DE10158078A1 - Suizidgensysteme - Google Patents

Abstract

The invention relates to suicide gene systems, in particular those comprising Herpes simplex virus type 1 thymidine kinase (HSV-tk), cytochrome P450 isoenzyme 4B1 (cyp4B1) or human folylpolyglutamate synthase (fpgs), in particular the above in combination with each other and corresponding DNA sections, vectors, infectious particles, transfected cells and medicaments containing the above. The invention further relates to the use of objects and suicide genes, also in combination with activated agents for the treatment of leukemia or graft-host reactions for example in donor lymphocytes.

Description

The invention relates to suicide gene systems with herpes simplex virus type 1 Thymidine kinase (HSV-tk), cytochrome P450 isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs), especially in Combination of these and corresponding DNA segments, vectors, infectious particles, transfected cells and medicines containing them as well as the use of the objects and suicide genes also in Combination with activated agents for the treatment of leukemia or graft-versus-host reactions (GvHD), for example, when Donor lymphocytes.

The development of multimodal therapy strategies with chemotherapy and Radiation treatment in leukemia Childhood has revolutionized in the past four decades and the past gloomy prognosis of these patients improved significantly. So today z. B. about 80% of children with acute lymphoblastic leukemia polychemotherapy can be permanently cured (Harms and Janka 2000; Schrappe et al. 2000). But it also works with the Polychemotherapy for many relapses and certain High-risk patients with acute leukemia and in patients with chronic forms no cure. Provides for these patients Allogeneic hematopoietic stem cell transplantation is the only one possible curative treatment approach. In the last Years have shown that less the intensive chemical and radiotherapy treatment as part of conditioning rather the immunological induced by the transplant Operations play the crucial role in forecasting after allogeneic Transplant plays. These as "graft-versus-leukemia" (GvL) effects designated immunological phenomena are based primarily on the Detection and destruction of the recipient leukemia cells by the transplanted donor T cells. Besides that are probably too other immune system cells, such as B. NK cells and dendritic Cells involved.

The GvL effect has a special relationship to the so-called Graft-versus-host disease; GvHD). The transplanted mature donor T cells react here also against other patient cells, particularly those in the skin that Liver, the gastrointestinal tract, in exocrine glands and in the Lung tissues are localized. The extent of this reaction assume life-threatening proportions. Chronic courses are also not infrequently. The emergence of severe acute or chronic GvHD is after for a large part of the morbidity and mortality allogeneic stem cell transplantation responsible. GvHD and GvL effect are not necessarily associated with each other. Many investigation in Mouse model and also in clinical studies rather prove that principle possibility of a dissociated inducibility of GvL effect and GvHD that respond to different immune responses (GvL: Th2 / Tc2- Polarization; GvHD: Th1 / Tc1 polarization; see Fowler for an overview and Gress 2000) and apoptosis mechanisms (GvL: Perform system; GvHD: Fas system; Schmaltz et al. 2001) seem to be based. The Control and stabilization of the narrow balance between GvL effect and GvHD is now a primary goal of many research efforts in the field of hematological transplantation immunology and immunopathology (as a review: Bordignon et al. 1999).

A special use of the GvL effect is the use of Donor lymphocyte infusion (DLI) for treatment recurrences of chronic myeloid leukemia (CML), one acute myeloid leukemia (AML), a multiple myeloma and one NHL lymphoma (Kolb et al. 1990, 1995; Porter et al. 1994; Tricot et al. 1996). The response rate is when treating a CML relapse after allogeneic stem cell transplantation with a complete remission up to 70% of patients treated with DLI are impressive (Kolb et al. 1995) and significantly higher than in the other listed applications. However, at least 20-30% of patients with AML relapse also appear after allogeneic stem cell transplantation of donor lymphocyte doses to benefit (Kolb et al. 1995; Mehta et al. 1997).

In the treatment of post-transplant relapse an acute lymphocytic leukemia (ALL), especially in children and Adolescents are usually found playing donor lymphocyte doses only positive in individual reports (Lawson and Darbyshire 1998; Saw et al. 1997; Atra et al. 1997; Chang et al. 1998). With larger ones Case numbers do not appear to have a significantly positive therapeutic effect from DLI the treatment of ALL recurrences after allogeneic To show bone marrow transplantation: In the study by Kolb et al. (1995) could out of 12 patients with post-transplant recurrence one ALL not a single remission from donor lymphocytes alone brought in by Mehta et al. (1997) it was also only one of six patients. However, an ALL recurrence in children seems to be allogeneic hematopoietic stem cell transplantation through prophylactic Donor lymphocytes have been shown to be avertable in a variety of cases. In a pilot study (Bader et al. 1999) a total of 5 children, the post-transplant due to an increasing mixed haematological chimerism a significantly increased risk of ALL recurrence had (Bader et al. 2000), by reducing the immunosuppressive agents and / or donor lymphocytes again gave a complete haematological chimerism induced. Three of the five children stayed permanently in remission, two appeared several months later Recurrence. In children with increased MDS (myelodysplastic Syndrome) and AML risks with increasing mixed chimerism treatment numbers were similarly good. Continuing this Pilot study confirmed as a prospective multi-center study (Beck et al. 2001) the therapeutic value of DL1 therapy for the prevention of a Relapses: In a group of 43 children with ALL, AML or MDS (myelodysplastic syndrome) and increasing mixed Chimerism after allogeneic stem cell transplantation resulted in 24 children treated prophylactically with donor lymphocyte doses, 19 children were not subjected to this immunotherapy. Eleven of the 24 prophylactically treated children remained free of recurrence in the follow-up period, in contrast, only one of the remaining 19 children who were not specifically treated.

The prophylactic value of adoptive immunotherapy with Donor lymphocytes may also be present on ALL patients with a minimal residual disease (MRD) Extend transplant. Patients with MRD have a significant increased risk of recurrence after transplantation (Bader et al. 2001). Currently at Pediatric oncology could thus be monitored by pre- and Post-transplant risk factors for an ALL relapse Patient groups are defined by prophylactic Donor lymphocyte donations would benefit.

The number of T cells applied remains an important prerequisite for the promising therapeutic and prophylactic use of donor lymphocytes. Here again the balance between GvL effect and GvHD induction is crucial. At low T cell doses, the desired therapeutic or preventive effect is not certain. With a higher DLI dose, however, the GvHD risk increases significantly. As a rule, approx. 50% of the patients develop moderate to severe GvHD after donor lymphocyte administration (GvHD II ⁰ -IV ⁰ ; Kolb et al. 1995; Collins et al. 1997). The presence of severe GvHD usually entails a further increase in immunosuppression. This in turn can, as described by Bader et al. (1999) described in a single patient, abolish the preventive value of donor lymphocyte administration and thus further promote the occurrence of a relapse.

The largest possible number of donor lymphocytes for one to be able to apply the curative GvL effect without losing the life of the Endangering patients with uncontrollable GvHD, were the donor T cells with suicide genes by some working groups transduced. Suicide genes code for pro- or eukaryotic enzymes Origin, which is an inert substance ("prodrug") into a cytotoxic Can transfer substance or an active metabolite. The Toxic metabolites are only formed in the transduced cells and specifically lead to the death of these cells. So far at Exclusively related suicide gene is the gene for herpes simplex virus type 1 thymidine kinase (HSV-tk), which like nucleoside analogues Can convert ganciclovir and acyclovir into cytotoxic metabolites (see e.g. US 6,048,525). Through in vitro gene transfer, the today's vector systems using specific transduction and selection procedures using more than 99% of all donor lymphocytes HSV-tk can be successfully transduced. Should follow up on a patient Transfusion of such genetically engineered donor lymphocytes severe GvHD occur, so should the HSV-tk positive effector cells GvHD induction killed by ganciclovir (GCV) treatment (see e.g. US 6,048,525). The principle of this procedure is in have been successfully demonstrated in some clinical studies (Bordignon et al. 1995; Bonini et al. 1997; Verzeletti et al. 1998; Tiberghien et al. 2001).

However, GvHD was not successfully treated in all patients become. This was mostly due to an annihilation of the genetics modified lymphocytes returned by an immune response that probably against the non-human gene product, the HSV-TK- Protein, is directed (Verzeletti et al. 1998). Tiberghien and co-workers (2001) wanted this immune response in their clinical study prevent the HSV-tk-transduced donor lymphocytes from failing only a few weeks after stem cell transplantation (e.g. in case of relapse a CML), but prophylactically in parallel with the administration of T cell depleted grafts were infused. This should make one Tolerance of the newly formed T cells to the HSV-TK protein be induced. In fact, despite the in vitro Manipulation of the T cells caused the GvHD rate to be around 25% in the Order of magnitude, as is the case with the administration of non-manipulated drugs Donor lymphocytes are expected. But not one anyway partial inactivation of donor lymphocytes by cytotoxic Immune responses to the HSV thymidine kinase did not take place be excluded. At least in the study by Tiberghien et al. (2001) in one patient the GvHD induced by DLI should not be contained by ganciclovir alone. First the additional corticosteroid administration brought the desired clinical Success.

An obvious ganciclovir resistance of the genetically modified Lymphocytes have also been observed in preliminary studies (Bonini and Bordignon 1997). In addition to immunological inactivation is also a genetic loss of function of the HSV-tk gene accepted. Garin et al. (2001) were able to demonstrate that Donor lymphocytes are not uncommon due to certain splice variants can be transduced with a non-functional HSV-tk gene, the one Deletion comprising 200 base pairs.

Furthermore, it is also quite possible that by switching off the viral Promoters ("silencing") that control HSV-tk expression, ganciclovir Resistance arises. Long term expression studies have shown that the 5 LTR promoter of retroviral vectors very often in mammalian cells is inactivated (Hoeben et al. 1991).

Partial ganciclovir resistance in the treatment of chronic GvHD was developed by Verzeletti et al. (1998) also on the proliferation-dependent mode of action of the HSV-tk / ganciclovir system recycled. A low proliferation activity of the T cells, as in chronic GvHD may not be enough to trigger an effective cytotoxicity with GCV.

Finally, of course, purely pharmacological ones Resistance to ganciclovir to weaken the Cytotoxicity effect. Here is z. B. inactivation events in downstream signal cascades of the cytotoxicity or Think apoptosis mechanism.

• 1. Immunologische Eliminierung der T-Zellen durch Immunisierung gegenüber dem HSV-TK-Protein
• 2. Genetische Inaktivierung durch Mutation/Deletion im HSV-tk-Gen
• 3. Genetische Inaktivierung durch Promotor-Silencing
• 4. Funktionelle Ineffektivität durch unzureichende Zellzyklusprogression
• 5. Pharmakologische Resistenz durch Inaktivierung auf der Ebene nachgeschalteter Zytotoxizitätsmechanismen

In summary, the following proven and suspected inactivation mechanisms of the HSV-tk / ganciclovir system in donor lymphocytes result:

• 1. Immunological elimination of the T cells by immunization against the HSV-TK protein
• 2. Genetic inactivation by mutation / deletion in the HSV-tk gene
• 3. Genetic inactivation through promoter silencing
• 4. Functional ineffectiveness due to insufficient cell cycle progression
• 5. Pharmacological resistance through inactivation at the level of downstream cytotoxicity mechanisms

• 1. Zur schnellen und effektiven Verhinderung und Behandlung einer lebensgefährlichen GvHD müssen die Spenderlymphozyten durch das Suizidgensystem zuverlässig und schnell abgetötet werden.
• 2. Die Substanz, die verwendet wird, soll wenig toxisch sein für die übrigen Körperzellen.
• 3. Das Suizidgensystem soll eine möglichst geringe Eigenimmunität aufweisen.

These inactivation mechanisms represent a clear disadvantage of the HSV-tk / ganciclovir system, that is to say of the prior art, so that the necessary biological safety is not satisfactorily guaranteed when donor lymphocytes are used. There is therefore a need for alternatives and improvements. It is therefore an object of the invention described below to develop new suicide gene strategies which are intended to take into account the different disadvantages of the HSV-tk / GCV system when used in donor T cells. The possible restrictions 1, 4 and 5, as can be seen from the above list of suspected inactivation mechanisms of the HSV-tk / ganciclovir system in donor lymphocytes, should be overcome by the choice of alternative suicide gene systems. In addition, restrictions 2 and 3 are overcome as far as possible by changing the vector design. The following requirements of the solution search were used:

• 1. For the rapid and effective prevention and treatment of life-threatening GvHD, the donor lymphocytes must be killed reliably and quickly by the suicide gene system.
• 2. The substance that is used is said to be little toxic to the rest of the body cells.
• 3. The suicide system should have the lowest possible immunity.

The invention therefore relates to a DNA section containing a first sequence for the herpes simplex virus type 1 thymidine kinase (HSV-tk), and a second sequence for the cytochrome P450 Isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs) encoded.

The invention also relates to a viral vector containing a first sequence for the herpes simplex virus type 1 thymidine kinase (HSV-tk), and a second sequence for the cytochrome P450 Isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs) encoded.

The invention also relates to a retroviral vector containing one first sequence for the herpes simplex virus type 1 thymidine kinase (HSV-tk), and a second sequence for the cytochrome P450 Isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs) encoded.

For all of these objects of the invention, the DNA Section, the viral vector according to the invention or the retroviral vector according to the invention applies that this at least two Promoters, preferably one, especially both, is / are of eukaryotic origin, and / or at least one selection or contains reporter gene or selectable marker gene, and / or the second sequence encoded for the cytochrome P450 isoenzyme 4B1 (cyp4B1).

The central solution of this invention is the choice of the cyp4B1 as an element cheaper suicide systems here, especially in combination with the already known HSV-tk / ganciclovir (GCV) system. As alternatives there were at least 12 more besides the HSV-tk / GCV system Enzyme gene systems with their substrates (Aghi et al. 2000). As the only system besides HSV-tk / GCV is the E. coli cytosine deaminase (cd) / 5-fluorocytosine (5FC) system (Mullen et al. 1994) already in clinical Tumor therapy studies tested (Crystal et al. 1997; Pandha et al. 1999; Harvey et al. 1999). That would have recommended this system. It turned out but surprisingly it turns out that the rabbit Cytochrome P450 4B1 (cyp4B1) system (Rainov et al. 1998a) as well the system with human folyl polyglutamate synthetase (fpgs) (Aghi et al. 1999a, b) was the most suitable here.

The cyp4B1 system can have two chemically different substances activate, 4-ipomeanol (4IPO) and 2-aminoanthracene (2AA). 4IPO is in higher doses used, a tested in phase I clinical trials Chemotherapy drug (Rowinsky et al. 1993; Kastun et al. 1998). The cyp4B1 / 4IPO and cyp4B1 / 2AA systems are already one possibility cancer therapy (see e.g. WO 99/05299 from the University of Boston, USA). The cyp4B1 / 4IPO and cyp4B1 / 2AA systems were developed in with regard to toxicity profile and kinetics and to the "bystander effect" in Rat glioma cells characterized in more detail. This showed up Surprisingly, that cyp4B1-transduced cells with both 2AA as well as 4IPO are killed much faster than HSV-tk- expressing cells that are incubated with GCV. Already short Incubate 15-30 minutes of treatment with 2AA or 4IPO significant cytotoxicity in the cyp4B1 system. After treatment of infected lymphocytes with different effective concentrations of 4IPO shows that with higher doses of 4IPO after only one day Treatment only 20-30% of the originally over 80% cyp4B1 / egfp- transduced cells live.

This is the minimum or even missing, even with high cell density "Bystander effect" is a great advantage of the suicide gene system with cyp4B1. This strong "bystander effect", the phenomenon that also nontransduced cells in the immediate vicinity of the transduced cells from the toxic metabolites of the activated ones Prodrug damage (Moolten and Wells 1990), however, is a clear limitation of the HSV-tk system. So are in cell culture only 10% HSV-tk positive cells necessary to cover the entire cell population by a ganciclovir treatment which is non-toxic for non-transducted cells kill. However, this requires close cell-to-cell contact.

Under similar cell culture conditions with tight intercellular In contrast, contacts were only found in the cyp4B1 system under 2-AA weaker, under 4IPO even no "bystander effect". A treatment with the cyp4B1 / 4IPO system shows a large therapeutic window in human lymphocytes. While under treatment with 50 µg / ml 4IPO the transduced cells die within the first two days, indicate the uninfected and the infected but not transducted Cells no significant cytotoxicity compared to untreated Lymphocytes.

Overall, the cyp4B1 / IPO system fulfills essential requirements for use in donor lymphocytes: the rapid effectiveness and low toxicity to untransduced cells, recognizable by the little or no "bystander effect". Add to that the eukaryotic origin with a consequent lower immunity than in the suicide system of viral or bacterial origin.

There are also preliminary results with human T- Lymphocytes that appear to prove that in rat glioma cells found results are transferred to human lymphocytes can (see examples).

Regarding the transfection rate after retroviral infection, Fibronectin with a transduction protocol according to Hanenberg et al. 1996; Hanenberg et al. 1997 and Pollok et al. 1998 in initial trials even without further selection using more than 80% of human T cells a cyp4B1-egfp fusion gene can be transduced. So that's it transduction result achieved significantly above that in literature transduction efficiencies described above without selection process of 10-50% (Di lanni et al. 1999; Movassagh et al. 2000; Maddens et al. 2000). This good result after infection alone results in a good starting point for the selection To increase the overall transduction rate to almost 100% and with that Risk of uncontrolled GvHD from initially non-transduced Almost exclude lymphocytes.

The second new system, the fpgs system, is not a suicide system in the proper sense. It does not activate any ineffective ones Precursor substance. Rather, this gene enhances the effect of the already potent chemotherapy drug methotrexate (MTX) by taking its intracellular length of stay prolonged. By transferring the fpgs- Genes in the rat brain tumor model the tumor effective methotrexate dose lowered and thus the side effect rate on normal body cells can be reduced (Aghi et al. 1999a). The fpgs / MTX system also shows no "bystander effect" and promises due to its human Origin significantly reduced immunity. Low dose MTX will already in the clinical routine for GvHD prophylaxis after allogeneic Stem cell transplantation used. The effectiveness of MTX on T- This has proven cells to suppress GvHD.

Is particularly preferred and is therefore at the center of this invention the combination of two suicide systems. That combination several suicide systems, i.e. the well-known HSV-tk system the cyp4B1 or fpgs system, has proven beneficial to the biological Backup of donor lymphocytes was exposed as it already did described genetic mechanisms of system inactivation can counteract effectively. By using two Suicide genes, especially those under the control of independent promoters, the risk of inactivation by mutation / deletion of the suicide gene or by switching off the Promoter activity significantly reduced: when one is inactivated Suicide genes still attack the second gene. The simultaneous Deactivating both systems can be relative in this context unlikely event to be viewed, at least by one Much less likely than inactivating a single system. The combination of different suicide systems also works the development of pharmacological resistance.

In a preferred embodiment of the invention retroviral vector therefore applies to the promoter, under its control the expression of the first sequence is carried out for the herpes simplex Virus type 1 thymidine kinase (HSV-tk) encodes itself from the promoter under his control distinguishes the expression of the second sequence done for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or the encoded human folyl polyglutamate synthase (fpgs).

• a) einen 5'-LTR und einen 3'-LTR vorzugsweise von einem Retrovirus,
• b) mindestens einen, vorzugsweise zwei, Promotoren, insbesondere ausgewählt aus der Gruppe bestehend aus: SV40-Promotor, 5'- LTR mit Promotor-Funktion, CD25-Promotor und CD69-Promotor, vorzugsweise 5'-LTR mit Promotor-Funktion, CD25-Promotor und CD69-Promotor;
• c) mindestens ein Selektions- oder Reportergen, bzw. selektierbares Markergen ausgewählt aus: dem vorzugsweise trunkierten Gen für den humanen "low affinity NGF Rezeptor" (LNGFR) und/oder dem vorzugsweise trunkierten humanen CD34-Gen (CD34),
• d) mindestens ein retrovirales Verpackungssignal (ψ) und
• e) gegebenfalls ein IRES-Element (IRES) mit dem die Expression des Selektions- oder Reportergens, bzw. selektierbares Markergens an die Expression der ersten oder zweiten Sequenz gekoppelt ist.

In a further preferred embodiment of the retroviral vector according to the invention, the vector applies in addition to the first sequence coding for the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the second sequence coding for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or encodes human folyl polyglutamate synthase (fpgs) containing the following functional elements:

• a) a 5'-LTR and a 3'-LTR, preferably from a retrovirus,
• b) at least one, preferably two, promoters, in particular selected from the group consisting of: SV40 promoter, 5'-LTR with promoter function, CD25 promoter and CD69 promoter, preferably 5'-LTR with promoter function, CD25 Promoter and CD69 promoter;
• c) at least one selection or reporter gene or selectable marker gene selected from: the preferably truncated gene for the human "low affinity NGF receptor" (LNGFR) and / or the preferably truncated human CD34 gene (CD34),
• d) at least one retroviral packaging signal (ψ) and
• e) if appropriate, an IRES element (IRES) with which the expression of the selection or reporter gene or selectable marker gene is coupled to the expression of the first or second sequence.

The CD25 and CD69 promoters in particular are possible Decrease in immunogenicity since it is specific for activated T- Cells are e.g. B. the CD69 promoter the promoter for the T cell Activation antigen CD69 or the CD25 promoter is the promoter for is the short chain of the IL-2 receptor. When using such Promoters e.g. B. for HSV-tk expression would be the expression of Suicide genes associated with T cell activation. A diminished one Expression of the foreign protein in the unactivated state of the T cells this reduces the likelihood of immunization. It will also be like this a viral promoter is replaced by a human promoter, which is the Reduced risk of promoter inactivation.

It is also advantageous to express the expression of one of the suicide genes via an IRES Element with the expression of the truncated human CD34 gene coupled. This coupling offers advantages. The CD34 antigen is of human origin and labeled hematopoietic progenitor cells, however no mature T cells. Clinically approved, commercially exist available selection kits (e.g. from Miltenyi Biotec, Bergisch-Gladbach), selected via the CD-34 positive cells using microbeads and can be enriched. This also applies to CD-34 negatives mature T-cells by retroviral transduction of the CD34 antigen express. The human origin of this selection and reporter gene concludes immunization against this additional genetic Material largely from. In addition, the cDNA for human CD34 Antigen e.g. B. commercially available from ATCC, Manassas, USA. The the second selection and reporter gene besides CD34 is this truncated gene for the human low affinity NGF receptor (= LNGFR). This gene has been used as a selection marker in clinical trials the group around Claudio Bordignon, Milan, Italy, with the HSV-tk / GCV- System successfully used in DLIs. The selection is made as for CD34 via the MACS system with the mediation of a specific antibody.

In a particularly preferred embodiment of the retroviral vector according to the invention, it is a mouse leukemia virus (murine leucemia virus / MuLV) vector, preferably with one of the following sequences of the functional elements:
5'LTR ψ-cyp4B1-IRES-CD34-CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-FPGS-IRES-CD34-CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-FPGS-IRES-LNGFR CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-cyp4B1-IRES-LNGFR CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-HSV-tk-IRES-CD34-CD69-LTR promoter cyp4B1-3'-,
5'LTR ψ-HSV-tk-IRES-CD34-CD69 promoter FPGS-3'-LTR,
5'LTR ψ HSV-tk-IRES-CD69 LNGFR promoter cyp4B1-3' LTR
or
5'LTR ψ-HSV-tk-IRES-LNGFR CD69 promoter FPGS-3'-LTR.

• a) eine Retrovirus-Verpackungszelle mit einem erfindungsgemäßen retroviralen Plasmid-Vektor transfiziert wird,
• b) die Retrovirus-Verpackungszelle in einem geeigneten Medium in vitro und/oder in vivo so kultiviert wird, daß infektiöse Retroviruspartikel gebildet werden.

Another object of the invention is a method for producing an infectious retrovirus particle, in which

• a) a retrovirus packaging cell is transfected with a retroviral plasmid vector according to the invention,
• b) the retrovirus packaging cell is cultivated in a suitable medium in vitro and / or in vivo in such a way that infectious retrovirus particles are formed.

Among the infectious particles are, in particular, packaged retrovirals Understand vectors in envelope proteins. Here are the Packaging cell lines PA3 17 and AMIZ with the amphotropic MuLV-env- Protein and pgl3 with the GALV (gibbon ape leukemia virus) -env protein on. These packaging cell lines are all becoming clinical for manufacturing Vectors used.

It is therefore particularly preferred if in the invention Method the retrovirus packaging cell of a cell line selected from the packaging cell lines PA317 and AMIZ with the amphotropic MuLV env protein and pg13 with the GALV (gibbon ape leukemia virus) -env- Protein comes from.

Another object of the invention is an infectious particle that by the inventive method described above is available.

Another object of the invention is a host cell containing at least one DNA section according to the invention, viral vector according to the invention, retroviral according to the invention Vector and / or infectious particle according to the invention.

In principle, this can be any type of pro- or eukaryotic Act cell. However, a mammalian cell is preferred, in particular a human cell. It is particularly preferred if the host cell according to the invention a - preferably human - Lymphocyte cell.

Another object of the application is a method for manufacturing a host cell according to the invention, wherein the cell with a first and a second retroviral vector according to the invention in the form of a infectious particle according to the invention is transfected, the first and the second retroviral vector according to the invention is equal to or can be different, with the fact that in the invention Process for the preparation of the infectious particles according to the invention Retrovirus packaging cells with different cell lines different packaging proteins can be used.

Simultaneous infection (cotransduction) with two different or same (combination) vectors has significant advantages. The Using two identical combination vectors has the advantage that by the double infection a higher expression of the respective Suicide genes can be achieved and stronger suicide gene expression also means a higher effectiveness of biological Security systems. Co-transduction of lymphocytes with different combination vectors in turn opens the Possibility of two instead of a single combination of two suicides different combinations of suicides can be used in parallel. For example, if the GvHD is not treated properly, it can be treated with the first combination of suicides in a simple manner to the second Combination can be changed to still be an effective GvHD To achieve control.

Basic requirement for the simultaneous infection of lymphocytes with two retroviral vectors, however, is that both retroviral vectors are of different packaging cell lines with different envelope Proteins are generated and packaged. In an earlier work (MacNeill et al. 1999) has been shown that when packaging different retroviral vectors with different envelope Proteins have no negative interference when transducing the same Cell population occur. It lends itself to those presented here Combination vectors the clinically used packaging cell lines PA317 and AMIZ with the amphotropic MuLV env protein and pg13 with the GALV (Gibbon Ape Leukemia Virus) -env protein.

It also lends itself to the two different Combination vectors with different selection or Reporter genes are equipped. So can with cotransduction be selected in two ways, what the Overall transduction efficiency certainly benefits. Also can the distribution of the vectors in the transduced lymphocyte population for each individual vector, but also in combination via FACS be visualized. In addition to CD34, the LNGFR comes into question.

Another object of the invention is a double-labeled cell containing a first DNA sequence for the herpes simplex virus type 1 thymidine kinase (HSV-tk) encoded, and one in a separate molecule second DNA sequence for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs), preferably cyp4B1, encoded, each in the form of a DNA segment, a viral vector, a retroviral vector and / or one infectious particle and or containing on the one hand recombinantly expressed HSV-tk and on the other recombinantly expressed cyp4B1 or fpgs, preferably cyp4B1.

In principle, this can be any type of pro- or eukaryotic Act cell. However, a mammalian cell is preferred, in particular a human cell. It is particularly preferred if the double-labeled cell according to the invention a - preferably human - Lymphocyte cell.

Another object of the invention is a method for manufacturing a double-labeled cell according to the invention, wherein the cell with a first and a second retroviral vector according to the invention is transfected in the form of an infectious particle according to the invention, wherein the first and the second retroviral vector according to the invention are the same or can be different, with the proviso that at Process according to the invention for the production of the invention infectious particles retrovirus packaging cells different Cell lines with different packaging proteins are used, or the cell with a first retroviral vector containing a first DNA Sequence for herpes simplex virus type 1 thymidine kinase (HSV-tk) encoded, and with a second retroviral vector containing a second DNA sequence necessary for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs), preferably cyp4B1, encoded, in each case in the form of an infectious particle, where applies that in the process for producing the infectious particles Retrovirus packaging cells with different cell lines different packaging proteins can be used.

Simultaneous infection (cotransduction) with two different or same vectors has significant advantages. The use of two same combination vectors has the advantage that by the Double infection achieved a higher expression of the respective suicide genes and a stronger suicide gene expression also means higher effectiveness of the biological safety systems. The Co-transduction of lymphocytes with different Combination vectors in turn open the possibility that instead of one individual two-suicide gene combination two different Suicide combinations can be used in parallel. So at inadequate treatment of a florid GvHD with the first Suicide combination in a simple way on the second combination be changed to still have an effective GvHD control to reach.

Basic requirement for the simultaneous infection of lymphocytes with two retroviral vectors, however, is that both retroviral vectors are of different packaging cell lines with different envelope Proteins are generated and packaged. In an earlier work (MacNeill et al. 1999) has been shown that when packaging different retroviral vectors with different envelope Proteins have no negative interference when transducing the same Cell population occur. It lends itself to those presented here Combination vectors the clinically used packaging cell lines PA317 and AMIZ with the amphotropic MuLV env protein and pg13 with the GALV (Gibbon Ape Leukemia Virus) -env protein.

It also lends itself to the two different Combination vectors with different selection or Reporter genes are equipped. So can with cotransduction be selected in two ways, what the Overall transduction efficiency benefits. In addition, the Distribution of the vectors in the transduced lymphocyte population for each individual vector separately, but also in combination via FACS be visualized. In addition to CD34, the LNGFR comes into question.

Another object of the invention is a human lymphocyte containing at least one DNA section, a viral vector, one retroviral vector and / or an infectious particle, the or each contains a DNA sequence which is isoenzyme 4B1 for the cytochrome P450 (cyp4B1) encoded, and / or containing recombinantly expressed cyp4B1.

This lymphocyte in particular is at the core of this invention because it the low "bystander effect" combined with the effectiveness of the Cyp4B1 the ideal, safe donor lymphocyte for a DLI is with a suitable one Suicide system is because 4IPO and 2AA can be used if necessary.

The invention further relates to medicaments containing at least one DNA section according to the invention, viral vector according to the invention, retroviral according to the invention Vector, infectious particle according to the invention, according to the invention Host cell, double-labeled cell according to the invention and / or human lymphocytes according to the invention and optionally at least one pharmaceutically compatible auxiliary and / or carrier.

In principle, the pharmaceuticals according to the invention can be in the form of liquid or solid dosage forms, in particular in the form of injection solutions Tablets, capsules or coated tablets can be administered. But is preferred either use as a therapeutic in gene therapy, in particular in vitro gene therapy, in which cells outside the body before the therapeutic use can be transfected, or use in suitable Form of pharmaceutically prepared cells according to the invention, the implanted or applied in another form. Suitable auxiliary and / or carriers are e.g. B. solvents or diluents, Stabilizers, suspending agents, nutrient solutions or nutrients, Transfection aids, salts, buffer substances, preservatives, as well as dyes, fillers and / or binders. The selection of the Auxiliaries and the amounts to be used depend on whether the drug is parenteral, intravascular, intravenous, intraperitoneal or to be applied intramuscularly. For all parenteral applications Preparations in the form of suspensions and solutions are suitable as well as easily reconstitutable dry preparations.

• a) eines erfindungsgemäßen DNA Abschnitts, eines erfindungsgemäßen viralen Vektors, eines erfindungsgemäßen retroviralen Vektors, eines erfindungsgemäßen infektiösen Partikels, einer erfindungsgemäßen Wirtszelle, einer erfindungsgemäßen doppeltmarkierten Zelle, eines erfindungsgemäßen humanen Lymphozyten und/oder eines erfindungsgemäßen Arzneimittels,
• b) eines DNA-Abschnitts, eines viralen Vektors, eines retroviralen Vektors und/oder eines infektiösen Partikels, der oder das jeweils eine DNA Sequenz enthält, die für das Cytochrom P450 Isoenzym 4B1 (cyp4B1) kodiert; einer Zelle enthaltend mindestens einen DNA-Abschnitt, einen viralen Vektor, einen retroviralen Vektor und/oder ein infektiöses Partikel, der oder das jeweils eine DNA- Sequenz enthält, die für das Cytochrom P450 Isoenzym 4B1 (cyp4B1) kodiert; einer Zelle enthaltend rekombinant exprimiertes cyp4B1; und/oder des Enzyms cyp4B1,
• c) von 4-Ipomeanol (4IPO), 2-Aminoanthracen (2AA), Methotrexat (MTX) und/oder einer Kombination von 4-Ipomeanol (4IPO), 2- Aminoanthracen (2AA) oder Methotrexat (MTX) mit Ganciclovir (GCV), vorzugsweise zusammen mit einem Gegenstand aus den Gruppen a) und/oder b),

zur Herstellung eines Arzneimittels zur Behandlung von bzw. zur Behandlung von Leukämie, insbesondere der chronischen oder akuten myeloischen Leukämie (CML oder AML) sowie der akuten lymphatischen Leukämie (ALL) wie auch des myelodysplastischen Syndroms (MDS), des Multiplen Myeloms und des NHL-Lymphoms, und/oder zur Behandlung von oder Prophylaxe gegen Graft versus Host-Reaktionen, insbesondere nach Applikation von Spenderlymphozyten. Another object of the invention is the use

• a) a DNA section according to the invention, a viral vector according to the invention, a retroviral vector according to the invention, an infectious particle according to the invention, a host cell according to the invention, a double-labeled cell according to the invention, a human lymphocyte according to the invention and / or a medicament according to the invention,
• b) a DNA segment, a viral vector, a retroviral vector and / or an infectious particle which each contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing at least one DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing recombinantly expressed cyp4B1; and / or the enzyme cyp4B1,
• c) of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA), methotrexate (MTX) and / or a combination of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA) or methotrexate (MTX) with ganciclovir (GCV) , preferably together with an object from groups a) and / or b),

for the manufacture of a medicament for the treatment or treatment of leukemia, in particular chronic or acute myeloid leukemia (CML or AML) and acute lymphoblastic leukemia (ALL) as well as myelodysplastic syndrome (MDS), multiple myeloma and NHL Lymphoma, and / or for the treatment of or prophylaxis against graft versus host reactions, especially after application of donor lymphocytes.

In general, it is within the scope of this invention that the use of a Object for the treatment of a disease or prophylaxis against this disease also as a use of this item for Manufacture of a medicine to treat this disease or Prophylaxis against this disease can be understood.

• a) eines DNA-Abschnitts, eines viralen Vektors, eines retroviralen Vektors und/oder eines infektiösen Partikels, der oder das jeweils eine DNA-Sequenz enthält, die für das Cytochrom P450 Isoenzym 4B1 (cyp4B1) kodiert; einer Zelle enthaltend mindestens einen DNA-Abschnitt, einen viralen Vektor, einen retroviralen Vektor und/oder ein infektiöses Partikel, der oder das jeweils eine DNA- Sequenz enthält, die für das Cytochrom P450 Isoenzym 4B1 (cyp4B1) kodiert; einer Zelle enthaltend rekombinant exprimiertes cyp4B1; und/oder des Enzyms cyp4B1,
• b) von 4-Ipomeanol (4IPO) und/oder von 2-Aminoanthracen (2AA), vorzugsweise zusammen mit einem Gegenstand aus der Gruppe a) in Kombination mit Reportergenen für klinische Genmarkierungsexperimente, in Kombination mit therapeutischen Genen in therapeutischen Gen-Ersatz-Ansätzen, zur Umwandlung von Donor-Lymphozyten für die Leukämie Rezidiv-Prophylaxe und zur Behandlung nach allogenetischer Knochenmarkstransplantation (allo-BMT).

Another object of the invention is the use

• a) a DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing at least one DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing recombinantly expressed cyp4B1; and / or the enzyme cyp4B1,
• b) of 4-ipomeanol (4IPO) and / or of 2-aminoanthracene (2AA), preferably together with an object from group a) in combination with reporter genes for clinical gene labeling experiments, in combination with therapeutic genes in therapeutic gene replacement approaches , for converting donor lymphocytes for leukemia relapse prevention and for treatment after allogenetic bone marrow transplantation (allo-BMT).

• a) eines erfindungsgemäßen DNA-Abschnitts, eines erfindungsgemäßen viralen Vektors, eines erfindungsgemäßen retroviralen Vektors, eines erfindungsgemäßen infektiösen Partikels, einer erfindungsgemäßen Wirtszelle, einer erfindungsgemäßen doppeltmarkierten Zelle, eines erfindungsgemäßen humanen Lymphozyten und/oder eines erfindungsgemäßen Arzneimittels oder
• b) von 4-Ipomeanol (4IPO), 2-Aminoanthracen (2AA), Methotrexat (MTX) und/oder einer Kombination von 4-Ipomeanol (4IPO), 2- Aminoanthracen (2AA) oder Methotrexat (MTX) mit Ganciclovir (GCV) zusammen mit einem Gegenstand aus der Gruppe a)

in der Gentherapie. Dabei kann es sich um In-Vivo- oder In-vitro- Gentherapie handeln. Besonders bevorzugt ist hier die In-vitro Gentherapie, bei der Zellen außerhalb des Körpers vor dem therapeutischen Einsatz transfiziert werden. Another object of the invention is the use

• a) a DNA section according to the invention, a viral vector according to the invention, a retroviral vector according to the invention, an infectious particle according to the invention, a host cell according to the invention, a double-labeled cell according to the invention, a human lymphocyte according to the invention and / or a medicament according to the invention or
• b) of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA), methotrexate (MTX) and / or a combination of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA) or methotrexate (MTX) with ganciclovir (GCV) together with an object from group a)

in gene therapy. This can be in-vivo or in-vitro gene therapy. In vitro gene therapy is particularly preferred, in which cells are transfected outside the body before therapeutic use.

Another object of the invention is a treatment method in to a person or animal in need of such treatment in a suitable form, a medicament according to the invention is applied.

Another preferred subject of the invention is a treatment method for the treatment of leukemia, in particular chronic or acute myeloid leukemia (CML or AML) and acute lymphoblastic leukemia (ALL) as well as myelodysplastic syndrome (MDS), multiple myeloma and NHL- Lymphoma, and / or for the treatment of or prophylaxis against graft versus host reactions, in particular after application of donor lymphocytes, in the first step
a) either a host cell according to the invention, a double-labeled cell according to the invention, a human lymphocyte according to the invention, a cell containing at least one DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence, which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); or a cell containing recombinantly expressed cyp4B1 can be produced by the customary methods,
or donor lymphocytes with a DNA section according to the invention, a viral vector according to the invention, a retroviral vector according to the invention, an infectious particle according to the invention or with a DNA section, viral vector, retroviral vector and / or infectious particles which each contain a DNA sequence , which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1), are (if possible completely) transfected,
these are then applied to the patient in a second step b),
and if necessary in a third step c), if serious graft versus host reactions make this appear necessary, the patient 4-ipomeanol (4IPO), 2-aminoanthracene (2AA), methotrexate (MTX) and / or a combination of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA) or methotrexate (MTX) with ganciclovir (GCV), in a suitable pharmaceutical preparation and in a suitable dose and form, in order to kill the cells applied in step b) again.

It is particularly preferred if the suicide gene system used comprises at least Cyp4B1 or cells are used which are associated with Sequences encoding Cyp4B1, or contain recombinantly expressed Cyp4B1. It is then also advantageous the differentiated use of the active ingredients to be activated 4IPO and 2AA in a therapy regimen that shows their differences in effects and benefits takes into account, i.e. the faster and stronger effect of 2AA on the one and 4IPO's missing bystander effect on the other. An example would be after a strong graft versus host response Application of donor lymphocytes transfected with Cyp4B1 Immediate use of 2AA to get the most effective and fast Combat symptoms followed by longer-term treatment with 4IPO to completely kill donor lymphocytes reach - but without damaging the remaining tissue.

Another object of the invention is a treatment method using a suicide gene system with Cyp4B1 for the treatment of Cancers in which a "bystander effect" is not desired in the long term in which explicitly cyp4B1 in combination with 2AA and in particular with 4IPO is used. It is particularly preferred if the activating active ingredients 4IPO and 2AA differentiated in one Therapy scheme can be used that their differences in effects and Benefits taken into account, i.e. the faster and stronger effect of 2AA on the one hand and 4IPO's missing bystander effect on the other side. An example would be the early use of 2AA, to the extent possible fight cancer cells effectively and quickly, followed by one long-term treatment with 4IPO to completely kill the Reach cancer cells without damaging the remaining tissue.

In the following section, the invention is further illustrated by examples, without limiting it to that.

Examples and illustrations pictures

Fig. 1 shows the retrovirally mediated transduction of human lymphocytes with the cyp4B1-egfp fusion gene.

Fig. 2 Persistence of vital transduced lymphocytes under 1-8 days of treatment with different doses of 4IPO.

Fig. 3 Kinetics of killing human lymphocytes under treatment with 4IPO [µg / ml] depending on the infection and transduction status.

Fig. 4 shows the cytotoxicity of 2AA and 4IPO in 9L cells (9L-4B1) transduced with cyp4B1 and of GCV in 9L cells (9I-tk) transduced with HSV-tk. The survival rate was calculated after one day in relation to the untreated cells (= 100%). The average percentage results of up to 5 different experiments are shown.

Fig. 5 shows the cytotoxicity of 2AA and 4IPO in 9L cells (9L-4B1) transduced with cyp4B1 and of GCV in 9L cells (9I-tk) transduced with HSV-tk. The survival rate was calculated after two days in relation to the untreated cells (= 100%). The average percentage results of up to 5 different experiments are shown.

Fig. 6 shows the "bystander effect" in different cocultures after treatment with 4IPO (A) and 2AA (B). The "bystander" kill rate was measured in cocultures with different ratios between untransduced and transduced 9L cells (9L / 9L-4B1) in the presence of 5 µg / ml 4IPO or 2AA. The survival rate was determined after 1 and 2 days of drug treatment. The results shown are the average of at least 2 independent experiments.

Examples

It should be noted that the full content of the literature cited of the present description. Especially regarding experimental conditions, these references are part of the Consider disclosure of this invention.

example 1 Retroviral mediated transduction of human lymphocytes with the cyp4B1-egfp fusion gene

For the transduction of T cells, the cyp4B1-egfp fusion gene (Rainov et al. 1998 b; Steffens et al. 2000) was inserted into a retroviral vector, so that the identification of successfully transduced cells in FACS (fluorescence-activating cell sorting) is very simple via the inherent fluorescence of the egfp component (egfp = enhanced green fluorescence protein). It has already been demonstrated in previous work (Steffens et al. 2000) that the cytotoxicity of the suicide gene and the intrinsic fluorescence of the egfp are retained in the fusion gene and are closely correlated with one another. After retroviral infection with fibronectin using a transduction protocol according to Hanenberg et al. 1996; Hanenberg et al. 1997 and Pollok et al. In 1998, in initial experiments, as shown in Fig. 1, more than 80% of human T cells could be transduced with the cyp4B1-egfp fusion gene without further selection. The transduction result achieved is thus significantly above the transduction efficiencies of 10-50% described in the literature without a selection process (Di lanni et al. 1999; Movassagh et al. 2000; Maddens et al. 2000). This good result after infection alone results in a good starting situation in order to increase the overall transduction rate to almost 100% by subsequent selection and thus to virtually exclude the risk of uncontrolled GvHD (graft versus host disease) from initially untransduced lymphocytes.

Example 2 Retroviral mediated transduction of 9L cells with fusion genes as well as investigations

9L rat glioma cells were cultured at 37 ° C and 5% CO ₂ in RPMI medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U / ml penicillin, 100 µg / ml streptomycin and 10% heat-inactivated fetal calf serum , 9L cells were produced which stably contain cyp4B1 (9L-4B1), HSV-tk (9L-tk) and egfp (enhanced green fluorescent protein) of the fusion genes (cyp4B1 / EGFP (9L-4B1 / EGFP) and egfp / HSV- tk (9L-tk / EGFP) The cell line 9L-4B1 / EGFP was prepared by liposomal transfection of 9L cells with the plasmid p4b1-egfp containing the cyp4b1 / egfp fusion gene and the neomycin resistance-mediating gene, followed by one Selection of individual cell clones by G4I8 treatment [1 mg / ml].

To determine cytotoxic effects, the cells were plated in 6-well plates at a density of 4 × 10 ⁵ cells per well. After 24 hours, the cells were treated with 2AA, 4IPO and CGV, the treatment preferably being carried out with 5 µg / ml [25 µM] 2AA, 5 µg / ml [30 µM] 4IPO or 10 µg / ml [39 µM] CGV. The medium containing the prodrug was removed after defined periods of between 15 minutes and 8 hours and the cells were left for a further 1 or 2 days on medium which did not contain any prodrugs. As controls, cells were left in medium containing prodrug (or no prodrug) for 1 or 2 days. In each case, the cells were trypsinized after 1 or 2 days and counted using a Coulter Counter Z1. The ratio of surviving cells to the number of untreated control cells (100%) was determined. The cell counts from up to 5 independent experiments were statistically evaluated, using the Student's t-test to determine significant differences between the different suicide gene therapy systems. The significance assessment was carried out for each time measurement point of the prodrug treatment and the statistical significance was set to p <0.05.

To differentiate cytotoxic effects from cytostatic depending on the length of the prodrug treatment, 9L-4B1 and 9L-tk cells were treated as described above. After one day the surviving cells were trypsinized, counted and plated again on 100 mm Petri dishes with a density of 10 ³ cells per dish in a medium containing no prodrug. 9 days later, after fixation with 80% ethanol and staining with Grams crystal violet, colonies were counted with a diameter of <1 mm.

Example 3 Effectiveness of the cyp4B1 / 4IPO suicide gene system

After treatment of the lymphocytes infected according to Example 1 with different active concentrations of 4IPO, it can be seen that with higher doses of 4IPO (25-50 mg / ml) only 20-30% of the originally over 80% cyp4B1 / egfp-transduced after one day of treatment Cells live. Even with lower doses of 5-10 µg / ml 4IPO, less than 10% vital transduced lymphocytes can be detected after about 4 days of treatment ( Fig. 2).

Example 4 Therapeutic window of the cyp4B1 / 4IPO suicide gene system

Fig. 3 documents the therapeutic window of a 4IPO treatment in human lymphocytes treated according to Example 1. While the transduced cells die within the first two days under treatment with 50 µg / ml 4IPO, the uninfected and the infected, but non-transduced cells show no significant cytotoxicity compared to untreated lymphocytes. The experiments with infected but non-transducted lymphocytes thus confirm the absence of a "bystander effect" when treated with the cyp4B1 / 4IPO suicide gene system.

Example 5 Cytotoxicity of 2AA and 4IPO compared with cyp4B1 to GCV with HSV-tk

Here the cytotoxicity of 2AA and 4IPO is transduced with cyp4B1 9L cells (9L-4B1) and by GCV in 9L cells transduced with HSV-tk (9L-tk), the transduction taking place according to Example 2. 9L 4B1- Cells were treated with 5 µg / ml 2AA [25 µM] or 4IPO [30 µM] given time treated, incubation in regular medium followed. The parallel experiments were carried out with 9L-tk cells that were treated with 10 µg / ml GCV [39 µM]. For control experiments 9L, 9L-4B1, 9L4B1 / EGFP, 9L-tk and 9L-tk / EGFP cells were without Cultivated drug treatment.

The survival rate was calculated after one day ( Fig. 4) and two days ( Fig. 5) in relation to the untreated cells (= 100%). The average percentage results of up to 5 different experiments are shown.

2AA showed the fastest and strongest effect, while 4IPO showed similar effects Effectiveness as shown by CGV.

Example 6 Bystander effect "in different cocultures after treatment with 4IPO (A) and 2AA (B)

The "bystander effect" in different cocultures after treatment with 4IPO (A) and 2AA (B) was investigated. In order to investigate the bystander effect, cocultivation was carried out with transduced (9L-4B1) and native 9L cells, the transduction taking place according to Example 2. Different ratios of 9U9L-4B1 cells were in 6-well plates with a density of 4 × 10 ⁵ cells were plated out per well and then incubated for 24 hours The cells were treated with 5 µg / ml 2AA [25 µM] or 4IPO [30 µM] for 1 to 6 days followed by the determination of the cell survival rate.

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Claims (20)

1. DNA section containing a first sequence for herpes encoded simplex virus type 1 thymidine kinase (HSV-tk), and a second sequence for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or the human folyl polyglutamate synthase (fpgs) coded. 2. Viral vector containing a first sequence for herpes encoded simplex virus type 1 thymidine kinase (HSV-tk), and a second sequence for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or the human folyl polyglutamate synthase (fpgs) coded. 3. Retroviral vector containing a first sequence for the Encoded herpes simplex virus type 1 thymidine kinase (HSV-tk), and a second sequence for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or the human folyl polyglutamate synthase (fpgs) coded. 4. DNA section according to claim 1, viral vector according to claim 2 or retroviral vector according to claim 3, characterized characterized in that it has at least two promoters, of which preferably one, especially both, eukaryotic Origin is / are, and / or at least one selection or Contains reporter gene or selectable marker gene, and / or the second sequence for the cytochrome P450 isoenzyme 4B1 (cyp4B1) coded. 5. Retroviral vector according to one of claims 3 or 4, characterized characterized in that the promoter under its control Expression of the first sequence takes place for the herpes simplex Virus type 1 thymidine kinase (HSV-tk) encodes itself from that Under his control, promoter differentiates the expression of second sequence is carried out for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs) coded. 6. Retroviral vector according to one of claims 3 to 5, characterized in that the vector in addition to the first sequence which codes for the herpes simplex virus type 1 thymidine kinase (HSV-tk), and the second sequence which isoenzyme for the cytochrome P450 4B1 (cyp4B1) or the human folyl polyglutamate synthase (fpgs) encoded, contains the following functional elements: a) a 5'-LTR and a 3'-LTR, preferably from a retrovirus, b) at least one, preferably two, promoters, in particular selected from the group consisting of: SV40 promoter, 5'-LTR with promoter function, CD25 promoter and CD69 promoter, preferably 5'-LTR with promoter function, CD25 Promoter and CD69 promoter; c) at least one selection or reporter gene or selectable marker gene selected from: the preferably truncated gene for the human "low affinity NGF receptor" (LNGFR) and / or the preferably truncated human CD34 gene (CD34), d) at least one retroviral packaging signal (ψ) and e) if appropriate, an IRES element (IRES) with which the expression of the selection or reporter gene or selectable marker gene is coupled to the expression of the first or second sequence. 7. Retroviral vector according to one of claims 3 to 6, characterized in that it is a mouse leukemia virus (murine leucemia virus / MuLV) vector, preferably with one of the following sequences of the functional elements:
5'LTR ψ-cyp4B1-IRES-CD34-CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-FPGS-IRES-CD34-CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-FPGS-IRES-LNGFR CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-cyp4B1-IRES-LNGFR CD69 promoter HSV-tk-3'-LTR,
5'LTR ψ-HSV-tk-IRES-CD34-CD69-LTR promoter cyp4B1-3'-,
5'LTR ψ-HSV-tk-IRES-CD34-CD69 promoter FPGS-3'-LTR,
5'LTR ψ HSV-tk-IRES-CD69 LNGFR promoter cyp4B1-3' LTR
or
5'LTR ψ-HSV-tk-IRES-LNGFR CD69 promoter FPGS-3'-LTR. 8. A method for producing an infectious retrovirus particle, characterized in that a) a retrovirus packaging cell is transfected with a retroviral plasmid vector according to any one of claims 3 to 7, b) the retrovirus packaging cell is cultivated in a suitable medium in vitro and / or in vivo in such a way that infectious retrovirus particles are formed. 9. The method according to claim 8, characterized in that the Retrovirus packaging cell of a cell line selected from the Packaging cell lines PA317 and AMIZ with the amphotropic MuLV env protein and pg13 with the GALV (gibbon ape leukemia virus) -env protein. 10. Infectious particle, characterized in that it is caused by a Method according to one of claims 8 or 9 is obtainable. 11. Host cell containing at least one DNA section according to one of claims 1 or 4, viral vector according to one of the Claims 2 or 4, retroviral vector according to one of the Claims 3 to 7 and / or infectious particle according to claim 10th 12. Double-labeled cell containing a first DNA sequence for encoding herpes simplex virus type 1 thymidine kinase (HSV-tk), and in a separate molecule, a second DNA sequence, which is for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or the human Folyl polyglutamate synthase (fpgs), preferably cyp4B1, encoded, each in the form of a DNA segment, a viral vector, one retroviral vector and / or an infectious particle and or containing on the one hand recombinantly expressed HSV-tk and other recombinantly expressed cyp4B1 or fpgs, preferably cyp4B1. 13. Host cell according to claim 11 or double-labeled cell according to Claim 12, characterized in that the cell has a - preferably human - lymphocyte cell. 14. A method for producing a host cell according to claim 11 or 13, wherein the cell has a first and a second retroviral Vector according to one of claims 3 to 7 in the form of a infectious particle is transfected according to claim 10, wherein the first and second retroviral vectors the same or different can be characterized in that the method for Production of the infectious particles according to one of claims 8 or 9 retrovirus packaging cells with different cell lines different packaging proteins can be used. 15. A method for producing a double-labeled cell according to one of claims 12 or 13, wherein the cell with a first and a second retroviral vector according to one of the claims 3 to 7 in the form of an infectious particle according to claim 10 is transfected, the first and second retroviral vector can be the same or different, characterized in that that according to the method for producing the infectious particles one of claims 8 or 9 retrovirus packaging cells different cell lines with different Packaging proteins are used or the cell with a first retroviral vector containing a first DNA sequence required for the herpes simplex virus type 1 Encoded thymidine kinase (HSV-tk), and with a second retroviral vector necessary for the cytochrome P450 isoenzyme 4B1 (cyp4B1) or human folyl polyglutamate synthase (fpgs), preferably cyp4B1, coded, each in the form of an infectious Particle is transfected, characterized in that the Process for the preparation of the infectious particles retrovirus Packing cells of different cell lines with different Packaging proteins are used. 16. Human lymphocyte containing at least one DNA section, a viral vector, a retroviral vector and / or a infectious particle, or a DNA sequence contains, for the cytochrome P450 isoenzyme 4B1 (cyp4B1) encoded, and / or containing recombinantly expressed cyp4B1. 17. Medicament containing at least one / a / a DNA section according to one of claims 1 or 4, viral vector according to one of claims 2 or 4, retroviral vector according to one of the Claims 3 to 7, infectious particle according to claim 10, Host cell according to one of claims 11 or 13, Double-labeled cell according to one of claims 12 or 13 and / or human lymphocytes according to claim 16 and optionally at least one pharmaceutically compatible one Auxiliary and / or carrier. 18. Use a) a DNA segment according to one of claims 1 or 4, a viral vector according to one of claims 2 or 4, a retroviral vector according to one of claims 3 to 7, an infectious particle according to claim 10, a host cell according to one of claims 11 or 13, a double-labeled cell according to one of claims 12 or 13, a human lymphocyte according to claim 16 and / or a medicament according to claim 17, b) a DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing at least one DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing recombinantly expressed cyp4B1; and / or the enzyme cyp4B1, c) of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA), methotrexate (MTX) and / or a combination of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA) or methotrexate (MTX) with ganciclovir (GCV) , preferably together with an object from groups a) and / or b), for the manufacture of a medicament for the treatment or treatment of leukemia, in particular chronic or acute myeloid leukemia (CML or AML) and acute lymphoblastic leukemia (ALL) as well as myelodysplastic syndrome (MDS), multiple myeloma and NHL Lymphoma, and / or for the treatment of or prophylaxis against graft versus host reactions, especially after application of donor lymphocytes. 19. Use a) a DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing at least one DNA segment, a viral vector, a retroviral vector and / or an infectious particle, each of which contains a DNA sequence which codes for the cytochrome P450 isoenzyme 4B1 (cyp4B1); a cell containing recombinantly expressed cyp4B1; and / or the enzyme cyp4B1, b) of 4-ipomeanol (4IPO) and / or of 2-aminoanthracene (2AA), preferably together with an object from group a) in combination with reporter genes for clinical gene labeling experiments, in combination with therapeutic genes in therapeutic gene replacement approaches , for converting donor lymphocytes for leukemia relapse prevention and for treatment after allogenetic bone marrow transplantation (allo-BMT). 20. Use a) a DNA segment according to one of claims 1 or 4, a viral vector according to one of claims 2 or 4, a retroviral vector according to one of claims 3 to 7, an infectious particle according to claim 10, a host cell according to one of claims 11 or 13, a double-labeled cell according to one of claims 12 or 13, a human lymphocyte according to claim 16 and / or a medicament according to claim 17 or b) of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA), methotrexate (MTX) and / or a combination of 4-ipomeanol (4IPO), 2-aminoanthracene (2AA) or methotrexate (MTX) with ganciclovir (GCV) together with an object from group a) in gene therapy.