WO2003004063A1

WO2003004063A1 - Genetically engineered therapy method for treating gnrh receptor-positive carcinoma by gnrh-induced tumor cell-specific activation of a therapeutic gene, corresponding nucleic acid constructs and vectors

Info

Publication number: WO2003004063A1
Application number: PCT/DE2002/002388
Authority: WO
Inventors: Günter EMONS; Carsten GRÜNDKER; Abdolhamid Huschmand Nia
Original assignee: Georg-August Universität Göttingen
Priority date: 2001-07-05
Filing date: 2002-07-02
Publication date: 2003-01-16
Also published as: DE10292896D2

Abstract

GnRH-induced NFKB activation can be used for the tumor cell-specific of an introduced effector gene under the control of an NFKB-dependent promoter. To achieve this, the tumor cells are transfected with a vector that contains the effector gene under the control of the NFKB promoter. Said gene is then activated in a target cell-specific way by the activation of NFKB through GnRH agonists so that an inactive pro-substance (prodrug) is reacted to a cytotoxic substance only in GnRH receptor-carrying tumor cells, thereby allowing for a target cell-specific cytotoxic therapy.

Description

Gene therapy method for the treatment of GnRH receptor-positive carcinomas by GnRH-induced tumor cell-specific activation of a therapeutic gene, associated nucleic acid constructs and vectors

The invention relates to nucleic acid constructs and vectors containing them for introduction into gonadotropin-releasing hormone (GnRH) -positive carcinomas and to their use for the treatment of GnRH-positive tumors. O

Endometrial cancer is the most common gynecological cancer in the western world. In most cases, endometrial cancer is diagnosed at an early stage, so surgery or chemotherapy offer a good chance of recovery. Steroid receptor negative tumors in the elderly

5 women or later stages of cancer have very poor prognoses. So far there is no therapy to improve these unfavorable prognoses.

Ovarian tumors are less common, but have much less favorable prognoses and often lead to death. Although ovarian cancer occurs relatively rarely, it causes more deaths than all other gynecological cancers combined. Effective surgical techniques and chemotherapy procedures are well established, but there is little chance of recovery in advanced stages or relapses.

: 5 Breast cancer is one of the most common malignancies in women. About 10% of all women develop breast cancer during their lifetime, which corresponds to about 25% of all cancer deaths for women. In the past 10 years, the entire treatment, both surgical and drug therapy, has undergone a significant change. This led despite the slightly increasing trend of

, o Breast cancer rate, although a reduction in the overall mortality rate of this disease, but there is little chance of a cure for breast cancer in advanced stages or for relapses.

Both for endometrial carcinoma, ovarian carcinoma and for breast new, well-tolerated and, above all, more efficient therapies are needed for cancer.

The object of the invention is to provide means for the treatment of the above. To provide tumor types.

This object is achieved by the provision of a nucleic acid construct which contains a therapeutic gene, the gene product of which triggers the production of a cytotoxic active substance in a cell, under the control of a nucleus factor kappa .0 B (NFB) -specific promoter. In tumor treatment, this construct is introduced into tumor target cells using gene therapy agents, the exact method being described in more detail below.

Over 80% of endometrial and ovarian cancers and over 50% of breast carcinomas express the gonadotropin releasing hormone (GnRH) and its receptor as part of an autocrine regulatory mechanism of cell proliferation, growth and differentiation control. In contrast, only a few reproductive organs as well as the hypothalamus and pituitary express the GnRH receptor. Therefore, the GnRH receptor is particularly suitable as a target for tumor cell-specific therapy.

The signal transduction of the GnRH receptor in gynecological tumors differs fundamentally from that in the hypothalamus and pituitary (Gründker et al. 2002). GnRH and its analogues induce only in endometrial and OVA rialkarzinomzellen GnRH receptor mediates a 5- to 8-fold increase in activated ^"5 tivity of the transcription factor nucleus factor B (NFB).

GnRH-induced NFB activation is now used according to the invention to express a tumor cell-specific expression of an introduced effector gene (hereinafter also referred to as a therapeutic gene) under the control of an NFB-dependent promoter. For this purpose, the tumor cells are constructed with a nucleic acid construct which contains the effector gene under the control of the NFB promoter, preferably in a vector, transfected. This gene can then be activated in a target cell-specific manner by activating NFB using GnRH analogs, so that an inactive active substance precursor (pro-drug) is only converted into a cytotoxic active substance in tumor cells carrying GnRH receptor and thus target cell-specific cytotoxic therapy 5 is made possible.

The proof of principle for this approach could be provided. In vivo experiments with tumor-bearing nude mice, which were transfected liposomally with the LacZ reporter gene under the control of the NFB promoter, showed that NFB was only activated by GnRH analogue triptorelin in GnRH receptor positive tumor cells. NFB was constitutively active in the ovary and uterus. In the case of corresponding carcinomas, the ovary and uterus are removed beforehand. All other organs showed no activation.

.5 This form of therapy can be used for all GnRH receptor-expressing carcinomas. In addition to the endometrial, ovarian (> 80% of tumors expressing the GnRH receptor) and breast cancers (> 50% of the tumors expressing the GnRH receptor) also include prostate cancers. For the latter, no exact numbers are known yet, what percentage of these tumors

: o express the GnRH receptor.

In principle, the therapeutic gene within the construct according to the invention must be able to produce a cytotoxic substance in the target cell, i.e. of a cytotoxic agent in any way. In general, 5 this is brought about by the fact that the gene product of the therapeutic gene is an enzyme which can convert an active substance precursor which is present in or supplied to the cell into the cytotoxic active substance.

The therapeutic gene is only activated in GnRH receptor-positive cells, in that an NFB-specific promoter induces the activation of the gene when GnRH or a GnRH analogue is released or delivered. The NFB-specific pro motor preferably contains at least one copy of the kappaB enhancer, in particular 4 to 6 copies, fused to a promoter suitable for activating the therapeutic gene.

5 The principle on which the invention is based is that a therapeutic gene or effector gene is under the control of the NFB promoter, which is specific for tumor cells via GnRH (LHRH) or GnRH analogs in GnRH receptor-positive tumor cells (ovary, endometrial , Breast and prostate tumors) is activated. The individual therapeutic gene is not important, so that the execution of the

.0 different promoter-gene / precursor-drug pairs are suitable.

For cancer therapy, the therapeutic genes are primarily suicide genes that trigger the death of the target cell, although therapeutic genes that enable real therapy of the target cell (reprogramming of the cancer cell) .5 are not excluded.

A summary description of currently common suicide genes can be found, for example, in: G. Coukos, "Gene Therapy for Ovarian Cancer", Oncology 2, 2001, 1197-1207.

! 0

Examples of suicide genes which can be used as therapeutic genes in the context of this invention are given in Table 1.

In a particularly preferred exemplary embodiment it is provided that the therapeutic gene is the herpes simplex virus (HSV) thymidine kinase gene. The HSV-TK gene is placed under the control of the HSV-TK promoter. HSV-TK converts non-toxic ganciclovir and its descendants into toxic ganciclovir triphosphate (or its descendants) (advantage: bystander effect).

It is provided in a further exemplary embodiment that the therapeutic gene is the varicella zoster V / ^' ti / s thymidine kinase gene. VZV-TK converts non-toxic 6-methoxypurine arabinonucleoside (araM) into toxic adenine Arabinonucleoside triphosphate (araATP).

Another exemplary embodiment provides that the therapeutic gene is the Eche chia co // cytosine deaminase gene. When activated by a suitable 5 promoter, E.coli-CD converts non-toxic 5-fluorocytosine into toxic 5-fluorouracil (advantage: bystander effect).

As a rule, the nucleic acid construct is present in a plasmid. The minimal configuration of the plasmid consists of at least one copy, preferably 4 to 60 copies, of the B enhancer, which is an NFB binding site, fused to a promoter which specifically induces the therapeutic gene. Fusing the KappaB enhancer with the promoter gives an NFB-specific promoter for the therapeutic gene. The construct also contains at least one PolyA sequence.

.5

In a development of the invention, the construct can additionally contain a sequence for the expression of the active substance precursor in the target cells. This generally includes at least one coding sequence for the active substance precursor and a suitable promoter. Furthermore, it is also possible to provide a separate plasmid or nucleic acid construct for the supply of the active substance precursor iθ which contains at least one sequence coding for the active substance precursor and a promoter.

It is possible to introduce the plasmid (construct) into the target cells using various non-viral! 5 (e.g. liposomal) techniques. Viral transfection options also exist, but they have a number of side effects. The certain tumor specificity that can be achieved by viral techniques (e.g. transfection of proliferating cells) is not necessary in the therapy method presented, since via the specific expression of the GnRH (LHRH) receptor IO and additionally via the mechanism of action specific for GnRH receptor-carrying [activation from NFB through GnRH (LHRH) and its analog ga] a much higher tumor specificity of the therapy is guaranteed. In principle, however, the use of viral vectors is also possible.

In a preferred embodiment, the construct is introduced into the tumor target cells in a liposomal vector. Kim et al. (Gynecologic Oncology 2002 Volume 84 pages 85 to 93) describe, for example, an efficient liposomal method that can also be used here to transfect human ovarian cancer cells.

For a preparation for the treatment of GnRH receptor-positive tumors, the nucleic acid construct or the vector according to the invention can be contained, for example, in an injection solution or an infusion solution. This preparation can additionally contain the required active substance precursor or be provided for co-application with it, provided the active substance precursor is not present in the cell or is coded on the nucleic acid construct.

5

The preparation with the nucleic acid construct or the vector which contains this construct is administered in chronological coordination with GnRH or a GnRH analog and optionally the active substance precursor or a plasmid coding for the active substance precursor. O

In a preferred embodiment of the invention, the tumor treatment is carried out according to the following scheme:

a) introducing the nucleic acid construct or the vector according to one of the claims: 5 claims 1 to 9 into tumor target cells; b) administration of GnRH or a GnRH analog at intervals of step a); c) administration of an active substance precursor which is converted into the active substance by the gene product of the therapeutic gene contained in the nucleic acid construct

(0 delt, in chronological order before, after or with step b), provided the active substance precursor is not already present in the cell as a natural cellular substance. The GnRH or the GnRH analog is given in an advantageous procedure 8 to 48 hours after the construct or the vector has been introduced into the target cells. Triptorelin is very advantageously given as the GnRH analog, preferably systemically.

The bare construct or the vector can be introduced by intraperitoneal injection. The treatable tumors are reached so well. GnRH or a GnRH analogue is then preferably administered intraperitoneally or intravenously. If necessary, the active substance precursor is preferably administered intraperitoneally after or with the GnRH or GnRH analog. Another possibility is that the active substance precursor is additionally encoded on the nucleic acid construct.

In a particularly preferred method example, the plasmid “pNFB-TK is used. The plasmid "pNFB-TK" was constructed to express the effector gene herpes simplex virus thymidine kinase (HSV-TK) by NFB, which is specifically activated via GnRH (LHRH) and its analogs in GnRH (LHRH) receptor positive tumor cells. The plasmid "pNFB-TK" contains 4 tandem copies of the B enhancer fused to the herpes simplex virus thymidine kinase promoter followed by the herpes simplex virus thymidine kinase gene with polyA tail.

The plasmid "pNFB-TK" is injected intraperitoneally together with a transfection reagent based on liposomes. After 24 hours, a GnRH (LHRH) analog is injected. The binding of the GnRH (LHRH) analogue to the GnRH (LHRH) receptor of the tumor cell leads to the activation of NFB. Activated NFB binds to the B enhancer of the plasmid "pNFB-TK" and thus induces the expression of the herpes simple virus thymidine kinase gene. The gene product, the herpes simplex virus thymidine kinase, now phosphorylates the administered precursor drug ganciclovir, which cannot be implemented by human enzymes. This ultimately creates the cytotoxic agent ganciclovir triphosphate, which kills the tumor cells. GnRH (LHRH) and its analogues cannot activate NFB on normal cells that do not express GnRH (LHRH) receptors. In addition, the mechanism of NFB activation by GnRH (LHRH) and its analogues is limited to tumors of reproductive organs.

The invention is described in more detail below with the aid of examples and reference is made to figures. Show in detail:

o Fig. 1 schematic representation of the functional principle of the invention

GnRH (LHRH) or GnRH analogs bind to the GnRH receptor on the cell surface of the tumor cell. This activates the tumor-specific nucleus factor kappa B (NFKB). Activated NFKB binds to the κB binding site of the plasmid and thereby induces the expression of the effector gene (e.g. herpes

.5 simplex virus thymidine kinase gene). The gene product is an enzyme (e.g. herpes simplex virus thymidine kinase), which catalyzes the conversion of the active ingredient precursor (e.g. ganciclovir) into an active ingredient (e.g. ganciclovir triphosphate). This leads to the death of the tumor cell.

2 schematic representation of the plasmid pNFB-TG

- TG = therapeutic gene = effector gene (here as an example herpes simple virus thymidine kinase)

- B ₄ - TK = 4 copies of the kappa B enhancer fused to a promoter suitable for activating the therapeutic gene (here TK = thymidine

> 5 nose promoter).

- SV40 poly A = poly A sequence

- resistance gene EXPERIMENTAL

Detection of GnRH receptors in cell lines and tumor tissue

In order to determine the frequency of the expression of the GnRH receptor in endometrial and ovary carcinomas, various endometrial and ovarian carcinoma cell lines as well as 40 endometrial and ovarian primary carcinomas were determined using a specific radioreceptor assay and RT-PCR for the expression of GnRH binding sites examined. Most of the ovarian (EFO-21, EFO-27, NIH: Ovcar-3, BG-1) and endometrial carcinoma cell lines (Ishikawa, HEC-1A, HEC-1B, KLE, AN-3-CA) each had a high-affinity binding site for the GnRH analog triptorelin (Kd 1, 5 - 8.2 nmol / L). The two ovarian carcinoma cell lines SK-OV-3 and SW 626 and the endometrial carcinoma cell line MFE 296 reacted negatively. When examining the primary tumors, more than 80% of the ovarian and endometrial carcinomas showed high-affinity binding sites (Kd 0.1 - 90 nmol / L) for GNRH. Due to this high frequency of GNRH receptor expression, the GnRH receptor is well suited as a target for target cell-specific therapy.

Most normal tissues do not express GnRH receptors:

A prerequisite for target cell-specific tumor therapy via the GnRH receptor is that normal tissue expresses no or only at a low frequency GnRH receptors. RT-PCR and radioligand assays showed that only very few normal tissues express the GnRH receptor. GnRH receptor expression was only found in the reproductive organs ovary, myometrium, endometrium, tube and cervix as well as in the placenta and breast except in the pituitary and hypothalamus (Kakar et al. 1994 and own unpublished data). All non-reproductive organs, including the lymphatic and hematopoietic systems, were GnRH receptor negative. The hypothalamus and pituitary gland are not affected by local therapy via the GNRH receptor (intra-tonal application). Furthermore, NFB becomes positive in pituitary GnRH receptor Cells not activated (see the next two sections). In addition, activated NFB would not be toxic in these non-proliferating cells. The reproductive organs such as the uterus and ovary are removed in the event of carcinomas. The breast tissue, like the pituitary and hypothalamus, is not affected. Because the vast majority of endometrial and ovarian carcinomas express GnRH receptors, but only very few normal tissues have GnRH receptors, targeting via GnRH receptor appears to be particularly suitable for target cell-specific therapy.

Signal transduction of the GnRH receptor in the tumor:

The following experiments were carried out to show the differences between signal transduction in the pituitary and hypothalamus as well as in gynecological tumors. Various key sites of GnRH receptor signal transduction that GnRH activates in the pituitary and hypothalamus were analyzed. Although phospholipase C (PLC), protein kinase C (PKC) or adenylate cyclase could be activated pharmacologically in the tumor cells, GnRH and its analogues had no influence on PLC, PKC or adenylate cyclase. This indicates that a different signal transduction mechanism must be present in the tumor cells. Unlike in the pituitary and hypothalamus, the signal of GnRH receptor activation in the tumor cells is passed on by G-protein i and not by G-protein q. An important mechanism is the interaction of the GnRH receptor with the mitogenic signal transduction mediated by growth factors. Using a p42 / p44 MAP kinase assay it could be shown that the MAP kinase activity induced by EGF can be completely suppressed by GnRH analogues. Quantitative RT-PCR showed that GnRH analogues are able to inhibit the EGF-induced expression of the immediate early response gene c-fos in a dose-dependent manner in all examined ovarian and endometrial carcinoma cell lines with GnRH receptor expression. Nanomolar concentrations are sufficient to push the c-fos expression to a basal level of non-proliferating cells. GnRH controls the growth of the tumor cells through interaction of the GnRH receptor with the mitogenic signal transduction of the growth factors. Activation of the NFB transcription factor by GnRH analogues in ovarian and endometrial cancer cells:

In the ovarian carcinoma cell lines EFO-21, EFO-27 (Gründker et al. 2000) and 5 NIH: OVCAR-3 as well as in the endometrial carcinoma cell lines Hec-1A, Hec-1 B and Ishikawa it was observed for the first time that GnRH analogues in the Are able to activate the transcription factor NFB receptor-mediated. The activity of NFB increases 5 to 8 times. This activation is mediated via the G protein i and can be inhibited by pertussis toxin. In control experiments with cell lines without GnRH receptor expression, NFB is not activated by GNRH. We have two different clones of the SK-OV-3 cell line, of which only one expresses the GnRH receptor. Only in this clone can NFB be activated by GnRH.

.5 Activation of the NFB transcription factor by GnRH analogues in normal cells:

The proof that NFB in the GnRH receptor positive gonadotropic pituitary cells cannot be activated by GnRH analogues was carried out with the immortalized mouse pituitary cell line T3-1. Since the signal transduction of the

; o GnRH receptor in the pituitary fundamentally differs from that in the tumor cells, we therefore assume that GnRH analogs in the pituitary cells have no effect on NFB. In the 3T3 fibroblast cell line without GnRH receptor expression, NFB was never activated by GnRH analogues. Only in the GnRH receptor positive tumor cells but not in GnRH receptor positive

! 5 Like negative normal cells, NFB can be activated by GnRH. This fact makes the therapeutic concept of activating NFB via the GnRH receptor in order to activate its control to activate a therapeutic gene looks promising.

In vivo transfection with NFB-LacZ and activation of the transcription factor 10 NFB by GnRH analogues in vivo:

The following experiments should show that the expression of a introduced effector gene under control of the NFB response element in vivo in GnRH receptor positive tumors can be induced by GnRH analogs. At the same time, the experiments should show that no NFB-mediated expression of the effector gene is induced in normal tissues. Immunodeficient nude mice

5 Hec-1 B endometrial carcinoma cells were administered intraperitoneally. After the tumor cells had grown, the reporter gene plasmid NFB-LacZ in combination with the transfection reagent Lipofect was administered intraperitoneally to the mice. After 24 hours, the control mice were injected with saline and the experimental mice with the GnRH agonist triptorelin intravenously. Another 24 hours later, the animals were killed, the organs and the tumors removed and then stained with X-Gal. Only tumors from mice treated with triptorelin were stained blue. NFB-LacZ was not activated without triptorelin. The ovary and uterus were colored blue both with and without triptorelin. All other organs (brain, heart, lungs, spleen, liver, kidney, stomach, intestine) were not stained with or without triptorelin.

5 This result shows that GnRH analogues only activate the NFB in the endometrial carcinoma cells. NFB appears to be constitutively active in the ovary and uterus. However, this is negligible, since these organs are removed anyway in ovarian and endometrial cancer. NFB is not activated in all other organs examined, as long as inflammation of these organs is avoided. The reporter gene LacZ could be expressed in the tumor solely by liposomal transfection mediated by NFB. All other organs with the exception of the ovary and uterus showed no expression. These results lead to the conclusion that the desired therapy concept will also work in vivo.

: 5 In vitro evaluation of the therapy concept with the therapeutic construct NFB tymidine kinase (TK) and the active ingredient (prodrug) ganciclovir:

The following experiments were intended to show that the expression of an introduced therapeutic gene under the control of the NFB response element in vitro in GnRH receptor positive tumor cells can be induced by GnRH analogues. In the GnRH receptor-positive ovarian carcinoma cell lines EFO-21, EFO-27 and NIH: OVCAR-3 and in the GnRH receptor-positive endometrial carcinoma Nom cell lines Hec-1A, Hec-1B and Ishikawa could be shown that in NFB-TK transfected tumor cells the thymidine kinase is expressed and activated by triptorelin. The thymidine kinase then phosphorylates gancicovir, which is then further phosphorylated by cellular enzymes. Only then does GancicIovir become toxic. As a result, the GnRH receptor positive tumor cells transfected with NFB-TK died after the administration of triptorelin (activation) and gancicovir (prodrug). Without triptorelin or without GancicIovir there was no effect. GnRH receptor negative cells were not damaged by this therapy.

Literature:

Gründker C, Schulz K, Günthert AR, Emons G (2000) Luteinizing hormone-releasing hormone induces nuclear factor B-activation and inhibits apoptosis in ovarian cancer cells. Journal of Clinical Endocrinology and Metabolism 85: 3815-3820

Gründker C, Günthert AR, Westphalen S, Emons G (2002) Biology of GnRH Systems in human gynecological cancers. European Journal of Endocrinology 146: 1-14

Table 1

IVDU = (E) -5- (2-iodovinyl) -2'-deoxyuridine.

Claims

claims:

1. Nucleic acid construct which contains a therapeutic gene whose gene product triggers the generation of a cytotoxic or therapeutic active substance in a cell under the control of an NFκB-specific promoter.

2. Nucleic acid construct according to claim 1, characterized in that the NFKB-specific promoter contains at least one copy of the kappaB enhancer, preferably 4 to 6 copies of the KappaB enhancer, fused to a promoter suitable for the activation of the therapeutic gene.

3. Nucleic acid construct according to claim 1 or 2, characterized in that the therapeutic gene is the herpes simplex virus (HSV) thymidine kinase gene.

5

4. Nucleic acid construct according to claim 1 or 2, characterized in that the therapeutic gene is the varicella zoster virus thymidine kinase gene.

5. Nucleic acid construct according to claim 1 or 2, characterized in that o the therapeutic gene is the Echerichia coli cytosine deaminase gene.

6. Nucleic acid construct according to one of claims 1 to 5, characterized in that it contains at least one polyA sequence downstream of the therapeutic gene

: 5

7. Nucleic acid construct according to one of claims 1 to 6, characterized in that it additionally contains a sequence for the expression of the active substance precursor in the target cell.

8. o nucleic acid construct according to one of claims 1 to 7, characterized in that the nucleic acid construct is a plasmid.

9. Vector for the transfection of target cells with the nucleic acid construct, characterized in that the vector is a liposomal vector which contains the nucleic acid construct according to one of claims 1 to 8.

5 10. Vector for the transfection of target cells with the nucleic acid construct, characterized in that the vector is a viral vector, preferably a retro-viral or adenoviral vector, which contains the nucleic acid construct according to one of claims 1 to 8.

11. Use of the nucleic acid construct or the vector according to one of claims 1 to 10 for the manufacture of a preparation for the treatment of GnRH receptor-positive tumors.

12. Use according to claim 11 or 12, characterized in that the

5 GnRH positive tumor is endometrial, ovarian, breast or prostate cancer.

13. Use according to claim 12, characterized in that the preparation comprises the nucleic acid construct or the vector with the nucleic acid construct in one injection.

: solution or infusion.

14. Use according to claim 12 or 13, characterized in that the preparation with the nucleic acid construct or the vector additionally contains an active substance precursor or is intended for co-application with the active substance precursor

! 5 is.

15. A method for treating a patient with GnRH-positive tumor disease, in particular endometrial or ovarian cancer, characterized by the following steps: ioa) introducing the nucleic acid construct or the vector according to one of claims 1 to 10 into tumor target cells; b) Administration of GnRH or a GnRH analog at intervals of step a).

16. The method according to claim 15, characterized in that the following further 5 step follows: c) administration of an active substance precursor which is not naturally present in the cell and which is converted into the active substance by the gene product of the therapeutic gene contained in the nucleic acid construct , in timing before, after or with step b). O

17. The method according to claim 15 or 16, characterized in that the introduction of the nucleic acid construct is carried out with the aid of a liposomal or viral vector.

5 18. The method according to claim 15 or 16, characterized in that triptorelin is given as GnRH analog, preferably systemically.

19. The method according to claim 15 or 16, characterized in that GnRH or the GnRH analog is given 8 to 48 hours after the introduction of the construct or ιo of the vector into the target cells.

20. The method according to claim 15 or 16, characterized in that the introduction of the bare construct or the vector is carried out by intraperitoneal injection.

! 5

21. The method according to claim 15 or 16, characterized in that the active substance precursor is administered intraperitoneally after or with GnRH or the GnRH analog.