EP1397163A2

EP1397163A2 - Method for the diagnosis and therapy of renal cell carcinoma

Info

Publication number: EP1397163A2
Application number: EP02748562A
Authority: EP
Inventors: Peter Daniel; Bernhard Gillissen
Original assignee: Daniel Peter Dr
Current assignee: Daniel Peter Dr
Priority date: 2001-05-31
Filing date: 2002-05-30
Publication date: 2004-03-17
Also published as: DE10126473A1; AU2002319080A1; WO2002096468A3; US20050053931A1; WO2002096468A2

Abstract

Die Erfindung betrifft ein neues Mittel zur Diagnose und Therapie des Nierenzellkarzinoms, sowie anderer Nierentumore, beispielsweise des Wilms-Tumors, oder anderer, nicht der Niere entstammender Tumor. Anwendungsgebiete der Erfindung sind die Medizin und die pharmazeutische Industrie. Der Erfindung liegt die Aufgabe zu Grunde, die bei dem heutigen Bearbeitungsstand dringend erforderlichen neuen Behandlungsformen und auch neue diagnostische Mittel für Nierenzellkarzinome bereitzustellen. Überraschenderweise wurde gefunden, dass das NBK-Protein, das im normalen Nierengewebe hoch exprimiert ist, im Tumorgewebe überhaupt nicht oder nur sehr schwach exprimiert ist. Die Proteinexpression und der Verlust von NBK wurde bei 80 Nierenzellkarzinom-Proben mittels Immunhistochemie und Mutationsanalytik untersucht. Das erfindungsgemässe Verfahren zur Diagnose ist dadurch gekennzeichnet, dass die NBK-Protein-Konzentration bzw. die NBK-RNA-Menge im Geweb bestimmt wird. Das erfindungsgemässe Mittel zur Therapie des Nierenzellkarzinoms und anderer Nierentumore ist dadurch gekennzeichnet, dass es die NBK-Proteinkonzentration in Nierenzellen oder anderen Geweben erhöht und in diesen Zellen direkt den therapeutischen Effekt auslöst.

Description

Method of diagnosis and therapy of renal cell carcinoma

description

The invention relates to a new agent for the diagnosis and therapy of renal cell carcinoma, as well as other kidney tumors, for example Wilms' tumor, or other non-kidney tumors. Areas of application of the invention are medicine and the pharmaceutical industry.

Surgical removal is the only treatment to date to cure non-metastatic renal cell carcinoma. The standard procedure is radical tumor nephrectomy with removal of the regional lymph nodes. The 5-year survival rate of organ-limited renal cell carcinomas after radical tumor phectomy is 67-92%, depending on the local tumor stage.

The prognosis of the metastatic (Nl and higher, Ml stages according to the TNM classification) is much worse. The average survival time in the presence of fermetastases is about half a year after diagnosis, the 1-year survival rate is 28%. It is not possible to cure metastatic renal cell carcinoma by chemotherapy, radiation therapy or hormone therapy. Compared to other tumors, renal cell carcinoma is extremely resistant to therapy and responds poorly to the therapies mentioned, which explains the poor prognosis of the patients with this disease. Limited therapy successes have been achieved with immunotherapy with interleukin-2 or interferons (complete plus partial remissions in 10 to 30% of the patients). But even these treatments cannot significantly extend patient survival (Ostendorf and Seeber, Hematology and Oncology, Urban and Schwarzenberg 1997).

New forms of treatment and new diagnostic tools are urgently needed.

The invention is based on the object of providing the new forms of treatment which are urgently required in the current state of processing and also new diagnostic agents for renal cell carcinomas.

The reasons for the extreme resistance to therapy of renal cell carcinoma are not yet known. To investigate this, a number of genes were tested, for which an influence on the prognosis of other tumor diseases has already been shown in the past. The gene and protein NBK, for which such a prognostic significance was not yet known, was also selected at random. Surprisingly, it was found that the NBK protein, which is highly expressed in normal kidney tissue (Daniel et al, 1999 and Figure 1), is not or only very poorly expressed in tumor tissue (Figure 1). The protein expression and the loss of NBK were examined in 80 renal cell carcinoma samples using immunohistochemistry (see Fig. 1) and mutation analysis (see below).

The loss of this cell death regulating protein in renal cell carcinomas is obviously responsible for the resistance of renal cell carcinomas.

In order to investigate the reason for the loss of expression, detailed mutation analysis methods were used. The known 5 exons of the NBK gene on chromosome 22ql3 (Verma et al. 2000) were analyzed by means of SSCP-PCR in DNA samples of the above. Patients with renal cell carcinoma examined. A conspicuous band was shown in 10 patients in the conformation-specific PCR analysis. These conspicuous samples (and, as a control, several patients without any abnormalities in the SSCP-PCR) were examined for changes in the genomic DNA sequence by sequencing the PCR products. In contrast to previous studies in colorectal cancer (Abdel-Rahman et al. 2000), point mutations were found. Furthermore, genetic changes were found in intronic sequences that can inhibit the splicing of the mRNA and the expression of the NBK protein.

In order to investigate the biological significance of NBK loss and also to investigate a possible therapy in terms of gene complementation, an adenoviral vector for the gene transfer of the wild-type NBK gene was constructed. Significant difficulties arose here because the vector-producing cells (293 cells; human embyronal kidney cell line) died when transfected with the constructs for constitutive NBK expression and, despite intensive efforts, no viruses could be produced using this conventional method.

For this reason, a technical development was necessary which aimed to enable gene transfer and conditional expression. This means that viruses had to be constructed which did not express NBK during the vector production, ie which were switched off, in order to enable the production of the viruses in the first place. Furthermore, such a conditional expression system after viral gene transfer also enables expression to be switched on and off, that is to say better control of the therapeutic transgene. For this purpose, the tet-off system was cloned into the El and E3 regions of an adenoviral vector system (see below). The gene transfer from NBK into a selection of renal cell carcinoma lines (Caki2, RCC-FG1, RCC-HS, RCC-KP, RCC-LR, RCC-MF, RCC-26) showed another surprising finding: these cells died rapidly as a result of the activation of apoptosis. This is in contrast to previous findings in T cell lymphoma cells (Daniel et al. 1999) and other cell lines (Daniel et al., Unpublished results), in which the constitutively high overexpression of the NBK gene sensitized the cells for apoptosis, but not per cell death. In control experiments with tumor cell lines of other carcinomas, in whose normal counterparts NBK expression could also be detected (Verma et al. 2000), namely tumor cells of the gastrointestinal tract, breast cancer and prostate, the adenoviral NBK gene transfer also triggered apoptosis, so that NBK gene transfer or activation of NBK gene expression represents a widely applicable new therapeutic principle for killing tumor cells.

The invention is implemented according to claims 1, 3 and 12, the subclaims are preferred variants.

The invention is illustrated by the following exemplary embodiments.

Abbreviations

PCMV: Complete "immediate early" promoter from cytomegalovirus, mediates efficient expression (Anderson et. Al 1989)

P _m ini _CMV: minimal "immediate early" promoter from cytomegalovirus This promoter does not have the strong CMV enhancer and is therefore not without the binding of tTA to the TRE-element active.

PhC _M C *: Combined promoter, the TRE-element is located in front of the P _m i _n i _CM V

TRE Tetracycline-Responsive Element, a regulatory sequence consisting of seven copies of the tet operator (42-bp, cis-regulatory DNA sequence from the bacterial tet operon, which corresponds to the binding site of the tet repressor) tTA: tetracycline-controlled transactivator, a fusion protein consisting of the

TetR and the VP16 AD (activation domain of the herpes simplex protein VP16)

BGHpolyA: Bovine growth hormone polyadenylation signal (Goodwin and Rottman, 1992)

Amp ^r ampicillin resistance gene (ß-lactamase)

ColElori: High-copy number origin of replication for E.coli

Construction of the conditional adenoviral vector system for the controllable overexpression of mycNBK

For the regulatable expression of the proapoptotic gene NBK in human cells by means of an adenoviral vector system, a recombinant adenovirus based on the Ad5 genome was produced, in which the E3 region by an expression cassette for tTA (tetracycline-controlled transactivator) and the El region by an expression cassette for mycNBK was replaced. The CMV promoter ensures constitutive expression of the tTA. In the absence of tetracycline or doxycycline, the tTA fusion protein specifically binds to the Tet-Responsive Element (TRE) of the promoter P _hCM v _* ι via the Tet repressor _component and activates the transcription of via the VP16AD component (Herpes simplex virus VP16 protein activation domain) mycNBK. Starting plasmids for the construction of adenoviruses pAdl-Del-El / E3 pAdl-Del-El / E3 contains an adenovirus 5 genome in which both the E3 and the El region were deleted (see FIG. 2). The E3 deletion corresponds to that of bed et al. (1994) described deletion from BHG10 and extends from 78.3 to 85.8 mu. Constructed by homologous recombination in E. coli: The Pacl site of BHG10 was filled in and an 8.7 kb Hapl-Notl fragment was recombined with pTG3602 (Chartier et al., 1996). The El Deletion extends from 0.9-9.8 mu and was introduced by homologous recombination of the 2.7 kb Pacl-Notl fragment from pHVAd2 into pAdl (Clal linearized) (see also pHVAd2).

pHVAd2

The left-end shuttle plasmid pAd2 is used to insert genes into the E1 region. It contains 2.6 kb of the left end of adenovirus 5 with a 3.2 kb deletion in the El region. This largest possible deletion and the polycloning region contained here correspond to that of Bett et. al (1994) for deletion described for pΔElsplA.

_P HVAd3

The shuttle plasmid pAd3 is used to insert genes into the E3 region. It contains a 4.6 kb BgHI fragment from BHG10 (Bett et. Al 1994). This corresponds to 2.9 kb downstream and 1.7 kb upstream sequences of that of Bett et. al described E3 deletion.

Construction of pAD-TreNBK-tTA

To construct pAD-TreNBK-tTA, the tetracycline-regulable promoter P _h CMv _* ι was first cloned as a Xhol EcoRI fragment (from pTre, Clontech) into the corresponding interfaces of pHVAd2. Subsequently, an NBK cDNA, including the BGH polyA signal (from pcDNA3, Invitrogene), fused with an ORF coding for a myc tag, was integrated into the Hindlll and Sall interface (see Fig. 2).

The resulting NBK expression cassette (PhCMv _* ι5 mycNBK; BGH polyA) was isolated with the flanking adenoviral DNA sequences as a Pacl Notl fragment and inserted by homologous recombination in the pAd-DelEl / E3 linearized with Clal (see Fig. 2). In order to integrate the expression cassette for the tetracycline-controlled transactivator tTA into the resulting plasmid pAd-TreNBK, the corresponding DNA fragment which contains the P _C Mv promoter, the reading frame for the tTA and a SV40polyA- Signal includes when the Xhol - PvuII fragment is isolated from the plasmid pTet-Off (Clontech) and cloned into the Sall and Nrul interface of the shuttle plasmid pHVAd3 (Fig.3).

From the resulting plasmid pAd3-tTA, the tTA expression cassette with 5 ^Λ and 3 ^λ flanking Ad5 sequences was cut out as a Nhel - StuI fragment and integrated into the plasmid pAdl-TreNBK by homologous recombination (Fig. 3)

The resulting plasmid pAD-TreNBK-tTA thus contains a complete Ad5 genome in which the El region is replaced by the NBK expression cassette and the E3 region by the tTA expression cassette (Fig. 4). The recombinant Ad5 genome can be cut out of the plasmid by cleavage with Pacl and used for transfection of 293 cells in which the recombinant adenoviruses are then produced.

Overexpression of mycNBK depending on the concentration of Dox

Infection of human cell lines with the recombinant adenovirus Ad-TreNBK-tTA (Fig. 5A) induces a strong overexpression of mycNBK depending on the Dox concentration. Figure 4B shows the overexpression of mycNBK in DU145 cells 24 hours after infection with 25 MOI Ad-mycNBK-tTA. In the absence of dox, strong expression occurs, which decreases with increasing dox concentration. At concentrations above 10 ng / ml, the mycNBK expression is almost completely represented (Fig. 5B). In the absence of dox or tetracycline, a strong overexpression of mycNBK was also found in a number of other cell lines 24 hours after infection with AdTreNBK-tTA (see below).

Induction of apoptosis by Ad-TreNBK-tTA mediated overexpression of mycNBK

The induction of apoptosis by adenoviral-mediated overexpression of mycNBK was investigated in renal cell carcinoma cells and other tumor cell lines. In the absence of Dox, the Western emblot analysis shows a strong overexpression of mycNBK in the corresponding cell lines 24 hours after infection with Ad-TreNBK-tTA (Fig. 6A). The modified cell cycle also shows a strong increase in the sub-Gl fraction, which represents the apoptotic cells, in the cells expressing mycNBK (Fig. 6B). Overexpression of mycNBK leads to the induction of apoptosis in the investigated renal carcinoma and other cell lines. In addition to the cell lines shown in Fig. 5, the induction of apoptosis in other renal carcinoma cell lines (RCC-LR, RCC-FG1, RCC-MF, RCC-26 Caki2), osteosarcoma cell lines (U2OS, Saos2), melanoma cell lines (Mel-2A, MeWo ) and in breast cancer cell line (MatuADR). Infection with Ad-TreNBK-tTA also led to the overexpression of mycNBK and the induction of apoptosis in these cell lines in the absence of Dox.

Role of endogenous NBK in tissues expressing NBK

Since NBK is an apoptosis-promoting, tissue-specific cell death-promoting gene, it is concluded on the basis of the findings outlined above that it can play a physiological role in the tissue death of kidney tissues and cells. The overactivity of such cell death genes can theoretically, as has been shown for other genes, trigger tissue decline and pathological conditions such as renal tissue degeneration. Such phenomena are involved in hereditary, somatic and exogenous damage, e.g. kidney damage caused by toxins and heavy metals or immune reactions such as infectious and non-infectious nephritis or graft rejection.

On the basis of this hypothesis, a further possible application of the invention is proposed, with which the effect of NBK is inhibited, for example by inhibiting the molecule itself, its effect in the downstream signal chain or down-regulation of the signal molecule itself. Another application of the knowledge according to the invention is to use it , in certain cases of hereditary or somatic kidney disease or kidney tissue degeneration caused by other causes, to lower the level of NBK protein or NBK RNA. This represents a new therapeutic principle for kidney diseases.

Claims

claims:

1. A method for diagnosing renal cell carcinoma, other kidney tumors, for example the Wilms tumor or other tumors not derived from the kidney, characterized in that the NBK protein concentration or the NBK RNA amount in the tissue is determined.

2. The method according spoke 1, characterized in that an analysis of mutations and polymorphisms in the NBK gene is carried out, preferably by DNA sequence analysis and measurement of modified NBK mRNA and NBK proteins.

3. Means for the therapy of renal cell carcinoma and other kidney tumors, for example the Wilms tumor, or other tumors not derived from the kidney, characterized in that it increases the NBK protein concentration in kidney cells or other tissues and directly triggers the therapeutic effect in these cells ,

4. Composition according spoke 3, characterized in that it increases the NBK protein expression by transfer of NBK cDNA or the NBK gene or its proportions.

5. Composition according spoke 3 and 4, characterized in that it increases the NBK protein expression using viral and non-viral expression vectors.

6. Composition according to approach 3 to 5, characterized in that it increases the NBK protein expression using a conditional adenoviral expression vector, preferably by means of the vector shown in Figure 4, constructed by cloning the expression cassette in pAD3 as shown in Figure 3.

7. Composition according spoke 3, characterized in that it achieves tissue-specific expression of NBK with preferential use of tissue-specific promoters and other, for example hormonal or other pharmacological regulators.

8. Composition according to claim 3, characterized in that it achieves tissue-specific expression of NBK under construction of chimeric molecules, consisting for example of NBK and transcription factor portions or signal peptides at the level of the NBK gene, the RNA or the protein.

9. Composition according to claim 3, characterized in that down-regulated endogenous NBK is brought back to expression, for example by means of pharmacological stimulation of NBK protein expression by activating gene expression and activating regulation of the NBK promoter.

10. Composition according spoke 3, characterized in that NBK protein expression is increased by stabilizing the NBK protein expression, for example by inhibiting NBK degradation.

11. Agent according spoke 3, characterized in that NBK activity is increased or decreased by activation or inhibition of regulatory portions of the protein.

12. Agent for the therapy of hereditary or somatic kidney diseases such as degenerative kidney diseases, infectious and non-infectious inflammatory or toxic kidney damage in which cells die by apoptosis, characterized in that inhibitors of the expression or activity of the NBK protein or the NBK RNA in Kidney cells.

13. Composition according spoke 12, characterized in that the activity of the NBK protein or the NBK RNA is reduced by activation or inhibition of regulatory portions of the NBK gene.

14. Composition according spoke 12, characterized in that the activity of the NBK protein or the NBK RNA is reduced by activation or inhibition of downstream signaling pathways.