EP1173225A2

EP1173225A2 - Agent for gene therapy and for the prevention of metastases, as well as for the gene therapy of tumors

Info

Publication number: EP1173225A2
Application number: EP00943552A
Authority: EP
Inventors: Karsten Brand; Andrew Baker; Michael Strauss
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-04-30
Filing date: 2000-05-02
Publication date: 2002-01-23
Also published as: WO2000066176A9; DE10022687A1; CN1367704A; AU5802200A; WO2000066176A3; WO2000066176A2

Abstract

The invention relates to an agent for the prophylaxis and treatment of primary tumors and metastases. The invention relates in particular to a gene therapy vector and a DNA sequence with an enhancer/promotor component and a transgene. The individual components of the agent are chosen such that they impregnate the normal tissue of potentially or already affected organs against invasive tumor cells. This prevents the formation of metastatic sites, induces the shrinkage of existing sites or limits the invasion of primary tumors.

Description

Means for gene therapy and for the prevention of metastases or for gene therapy of tumors

description

By far the most common cause of death from malignant tumor diseases is organ metastasis. While the primary tumor can be surgically resected at least in the early and middle stages, this is rarely possible in the case of metastasis. With a few exceptions, the conventional methods of chemotherapy and radiation can, if at all, only achieve temporary improvements in the clinical picture of metastatic tumors.

The most common tumors include colorectal cancer. Liver metastases of colorectal origin are the leading cause of death for patients with colorectal cancer. Since they are often the only manifestation of the disease over a long period of time after surgical removal of the primary tumor, they are a possible target for curative therapeutic approaches (Dreben, JA and Niederhuber, JE. (1993) Cancer of the lower gastrointestinal tract - colon cancer. In : Niederhuber, JE ed. Current Therapy in Oncology. St. Louis, MO: Decker, 426-431). The potentially curative surgical removal of liver metastases is only possible for a small percentage of the patients and temporary remissions are shown for chemotherapy but no life extension. There is therefore an urgent need for alternative forms of therapy. Due to their complexity, the gene therapy approaches that have been in development for around 10 years have a dramatically increased level of controllability compared to conventional forms of therapy. In the area of cancer gene therapy, this can be used, among other things, for tumor-specific targeting of the vectors or restriction of gene expression to tumor tissue and for the specific adaptation of the transferred transgenes to points of attack of the respective tumor type.

Apart from immunological approaches, the gene therapy approaches pursued so far, like the conventional methods, almost exclusively focus on the infiltrating tumor tissue. This

SPARE BLADE (RULE 26) harbors the problem of inadequate reaching of the tumor, which presents itself with increased intratumoral pressure and limited blood supply and is insufficiently met even with the direct intratumoral injection of gene therapy transfer vehicles which is usually carried out. In the case of multiple metastases, it has been shown that the systemic or regional administration of the vectors then required makes it much easier to infect the surrounding normal tissue than the tumor cells.

The invention aims to improve the prophylaxis and therapy of tumor metastases and primary tumors.

The invention is implemented according to the claims. The strategy developed according to the invention circumvents the problem of the difficult accessibility of the tumor tissue by declaring the easily accessible normal tissue to be the primary gene therapy target. The method is characterized in that the normal organ tissue is equipped with defense functions directly at the site of a potential or existing metastasis, which prevent the establishment or further growth of the metastases. The further spread of an inoperable primary tumor can also be prevented. This strategy of impregnating healthy tissue differs fundamentally from all gene therapy and non-gene therapy approaches carried out to date. Compared to systemic or intraperitoneal application e.g. Synthetic protease inhibitors (Nelson, NJ (1998) Inhibitors of Angiogenesis enter phase III testing, J Natl. Cancer Inst., 90, 960-963), the advantage of the gene therapy approach is to achieve very high concentrations of active substance locally in the target organ with the Advantage of the reduction of side effects and the possibility of simultaneous application of several inhibitors (see below). In addition, permanent gene expression after a single vector application is less expensive and has fewer side effects than repeated administration of synthetic substances over a longer period of time.

The approach described above can be inserted into the following clinical scenarios: 1. The preoperative or intraoperative vector application in target organs in order to prevent the extravasion of any metastatic tumor cells detached during operation of the primary tumor.

2. The continuous, continuous expression of inhibitors in the target tissue over years to prevent the growth or killing of occult micrometastases, especially in high-risk groups such as operated breast cancer with lymph node involvement.

3. Limiting the growth of already established metastases or inoperable primary tumors.

Embodiment 1: Gene therapy of colorectal liver metastases by adenoviral transfer of metalloproteinase inhibitors (TIMPs) into the liver parenchyma

Colorectal cancer metastases produce various proteases that they need for intravasion and extravasion, as well as for invasion into the target tissue. These include various metalloproteinases (MMPs) and here in particular the MMP-2 and MMP-9, which are responsible for the degradation of components of the extracellular matrix (ECM) (Duffy, MJ and McCarthy, K. (1998) Matrix metalloproteinases in cancer: Prognostic markers and targets for therapy (review), Int. J. Oncol., 12, 1343-48). MMPs-2 and -9 preferentially break down collagen IV as the main component of the basement membranes. In the course of these findings, synthetic protease inhibitors have been developed which are already in clinical use and in some cases have already reached phase III of clinical testing (Nelson, NJ (1998) Inhibitors of Angiogenesis enter phase III testing, J Natl. Cancer Inst., 90 , 960-963).

The basic therapeutic idea developed according to the invention is to have metalloproteinases secrete through the liver parenchyma in order to inhibit metastatic tumor cells from their extravasion, to prevent further infiltration of already established metastases and to prevent the supply of the tumors with vessels by inhibiting vascular development . First, the tissue inhibitor of metalloproteinase 2 (TIMP-2) was selected.

REPLACEMENT3LÄΪT (RULE 26) This inhibitor, like several related substances, physiologically limits MMP activity in remodeling processes. By binding to MMP-2, TIMP-2 can inhibit their activity.

First-generation adenoviruses, which are among the most established gene transfer systems, were used as vectors for gene transfer (Brand, K. and Strauss, M (1998) Molecular basis of gene transfer and application for gene therapy. In: Ruckpaul, D. and Ganten, D. (ed .) Handbook of Molecular Medicine, Vol. 2 Tumor Diseases, Springer, Berlin, Heidelberg, New York). These vectors, which cannot replicate due to the lack of the adenoviral E1 gene, infect epithelial cells with high efficiency and can be generated in the necessary large amounts.

a) To detect the secretion of TIMP-2 by hepatocytes in vitro, A2 cells (hepatocyte cell line from p53 knockout mice) were infected with different multiplicities of infection (MOIs) Ad-TIMP-2. The construction of Ad-TIMP-2 has been described (Baker, AH, Wilkinson, GW, Hembry, RM, Murphy, G., and Newby, AC (1996) Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase- 9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells. Matrix Biol. 15: 383-395). The virus contains the human TIMP-2 cDNA under the control of the CMV promoter. The cell culture supernatant (ZKÜ) was obtained 24 hours or 48 hours later. 10 μl were applied to a 10% polyacrylamide gel and, after electrophoretic separation, transferred to a nitrocellulose membrane. A monoclonal antibody (T2-101, Ab-1 from Dianova, Hamburg, Germany) was used for the immunological detection.

The Western blot showed a strong TIMP-2 band in the supernatant from Ad-TIMP-2 infected cells and no bands in uninfected and control virus-infected supernatants.

b) The functionality of TIMP-2 was determined using reverse zymography (according to Baker, AH, Wilkinson, GW, Hembry, RM, Murphy, G., and Newby, AC (1996) Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells. Matrix Biol. 15: 383-395). Cells were infected as described in (a). The ZKÜ was concentrated using an Amicon concentrator (Lexington, MA, USA). NaN ₃ , Brij and CaCl ₂ were added to give a final concentration of 0.1%, 0.05% and 5 mM, respectively. The samples were mixed with non-reducing buffer and loaded onto a 10% polyacrylamide / SDS gel containing 1 mg / ml gelatin (porcine skin type I, bloom 300, SIGMA G2500) and 107 ng / ml active MMP-2 protein (Oncogene, Cambridge, MA, USA) contains. After electrophoresis, the SDS was removed from the gel by incubation for 2 h in 2.5% Triton X 100. The gel was incubated overnight in 50 mM Tris HCl, pH 8.0, 50 mM NaCl, 10 mM CaCl ₂ , 0.05% Brij-35, and 0.02% NaN ₃ at 37 ° C. The gel was stained with 0.5% Coomassie Brilliant Blue (SIGMA R250, Deisenhofen, Germany) and bands of gelatinase inhibitory activity representing TIMP-2 then appear dark against the digested background. Reverse zymography showed gelatinase inhibitory activity only in the supernatants of Ad-TIMP-2 infected cells and not in the supernatants of uninfected or control virus infected cells.

c) The production of MMP-2 by tumor cells was demonstrated by gelatin zymography. The CCT of various cell lines was collected and conditioned as described in (b). The zymography was carried out as described under (b), only MMP-2 was not added. Bands of lysis indicating gelatinolytic activity were visible against the dark background. Among several cell lines, LS 174 colon tumor cells showed the strongest MMP-2 activity.

d) To detect recombinant human TIMP-2 in the blood of nude mice, different doses of Ad-TIMP-2 were injected intravenously into the tail vein of nude mice. Blood was drawn at various times and serum was obtained. The samples were checked for TIMP-2 content using a TIMP-2 ELISA (RPN 2618, Amersham Buchler, Braunschweig, Germany). The results show a dependence of the TIMP-2 expression on the amount of virus applied with a TIMP-2 concentration of 45 μg / ml at a dose of 3x10 ¹⁰ pfus (plaque forming units). The serum concentration of TIMP-2 was over 2 weeks stable. These results show that TIMP-2 is secreted into the blood after intravenous injection.

e) To test the extent of gene transfer to the liver, adenoviruses were injected into the tail vein of nude mice. After three days, the animals were sacrificed and the livers frozen in liquid nitrogen. A monoclonal mouse antibody against human TIMP-2 (1:10, T2-101, Ab-1, Dianova, Hamburg, Germany) was used for the immunohistochemical detection of TIMP-2. A FITC conjugated sheep anti-mouse antibody was used as the secondary antibody. The result of the staining resulted in expression of TIMP-2 by 40% of the hepatocytes at a dose of 3x10 ¹⁰ pfu and> 80% at a dose of 6x10 ¹⁰ pfu. A similar gene transfer efficiency was documented after the application of Ad-ßgal and subsequent X-gal staining.

f) The effectiveness of intravenous administration of Ad-TIMP-2 against LS 174 derived liver metastases was tested with the following experiments. Adenoviruses were administered 1 day before or 10 days after metastasis induction. The metastasis was induced by applying 2x10 ⁶ LS174 cells in the spleen of the animals. The animals were sacrificed 5 weeks after metastasis induction and the tumor weights were determined. In the preventive test approach, Ad-TIMP-2 or Ad-ßgal control virus was administered via the tail vein in a dose of 3x10 ¹⁰ pfu on day 0, which leads to approximately 40% liver cell infection. After 3 days, the metastasis was induced by the spleen injection of tumor cells described above. The experiment was terminated after 4 weeks and the volumes of the liver tumors were determined. It was found that both in the untreated control group and in the control virus-treated group, the majority of the animals had metastasis which almost completely consumed the liver, while the majority of the animals treated with AD-TIMP-2 were macroscopically tumor-free (Figure 1: Untreated, Ad-ßgal treated (middle) and Ad-TIMP-2 treated animal (right)).

The quantitative evaluation showed a ratio of the mean of the tumor weights in the test groups of 1:20 (Ad-TIMP-2: Ad-ßgal or untreated, Figure 2, points correspond to individual animals, bars correspond to the mean values). The histopathological examination only showed isolated micrometastases in the macroscopically tumor-free animals of the Ad-TIMP-2 group. It can thus be established that the gene therapy mediated secretion of TIMP-2 by hepatocytes limits both the number and the size of colorectal metastases in the nude mouse model in a highly significant manner. This finding was surprising in its uniqueness and speaks for a central, irreplaceable function of the MMP-2. Since only about 40% of the hepatocytes were transduced in the described approach, there is obviously a highly effective therapy here, which is presumably due to local high concentrations of the active substance with antimetastatic activity on several levels (extravasion, infiltration and angiogenesis).

In a second experiment, the therapeutic vectors were applied one week after the induction of metastases. In this case the therapy efficiency was lower than with preventive administration of the vectors. The differences between treatment group (TIMP-2) and control groups (ad-ßgal or untreated) were highly significant.

g) The livers of the animals of both experimental approaches were processed histologically in order to examine them for the standard tumor parameters apoptosis and proliferation as well as the extent of angiogenesis.

To determine the angiogenesis, proliferation and apoptosis, paraffin sections of the liver were prepared and stained with the following antibodies: A CD31 antibody (Daco, Hamburg, Germany) was used to detect the angiogenesis, and an MIB-1 (Ki- 67) Antibodies (dia 505, Dianova). The detection was carried out using a biotinylated second antibody and a horse-radish-conjugated avidin (Dako). An ApopTag fluorescein in situ apoptosis kit (Intergen, Oxford, UK, TUNEL method) was used to detect apoptosis. Deparaffinized sections were pretreated with Proteinase K. Terminal deoxynucleotidyl transferase (TdT) was used for primary staining and anti-digoxigenin conjugated to fluorescein was used as the reporter molecule. Propidium iodide was used as a counterstain. Mitoses were counted on hematoxylin / eosin stained sections. 10 microscopic fields per animal were measured at a magnification of x400 (MIB, mitoses, CD31) or with an oil immersion objective (x1000, TUNEL) using a Zeiss (axioscope) fluorescence microscope (Carl Zeiss, Jena,

REPLACEMENT BUTT (RULE 26) Germany) were counted and the mean values were calculated. The mean values of all data from tumor-bearing animals in the individual treatment groups were calculated and Student's t-test was used for statistical analysis. There was a significantly to highly significantly reduced proliferation rate and number of blood vessels and an increased apoptosis rate in animals treated with Ad-TIMP-2 compared to untreated or control virus-treated animals.

Further embodiments of the invention relate to vectors, promoters / enhancers, transgenes, transmembrane anchors and target organs

Vectors

Especially in the presence of occult metastases, which can only be reactivated after years of latency, and in preoperative metastasis prevention, long-term expression of the transferred gene is essential. When using first generation adenoviruses, the therapeutic effects are limited to a few weeks. Immunological defense reactions of the recipient are held responsible for this. These seem to be caused by the residual expression of adenoviral genes (Yang, Y. et al. (1994) Cellular immunity to viral antigens iimits E1-deleted adenovirus for gene therapy. Proc. Natl. Acad. Sci. USA 91, 4407-4411. ). A promising approach to reducing immunogenicity is the outsourcing of all coding adenoviral genome sections from the therapeutic vector (Chen, HH et al. (1997) Persistence in muscle of an adenoviral vector that lacks all viral genes, Proc. Natl. Acad. Sei. USA 94, 1645-1650). At the same time, the transport capacity of such viruses is increased considerably, so that several transgenes have space on one vector, which enables an attack at different points in the metastasis cascade. Such TIMP-2-bearing, so-called helper dependent (HD), gutless, or minimal adenoviruses can presumably offer long-term protection against organ metastasis. Retroviruses and adeno-associated viruses (AAVs) are further vectors that allow longer foreign gene expression. Both viruses are not immunogenic and necessarily integrate (retroviruses) or potentially (AAVs) into the genome of the host cell. While with retroviruses the need for replication of the target cells and the difficulty of the generation of high-titer virus suspensions still exist If problems arise, modern AAV vectors could already be used in the medium term for the gene transfer of protease inhibitors. Promising vectors are also lentiviruses, hybrid constructs and herpes simplex viruses, which have a high affinity for neuronal tissue and are therefore particularly suitable for the treatment of brain metastases and glioblastomas. Among the non-viral vector systems, liposomes should be emphasized. A preferred embodiment of the invention is the modifications in the surface structure of viruses, which enables retargeting of the vectors. This is achieved by expressing a suitable ligand on viral spikes, which enables a specific transduction of certain normal tissues. For example, by incorporating a heparin domain, heparan-expressing cells can be specifically transduced adenovirally.

Enhancers / promoters

Enhancers / promoters can be used which are active in the normal tissue to be protected. In most cases, this includes the organ parenchyma. In individual cases, expression of antitumor transgenes by underrepresented cells of the organ can also be useful, e.g. secretion of collagen e.g. through fibroblasts.

Another possibility is to use promoters that are only activated after the addition of a foreign substance. With such promoters, such as. B. tetracycline-dependent promoter elements or steroid responsive elements, you have the option to impregnate only sporadically or to select the most dangerous times for metastasis.

Transgenes

1. TIMP-2 is the appropriate protein for the treatment of LS174 cell derived metastases. MMP-2, which is inhibited by TIMP-2, is one of the most relevant proteases for tumor cell invasion. However, other cell lines also produce other MMPs, and this is also reflected in the protease pattern of human tumors. The extracellular matrix of the target organs is also different

SPARE BLADE (RULE 26) constructed which places different demands on the tumor cell proteases. A general approach must therefore include protease inhibitors other than TIMP-2, such as TIMP-1, PAI-1 or PAI-2. Modifications in the structure of the naturally occurring inhibitors lead to an increase in effectiveness or a reduction in any side effects. Such modifications consist of shortening the molecule or changing the sequence by exchanging individual bases of the DNA. For example, by removing a terminal (C-terminal) part of the TIMP-2 molecule, it is possible to remove its unwanted protease-activating function.

2. An alternative to inhibiting the degradation of the extracellular matrix (ECM) is to amplify or modify it. Naturally occurring components of the extracellular matrix can be overexpressed here. This includes the genes for the various collagens, fibronectin, laminin and genes whose products are responsible for the synthesis of non-protein components of the ECM. Furthermore, components of the ECM, which are usually not expressed in the organ in question, can be expressed in a non-local manner and thus the organ specificity of metastases can be changed. Furthermore, non-degradable or poorly degradable substances that are an insurmountable obstacle for metastatic cells can be expressed.

Example 2:

It has long been known that metastases occur less frequently in cirrhotic livers than in normal livers. The reason for this is probably the inhibition of the spread of metastatic cells in fibrotic tissue. This relationship can be used therapeutically and expanded by causing hepatic normal tissue to change its microanatomy by gene transfer in such a way that a kind of restricted artificial cirrhosis / fibrosis is generated and thus metastatic cells are prevented from expanding. Collagen IV is the main component of basement membranes. There is no such between hepatocytes and sinusoids. There is little collagenous tissue in the Disseschen area. A slight increase in the collagen content will severely limit the ability to metastasize. Vector construction: The two polypeptide chains that generate the triple helix of the collagen fibril are cloned into a minimal adenovirus shuttle vector. A tet activator-responsive and doxycycline-dependent promoter is used as the promoter in order to keep the extent of transgene expression controllable. The tet activator is also brought onto the shuttle plasmid. A minimal adenovirus is produced using any packaging system (= HDAD-tetColl)

Vector testing: In nude mice, liver metastases are induced by injection of LS 174 cells. HDAd-tetColl is administered in analogy to the methodology in exemplary embodiment 1. The same applies to the evaluation.

3. Another way to block tumor cell invasion and motility is to strengthen cell-cell and cell-matrix adhesions. Worth mentioning are the tight junetions with the proteins Claudin and Occludin, the Desmosomes and Adherence junetions with the main protein Cadherin, Integrine, which above all react with components of the ECM, the immunoglobulin superfamily, the Selectine and the Muzine.

Example 3:

It has already been shown that E-cadherin is responsible for the interaction of epithelial cells and a loss of E-cadherin by cells in the primary tumor

Metastasis promotes (Birchmeier, W. (1995) E-cadherin as a tumor (invasion) suppressor gene. Bioessays 17, 97-99). Expression of E-Cadherin by

On the one hand, normal cells lead to adhesion with the tumor cells and to one

Inhibiting their motility and further enhancing adhesion within normal cells.

Vector construction: A first-generation adenovirus is consecrated which

Cadherin gene under the control of the RSV promoter carries: Ad-RSV-E-Cad.

In vitro testing: A2 cells are transduced with Ad-RSV-E-Cad for functional testing. An increase in adhesion is determined by determining the amount of time it takes for trypsin to separate the cells.

Vector testing: Nude mice are injected with LS 174 cells

Liver metastases induced. Ad-RSV-E-Cad is administered in

Analogy to the methodology in exemplary embodiment 1. The same applies to the evaluation. Transgenes with membrane anchor sequence

For the purposes of the invention, suicide genes are used to impregnate normal tissue. To do this, they must be equipped with a membrane anchor sequence in order to be extracellularly effective and to be able to also toxically apply an applied prodrug extracellularly.

Example 4:

The gene for the suicide gene cytosine deaminase under the control of the

HNFAIbumin promoter is provided with a membrane anchor sequence so that it is expressed in the membrane after transfection. This expression cassette is cloned into an AAV shuttle plasmid and an AAV is produced using any helper system (= AAV-HNFAIb-CD-Tm).

In vitro testing: A2 cells are transduced with AAV-HNFAIb-CD-Tm. 24 hours after

Transduction, the Prodrug 5-FC is added to the cell culture supernatant (ZKÜ). 5-FC is now transferred to the cytotoxic 5-FU through the membrane-bound CD. The

The supernatant is collected after 24 h and applied to the cell line LS174 and to resting primary hepatocytes. After a further 72 hours, cell counts are made and the extent of apoptosis is determined.

In vivo testing: Vector application and determination of the therapy efficiency run in

Analogy to embodiment 1.

Target organs

Due to the excellent infectability of the liver with systemic administration of adenoviruses and the high clinical relevance of the treatment of colorectal metastases, the method according to the invention was developed using the disease model described above. The model can also be applied to other tumor diseases. The most common clinical pictures include liver, lung, bone and brain metastases in breast cancer with latency periods of up to 10 years after removal of the primary tumor, a period that has so far elapsed, brain metastases in bronchial cancer and bone metastases in prostate cancer. There are also primary tumors that are primarily inoperable due to their advanced stage, such as frequent glioblastomas and hepatocellular carcinomas. As a life-prolonging measure, the protection of the surrounding normal tissue according to the above-mentioned principle is conceivable.

Legend for the pictures:

Figure 1: Prevention of liver metastasis by systemic application of Ad-TIMP-2. On day 0, 3x10 ¹⁰ pfu Ad-TIMP-2 or Ad-ßgal were applied to the tail vein of nude mice. 3 days later, the animals received an intrasplenic injection of LS174 colon carcinoma cells to induce liver metastases. Representative in situ photographs of untreated (left), Ad-ßgal-treated (middle) and Ad-TIMP-2 treated (right) animals after 5 weeks.

Figure 2: Methodology as in Figure 1. After 5 weeks, the animals were sacrificed and the tumor masses were determined. Points correspond to individual animals, bars correspond to the mean values.

Claims

claims

1. Means for gene therapy prophylaxis and therapy of tumor diseases comprising one

- Vector in the sense of a gene transfer vehicle

- enhancer / promoter

- Transgene whereby at least one of the listed components is aimed at the impregnation of normal tissue.

2. Use of the agent according to claim 1 for gene therapy prophylaxis and therapy of tumor diseases comprising a

- Vector in the sense of a gene transfer vehicle

- enhancer / promoter

3. Use of a gene transfer vector comprising a transgene in operative association with an enhancer / promoter for the production of an agent for the gene therapy prophylaxis and therapy of tumor diseases by administration to normal tissue.

4. A method for gene therapy prophylaxis and therapy of tumor diseases, characterized in that an agent comprising a

- Vector in the sense of a gene transfer vehicle

- enhancer / promoter

- Transgene wherein at least one of the listed components is aimed at the impregnation of normal tissue, a subject who needs prophylactic or therapeutic tumor treatment, administered in such a way that the vector is essentially absorbed by normal cells.

5. Composition according to claim 1, with a promoter and / or enhancer which is regulated by transcription factors which are active in normal tissue.

6. Composition according to claim 5 containing the CMV promoter or the SV 40 promoter or the RSV promoter, or liver-specific promoters, such as the albumin promoter or lung-specific promoters or rasp tissue-specific promoters or bone-specific promoters or promoters, which occur in potential metastatic target organs or organs of primary tumors are active.

7. Composition according to claim 5 containing an enhancer / promoter which is activated by adding an applicable substance.

8. Composition according to claim 5 and 7, which is a tetracycline-dependent or a steroid hormone-dependent promoter.

9. Composition according to claim 1 containing transgenes for substances,

- which limit the growth of the tumor

- destroy the tumor

- protect normal tissue from tumor invasion.

10. Agent according to claim 1 containing genes of metalloprotease inhibitors

11. Agent according to claim 1 and 10 containing an antitumor transgene coding for:

TIMP-1 or TIMP-2

12. Composition according to claim 1 containing a protease inhibitory transgene coding for:

TIMP-3 or TIMP-4 or PAI-1 or PAI-2.

13. Composition according to claim 11 or 12 containing a modified transgene, the antitumor effect of which has been enhanced by this modification.

14. Composition according to claim 13 which contains the relevant transgene C-terminally truncated TIMP-2.

15. Composition according to claim 1, 2 or 3 containing a transgene of the extracellular matrix.

16. Composition according to claim 15 containing at least two polypeptide chains of collagen or fibronectin or laminin or genes whose products are responsible for the synthesis of non-protein components of the ECM.

17. Composition according to claim 15 or 16 containing a modified transgene of the extracellular matrix that it is difficult or not degradable.

18. Composition according to claim 1 containing a transgene coding for an adhesion molecule.

19. A composition according to claim 18, in which the adhesion molecule in question is the claudin or occiudin or a cadherin or an integrin or a gene from the immunoglobulin superfamily a selectin or a mucin.

20. Agent for the prophylaxis and treatment of tumor diseases containing an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence.

21. Use of a gene transfer vector for the preparation of an agent for the prophylaxis and treatment of tumor diseases containing an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence.

22. A method for the prophylaxis and treatment of tumor diseases in which an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence, is transferred.

REPLACEMENT BUTT (RULE 26)

23. A composition according to claim 20 which contains a suicide gene or an otherwise chemotherapeutically active gene as the relevant transgene

24. Means according to claim 23 in which the transgene in question

Cytosine deaminase or active partial sequences thereof or nitroreductase or active partial sequences thereof.

25. Composition according to claim 1, in which the vector is a virus.

26. A composition according to claim 25, in which the virus is a first generation adenovirus or an adeno-associated virus or a minimal adenovirus or an HSV or a lentivirus.

27. A composition according to claim 26, in which the virus is a lentivirus / minimal adenovirus hybrid.

28. A composition according to claim 27, in which the vector is a non-human mammalian adenovirus.

29. Composition according to claim 1, in which the vector is not a virus.

30. Composition according to claim 29, in which the vector is a liposomal formulation or carrier proteins are used.

31. Composition according to claim 25 and 29, in which the surface is modified so that a specific gene transfer is achieved in normal tissue.

32. Agent according to claim 1 containing a minimal adenovirus and TIMP-2.

33. Agent according to claim 1 containing a minimal adenovirus and C-terminally truncated TIMP-2.

34. Composition according to claim 1 containing an AAV and TIMP-2

ERSATZBUπ (RULE 26)

35. Agent according to claim 1 containing a first generation adenovirus and TIMP-2.

36. Agent according to claim 1 containing a lentivirus / minimal adenovirus hybrid and TIMP-2.

37. Agent according to claim 1 containing an AAV and C-terminally truncated TIMP-2.

38. Composition according to claim 1 containing a minimal adenovirus and E-cadherin.

39. Composition according to claim 1 containing an AAV and E-cadherin.

40. Agent according to claim 1 containing a minimal adenovirus and at least two polypeptide chains of collagen.

41. Agent according to claim 1 for gene transfer into liver tissue.

42. Agent according to claim 1 for the therapy and prophylaxis of liver metastases.

43. Agent according to claim 1 for the therapy of brain tumors.

44. Agent according to claim 1 for the therapy of lung metastases.

45. Agent according to claim 1 containing the HNFAIbumin enhancer / promoter, AAV and TIMP-1.

46. Agent according to claim 1 containing an enhancer / promoter which is activated by a foreign substance and at least two polypeptide chains of collagen.

47. Agent according to claim 1 containing a liver-specific promoter, an AAV and a metalloprotease inhibitor.

48. Composition according to claim 1 containing a liver-specific promoter, a minimal adenovirus and a metalloprotease inhibitor.

REPLACEMENT BUTT (RULE 26)

49. Agent according to claim 1 and 9 containing a liver-specific promoter and a minimal adenovirus.

50. Agent according to claim 1 and 9 containing a liver-specific promoter and an AAV.

51. Composition according to claim 1 and 9 containing a liver-specific promoter and a lentivirus / minimal adenovirus hybrid.

REPLACEMENT BUTT (RULE 26)