNA isolated from these tissue samples. Expression profiles of a total of 52 breast tissues were examined using a DNA chip based on Affymetrix technology. The results are summarized in FIG. 4. MIMPD mRNA was widely expressed in 84% ^' (26/31) of the analyzed invasive ductal breast cancers (IDC), while the MIMPD mRNA expression in Normal tissue was below the detection limit in virtually all cases (12/13). Expression in invasive lobular breast cancer (ILC) and ductal cancer in situ (DCIS) was found, but is not as prevalent as in the case of IDC. In detail, the results of paired tissue samples from nine different patients can be seen on the left in FIG. 4, the low values all coming from normal tissue and the high values from paired tumor tissue. The right side of FIG. 4 shows results from unpaired samples, as already explained above. NE is normal epithelium, N1 are breast carcinomas with lymph node metastases, NO are breast cancer without lymph node metastases, Nx are samples whose lymph node status is unknown.

Example 2: Overexpression of MIMPD in various tumor tissues.

A cancer profiling array (commercial product from Clontech) with 240 applied human cDNAs from tumor and corresponding normal tissues of one patient each was analyzed using a 32P-labeled MIMPD-specific probe using a phosphoimager. FIG. 5 (N = normal tissue, T = tumor tissue) shows a comparatively strong overexpression in almost all examined tumor tissues. The right column shows controls and expression in various human tumor cell lines. A labeling control for the hybridization is shown at the top right; 1 pg MIMPD plasmid DNA was easily detected using the labeled sample. In detail, the results from FIG. 5 were quantified in accordance with FIGS. 6a to 6g and Table 1. A differential expression (pos) was assumed with at least 2-fold overexpression of MIMPD in tumor tissue compared to normal tissue.

Table 1

Tumor tumor: tression abs. Overexpression rel ppooss ANumber / Total [%]

Chest 43/50

Colon 25/35 71

Lungs 12/21 48 ovary 5/14 36

Endomet] trium 19/42 45

Stomach 16/27 59

Rectum 13/18 72

Figures 6a to 6g show the above results in a graphical representation and in tabular order.

Example 3: In vivo diagnosis of MIMPD using 7 antibodies

In this example, the labeling of a tumor or its metastases by an anti-MIMPD antibody in vivo (mouse model) is described. 2,000,000 MIMPD-transfected human cells or endogenously expressing cells are transplanted into NMRI nude mice. The mice are labeled with antibodies 30 days after the transplant injected. The control animals are treated with an irrelevant antibody. A few hours after the antibody application, the animals are sacrificed and all organs removed. The relative accumulation in metastases or other organs is measured. Enrichment should mainly take place in the tumor.

The anti-MIMPD antibodies are monoclonal antibodies against human MIMPD protein, conjugated to a carrier protein, in a non-human mammal. The production of monoclonal antibodies is known to those of ordinary skill in the art. MIMPD peptides, recombinant MIMPD protein and cells transiently or stably transfected with the MIMPD cDNA or the MIMPD cDNA can be used for the immunization.

Example 4: Immunohistochemical detection of MIMPD in tumor cells.

Primary tumors are isolated from the patients with tumors and prepared as paraffin or frozen sections. These sections are examined with an anti-MIMPD antibody for the overexpression of MIMPD in tumor cells. In addition, 2 million MIMPD-transfected human cells or endogenously expressing human cells are transplanted into NMRI nude mice. One month after the transplant, the mice are sacrificed and tumor tissue removed and prepared as paraffin or frozen sections.

The immunohistological examination with the MIMPD antibody shows higher expression of MIMPD in the tumor cells compared to surrounding normal tissue. The The investigation is carried out in detail by incubation with the anti-MIMPD antibody as primary antibody, a biotinylated secondary antibody, which is directed against the species from which the primary antibody was obtained, and a streptavidin-coupled horseradish peroxidase. The coloring is done with DASS as a chromogenic substrate (brown coloring). Counterstaining is done with hemalaun solution (blue staining). Malignant and non-malignant cells can be distinguished, the malignant cells having a strong staining, ie high MIMPD content, while the non-malignant cells are only moderately stained.

In addition, the tissue sections can also be examined using immunofluorescence. For this purpose, the anti-MIMPD antibody is either labeled directly with a fluorescent dye or with a secondary fluorescence-labeled antibody which is directed against the species from which the primary antibody was obtained. The evaluation is carried out by means of fluorescence microscopy or by means of laser scanning microscopy.

Example 5: RNA inhibitors

FIG. 8 shows a hammerhead ribozyme which cuts MIMPD at the point shown and thus inhibits or at least reduces the activity of any translation products. Suitable antisense sequences are, for example, those of FIG. 9 which lead to their degradation after hybridization to the MIMPD mRNA.

Example 6: RNA in situ hybridization, ovarian carcino Tissue samples from ovarian cancer tissue were incubated with a MIMPD-specific probe and stained with BM-Purple. The counterstaining was done with Kernecht-Rot. Malignant and non-malignant epithelial cells were distinguishable, the malignant cells having a strong BM-Purple staining, which indicates a strong expression of MIMPD RNA in the stained tumor cells. In contrast, the staining and thus expression of MIMPD in normal breast epithelial cells was only very weak. The RNA in situ hybridization thus confirms the overexpression of MIMPD RNA in tumors compared to corresponding normal tissues (see FIG. 7a).

Example 7: RNA in situ hybridization, breast cancer

The procedure was as in Example 6, except that breast tissue was used. 7b, tumor cells of an invasive ductal breast cancer can be seen which have a strong BM-purple staining, which is evidence of a strong MIMPD RNA expression in these tumor cells. the second picture shows the corresponding hybridization with the sense RNA as a control. As expected, no coloration can be seen here. The third picture shows a hematoxylin / eosin staining of the corresponding tumor area.

Example 8: additional experiments, results and sequences.

Some additional experiments, results and sequences are described below. FIG. 7c: for exemplary embodiments 6 and 7 (in situ hybridization, breast cancer)

7c summarizes the evaluation of RNA in situ hybridizations of breast cancer tissue: Samples of normal and tumor tissue from the same patient were analyzed, a total of material from 34 patients, each with 2 normal and 2 tumor samples. Expression of MIMPD RNA in the tumor tissue is detectable in 50% of the cases, while the associated normal tissue of the patient has no MIMPD RNA expression.

FIG. 8: RNA in situ hybridization, ovarian carcinoma. In FIG. 8, tumor cells of a serous papillary adenocarcinoma of the ovary can be seen, which have a BM-Purple staining, which is evidence of MIMPD RNA expression in these tumor cells. The second picture shows the corresponding hybridization with the sense RNA as a control. As expected, no coloration can be seen here. The third picture shows a hematoxylin / eosin staining of the corresponding tumor area.

Figure 9: RNA in situ hybridization; lung cancer

In FIG. 9, cells of a squamous cell carcinoma of the lungs can be seen, which have a BM-Purple staining, which is evidence of MIMPD RNA expression in these tumor cells. The second picture shows the corresponding hybridization with the sense RNA as a control. As expected, no coloration can be seen here. The third picture shows a hematoxylin / eosin staining of the corresponding tumor area.

Figure 10: RNA in situ hybridization, xenografts. In addition, xenografts of the breast tumor cell line MDA-MB-468, which are orthotopic in the breast of NMRI nude mice were injected, incubated with a MIMPD-specific probe and stained with BM-Purple. There was a counterstaining with Kernecht-Rot. The sections obtained are shown in FIG. It can be seen in the MDA-MB-468 xenografts that MIMPD is overexpressed in xenografts at the RNA level. Tumor cells show a strong expression of MIMPD (dark blue staining). The control hybridization with the sense RNA in the second picture shows no staining. The third picture shows a ha-toxilin / eosin staining of the sample.

Figure 11: Phosphatase assay

To analyze the phosphatase activity of MIMPD, inorganic phosphate is quantified by enzymatic reaction from substrates such as lipid phosphates (phosphatidyl acid, lysophosphatidyl acid, ceramide 1-phosphate, sphingosine 2-phosphate, diacylglycerol pyrophosphate) or isoprenyl pyrophosphates (isopentenyl) Pyrophosphates, geranyl pyrophosphates, farnesyl pyrophosphates, geranyl geranyl pyrophosphates) is released.

For this purpose, the MIMPD cDNA (in the expression vector pcDNA3.1) is introduced stably or transiently into HEK293 cells and the membrane fraction is prepared from these cells. For the assay, 100 μg membrane protein of these cells (taken up in 50 mM Tris, pH 7.5 / 0.1% Triton X-100) with 50 mM Tris (pH 8.0), 5 mM Triton X-100, 1 mg / ml BSA and 0.5 mM Incubated in a total volume of 0.125 ml at 37 ° C. After 15-60 min, the reaction is stopped in 25 μl of the mixture with the same volume of 12% SDS. For the color reaction, 50 μl of 0.5% ammonium molybdate / 30 g / 1 L-ascorbic acid are added, incubated for 5 minutes, the color reaction is stopped with 75 μl of 2% sodium citrate / 2% sodium meta-arsenite / 2% acetic acid and then the absorption at λ = 750 nm was measured for a further 15 minutes. The released phosphate is quantified using a K ₂ HP0 ₄ calibration line (80-560 nmol / ml). The specific activity is calculated from the amount of ^J released phosphate per minute and per microgram protein (pmol / min * μg).

FIG. 11 shows the result of such a phosphatase assay with HEK293 cells which transiently express MIMPD. The membrane fraction of the cells was as described above in the presence of 500 .mu.m ⁰ LPA incubated for 45 minutes. A specific activity of 2.2 pmol / ug * min is measured in HEK293 cells transfected with MIMPD, while no phosphatase activity can be measured in HEK293 cells transfected with the empty vector. This result shows that ⁵ MIMPD is a lipid phosphate phosphatase.

Figure 12: Inhibitors for lipid phosphate phosphatases The activity of lipid phosphate phosphatases such as MIMPD can be inhibitors such as free divalent cations (Mg ²⁺ , ⁰ Ca ²⁺ , Zn ²⁺ , Mn ²⁺ ), sodium fluoride (NaF), N-ethylmaleimides (NEM), Propranolol Hydrochloride, Metoprolol tartrate, Sphingosine, Desipramine, Chlorpromazine, Phenylglyoxal, Di-Ethylpyrocarbonate (DEPC), 2, 4-Dinitrofluorobenzene, 7-Chloro-4-nitrobenz-2-oxa-l, 3-diazole, Spermine , Pyro- ^ phosphate, p-chloromercuriphenylsulfonic acid can be inhibited. The inhibition of the lipid phosphate phosphatase activity of MIMPD by propranolol is shown as an example in FIG. For this, the phasphatase activity of MIMPD was determined in the presence of 10 mM propranolol hydrochloride as ^{0 described} in the previous example. Figure 12 shows that the phosphatase activity of MIMPD drops to 31% of the untreated sample in the presence of propranolol. Figure 13: Antisense experiments

Transfection of MIMPD-specific antisense oligonucleotides (eyes) in cells reduces the intracellular amount of MIMPD mRNA. For such a knock-down experiment, MDA-MB-231 breast tumor cells are transfected with 10 nM oligonucleotide, after 72 hours the cells are lysed, RNA is prepared and the amount of MIMPD mRNA is quantified using real-time PCT analysis. As shown in FIG. 13, the transfection of this MIMPD-specific antisense oligonuleotide causes a reduction in the MIMPD RNA amount by 88.5% to 11.5% compared to the control which was transfected with a non-specific oligonucleotide (mismatch oligo).

Peptides for immunization

The derived peptide sequences for immunization are shown in figures.

Partial sequences from the MIMPD sequence (amino acids and nucleic acids) which are functionally essential for MIMPD

To screen MIMPD inhibitors, the entire MIMPD sequence (FIG. 1) or partial sequences of MIMPD can be cloned into an expression vector such as pcDNA3.1 and these plasmids can then be stably transfected in cell lines such as HEK293. The partial sequences are shown in the figures as amino acid sequences including the corresponding nucleic acid sequences.

Claims

24 patent claims:

1. MIMPD nucleic acid containing at least one partial sequence with a length of at least 12 nucleotides from SEQ.-ID 1 or SEQ.-ID 16 or from the further disclosed sequences, or consisting thereof.

2. MIMPD peptide or protein containing at least one partial sequence with a length of at least 4 amino acids from SEQ-ID 2 (MIMPD), or from the further disclosed sequences, or consisting thereof.

3. Peptide or protein according to claim 2 containing at least one partial sequence of at least 4 to 8 amino acids in length from one or more of the sequences SEQ.ID 3-9 (membrane-external regions) and / SEQ.ID 10-15 (conserved regions ), or consisting of one or more such partial sequences.

4. Use of a nucleic acid coding for MIMPD and / or a MIMPD peptide or protein for the detection of cancer or for the detection of a risk of the disease of cancer, wherein a tissue sample is examined for over-transcription of MIMPD RNA or for over-expression of a MIMPD protein or peptide.

5. Use according to claim 4, wherein a nucleic acid coding for MIMPD or a binding substance to MIMPD protein or peptide, preferably 25

containing a reporter group is used, wherein binding of said nucleic acid and / or said protein or peptide to the detector substance is detected semi-quantitatively or quantitatively.

6. Use of a MIMPD RNA or a MIMPD 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 according to prospective detector substances, whereby a prospectively binding substance or a mixture of such substances is contacted with said RNA or said protein or peptide, whereby binding events are determined with a binding assay, and wherein a binding prospective substance, optionally after deconvolution, as for detection or a substance suitable for the treatment of cancer is selected.

7. Use of a MIMPD inhibiting or binding substance for the production of a pharmaceutical composition for the detection and / or treatment of cancer.

8. Use according to claim 7, wherein the substance is an antibody which is obtained by immunizing a non-human mammal with a MIMPD peptide or protein, or with cells transfected with MIMPD cDNA, or with tumor cells expressing endogenously MIMPD, or with MIMPD produced in insect cells available 26

is, or a phage display antibody or an anti-idiotypic antibody.

5 9. Use according to claim 7, wherein the substance

Mimicry compound of an antibody against a MIMPD peptide or protein.

10. Use according to claim 7, wherein the substance, an aptamer, an antisense RNA, an inhibitory RNA, or a ribozyme against MIMPD.

15. Use according to one of claims 7 to 10, wherein the substance additionally carries a cytotoxic, radioactive and / or immunostimulating component.

12. Use according to one of claims 7 to 11, wherein the pharmaceutical composition is prepared for local or systemic application in tissue containing tumor cells.

25

13. A method for diagnosing a cancer, wherein a detector substance according to one of claims 4 to 12 in one embodiment with a reporter group in tissue to be examined, in vitro or in vivo,

30 is applied, 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 minimum value 27

the reporter group in the tissue is qualified as containing the tumor cells.

14. A method of treating a cancer, wherein a pharmaceutical composition according to any one of claims 4 to 12 is administered to a patient in a physiologically effective dose.

15. Use or method according to any one of claims 4 to 14, wherein the cancer is breast cancer, uterine cancer, colon cancer, stomach cancer, ovarian cancer, lung cancer, and / or rectal cancer.