WO1996010087A1

WO1996010087A1 - Method for treatment of cancers by regulating the activity of ras proteins

Info

Publication number: WO1996010087A1
Application number: PCT/FR1995/001227
Authority: WO
Inventors: Bruno Tocque; Bohdan Wasylik
Original assignee: Rhone-Poulenc Rorer S.A.; Institut National De La Sante Et De La Recherche Medicale
Priority date: 1994-09-27
Filing date: 1995-09-22
Publication date: 1996-04-04
Also published as: FR2724846A1; AU3524995A; FR2724846B1

Abstract

The present invention relates to a method for the treatment of cancers by regulation of the activity of ras proteins. More particularly, it relates to a method for treating cancers by means of a compound capable of antagonizing the activity of ETS proteins.

Description

METHOD OF TREATING CANCER BY REGULATING THE ACTIVITY OF RAS PROTEINS

The present invention relates to a new method for the treatment of cancers. More particularly, it relates to a method of treating cancers by regulating the activity of ras proteins. It also relates to gene therapy vectors making it possible to regulate the activity of the ras protein, as well as the pharmaceutical compositions containing them.

The products of the ras genes, generally designated p21 proteins, play a key role in the control of cell division in all the eukaryotic organisms where they have been sought. Certain specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic. Thus, a large number of human tumors have been associated with the presence of modified ras genes. Likewise, overexpression of these p21 proteins can lead to disruption of cell proliferation. The elucidation of the different biological functions of these p21 proteins in cells, their mode of operation and their characteristics is therefore of major interest for the understanding and the therapeutic approach of carcinogenesis.

The ras gene is a key component of a highly conserved signaling pathway from yeast to humans, connecting the outside of the cell to effectors of cell growth and differentiation. This signaling pathway involves a certain number of identified components, and in particular tyrosine kinase receptors, intermediate binding proteins (GRF, SOS, etc.), ras proteins, then a cascade of kinases including in particular Raf, MEKK, MEK and MAPK. However, we do not yet know the effectors located downstream of this signaling pathway. It is clear that each of these components constitutes a potential therapeutic target for the treatment of pathologies involving abnormal cell proliferation. This is all the more true for the components located downstream of the signaling channel, which deliver a more specific message.

The present invention results in part from the demonstration that the ETS proteins constitute a mediator of the cellular transformation induced by the ras proteins. It also results from the demonstration that it is possible to counteract the transforming effects of ras proteins by using compounds capable of antagonizing the activity of ETS proteins. The present invention also describes particularly effective systems enabling delivery in vivo, directly in tumors, such compounds and thus fight against the development of cancers. The present invention thus offers a particularly effective new approach for the treatment of tumors having an activated ras oncogene such as non-small cell lung tumors, pancreatic and colonic carcinomas, etc.

A first object of the invention therefore lies in the use of a compound capable of antagonizing the activity of the ETS proteins for the preparation of a pharmaceutical composition intended for the treatment of cancers.

More particularly, the compounds capable of antagonizing the activity of the ETS proteins within the meaning of the present invention are compounds capable of at least partially inhibiting the attachment to DNA of the ETS proteins. Such compounds are preferably double-stranded oligonucleotides corresponding to the site of attachment of ETS proteins on DNA or derivatives of ETS proteins. In a very particularly advantageous manner, the compounds used in the context of the present invention are derivatives of the ETS proteins, capable of binding to the site of attachment to DNA, but devoid of transactivating function.

ETS proteins form a family of transcription factors, that is to say proteins capable of interacting with DNA at specific binding sites, and of stimulating the transcription of genes. ETS proteins are proteins of around 400 amino acids, comprising several functional domains: one, responsible for the binding of the protein to DNA, others, responsible for transactivation. This organization is found in human ETS proteins, rats, chicken, etc. Different derivatives of ETS proteins can be used in the context of the present invention. It may in particular be any molecule obtained from an ETS protein by modification (s) of genetic and / or chemical nature, retaining the capacity to bind to the site of attachment to DNA, but devoid of transactivating activity. By modification of a genetic and / or chemical nature is meant any mutation, deletion, substitution, addition, and / or modification of one or more amino acids. As indicated above, the compounds used in the context of the present invention advantageously have one or more modifications in the region responsible for transactivation. Advantageously, these modifications are made at the level of residues 1-160 for PU. l, 1-374 for c-Ets-1 and 1-363 for c-Ets-2. Such modifications can be made, for example, by in vitro mutagenesis, by introduction of additional elements or synthetic sequences, or by deletions or substitutions of the original elements. When a derivative as defined above is produced, its activity as a partial inhibitor of the binding of the ETS proteins at their DNA binding site can be demonstrated in several ways, as described in the examples. Any technique known to those skilled in the art can obviously be used for this purpose.

As examples of derivatives of ETS proteins according to the invention, mention may be made in particular of the following compounds: - ΔPU.1, comprising residues 160 to 266 of the PU protein. l of mice, corresponding to the DNA binding site;

- N70, comprising residues 374 to 485 of the chicken c-Ets-1 protein, containing the DNA binding site;

- N70-DB, comprising residues 374 to 472 of the chicken c-Ets-1 protein, containing the DNA binding site;

- NM (2), comprising the DNA binding site of the protein c-Ets-2 (residues 364 to 479).

The nucleotide and peptide sequences of these compounds are accessible on databases (Databank in particular, access number X07202 for example) Furthermore, such derivatives may include additional modifications, in particular at the level of the DNA binding site, allowing in particular to reduce the size of the sequence to facilitate its cell penetration, to increase its inhibition activity, to target its cell localization, or also to construct sequences suitable for expression in a particular type of vector or host. In the context of the present invention, these compounds derived from proteins

ETS can be used as such or, advantageously, in the form of genetic constructs allowing their expression in vivo.

A particularly advantageous embodiment of the present invention in fact consists in transferring into the tumors a nucleic sequence coding for an ETS protein derivative as defined above. This approach is particularly suitable for the treatment of cancers with an activated ras oncogene, such as colonic or bronchial carcinomas for example.

The nucleic acid sequence used in the context of the present invention can be administered as it is, in the form of naked DNA according to the technique described in WO 90/1 1092. It can also be administered in complex form, for example with DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), with nuclear proteins (Kaneda et al. , Science 243 (1989) 375), with lipids (Felgner et al., PNAS 84 (1987) 7413), in the form of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc. . Preferably, the sequence used in the context of the invention is part of a vector. The use of such a vector in fact makes it possible to improve the administration of the nucleic acid in the cells to be treated, and also to increase its stability in said cells, which makes it possible to obtain a lasting therapeutic effect. In addition, it is possible to introduce several nucleic acid sequences into the same vector, which also increases the effectiveness of the treatment.

The vector used can be of various origins, since it is capable of transforming animal cells, preferably human cancer cells. In a preferred embodiment of the invention, a viral vector is used, which can be chosen from adenoviruses, retroviruses, adeno-associated viruses (AAV) or herpes virus.

In this regard, the subject of the present invention is also any recombinant virus comprising, inserted into its genome, a nucleic acid coding for a compound capable of antagonizing the attachment to DNA of ETS proteins. More particularly, it relates to any recombinant virus comprising a nucleic acid sequence coding for an ETS derivative capable of binding to DNA but lacking transactivating activity.

Preferably, the viruses used in the context of the invention are defective, that is to say that they are unable to replicate autonomously in the infected cell. Generally, the genome of the defective viruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the sequence coding for the ETS inhibitor. Preferably, the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.

With regard more particularly to adenoviruses, various serotypes, whose structure and properties vary somewhat, have been characterized. Among these serotypes, it is preferred to use, within the framework of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see request FR 93 05954). Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example]. Preferably, in the context of the invention, adenoviruses of human or canine or mixed origin are used.

Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence allowing the encapsidation and the sequence coding for the calpain modulator. Even more preferably, in the genome of the adenoviruses of the invention, the El gene and at least one of the E2, E4, L1-L5 genes are non-functional. The viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution, partial deletion, or addition of one or more bases in the gene or genes considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents.

The defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the DNA sequence coding for the ETS inhibitor. Homologous recorabination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. As an example of a line, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%). Strategies for the construction of vectors derived from adenoviruses have also been described in applications Nos. FR 93 05954 and FR 93 08596.

Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples. Concerning adeno-associated viruses (AAV), these are DNA viruses of relatively small size, which integrate into the genome of the cells they infect, in a stable and site-specific manner. They are capable of infecting a wide spectrum of cells, without inducing an effect on cell growth, morphology or differentiation. Furthermore, they do not seem to be involved in pathologies in humans. The AAV genome has been cloned, sequenced and characterized. It comprises approximately 4,700 bases, and contains at each end an inverted repeat region (ITR) of approximately 145 bases, serving as an origin of replication for the virus. The rest of the genome is divided into 2 essential regions carrying the packaging functions: the left part of the genome, which contains the rep gene involved in viral replication and the expression of viral genes; the right part of the genome, which contains the cap gene coding for the capsid proteins of the virus.

The use of vectors derived from AAVs for gene transfer in vitro and in vivo has been described in the literature (see in particular WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488,528). These applications describe various constructs derived from AAVs, in which the rep and / or cap genes are deleted and replaced by a gene of interest, and their use for transferring in vitro (onto cells in culture) or in vivo (directly into an organism ) said gene of interest. The defective recombinant AAVs according to the invention can be prepared by co-transfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing the sequence coding for the ETS inhibitor bordered by two inverted repeat regions (ITR) of AAV, and of a plasmid carrying the packaging genes (rep and cap genes) of AAV. The recombinant AAVs produced are then purified by conventional techniques. Concerning herpes viruses and retroviruses, the construction of recombinant vectors has been widely described in the literature: see in particular Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220, Bernstein et al. Broom. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, etc. In particular, retroviruses are integrative viruses, selectively infecting dividing cells. They therefore constitute vectors of interest for cancer applications. The genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted, in whole or in part, and replaced by a heterologous nucleic acid sequence of interest. These vectors can be produced from different types of retroviruses such as in particular the MoMuLV ("murine moloney leukemia virus"; also designated MoMLV), MSV ("murine moloney sarcoma virus"), HaSV ("harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("rous sarcoma virus") or the Friend virus. To construct recombinant retroviruses comprising a sequence of interest, a plasmid comprising in particular the LTRs, the packaging sequence and said sequence of interest is generally constructed, then used to transfect a cell line called packaging, capable of providing in trans retroviral functions deficient in the plasmid. Generally, the packaging lines are therefore capable of expressing the gag, pol and env genes. Such packaging lines have been described in the prior art, and in particular the line PA317 (US4,861,719); the PsiCRIP line (WO90 / 02806) and the GP + envAm-12 line (WO89 / 07150). Furthermore, the recombinant retroviruses may include modifications at the level of the LTRs to suppress transcriptional activity, as well as extended packaging sequences, comprising a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639). The recombinant retroviruses produced are then purified by conventional techniques.

For the implementation of the present invention, it is very particularly advantageous to use a defective recombinant adenovirus or retrovirus. These vectors indeed have properties which are particularly advantageous for the transfer of genes into tumor cells.

Advantageously, in the vectors of the invention, the sequence coding for the ETS derivative is placed under the control of signals allowing its expression in tumor cells. Preferably, these are heterologous expression signals, that is to say signals different from those naturally responsible for the expression of the inhibitor. They may in particular be sequences responsible for the expression of other proteins, or synthetic sequences. In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus, including the virus used. In this regard, mention may be made, for example, of promoters El A, MLP, CMV, LTR-RSV, etc. In addition, these expression sequences can be modified by adding activation, regulation sequences or allowing tissue-specific expression. It can indeed be particularly interesting to use signals of expression active specifically or predominantly in tumor cells, so that the DNA sequence is not expressed and produces its effect only when the virus has effectively infected a tumor cell.

In a particular embodiment, the invention relates to a defective recombinant virus comprising a cDNA sequence coding for an ETS derivative under the control of a viral promoter, preferably chosen from LTR-RSV and the CMV promoter.

Still in a preferred embodiment, the invention relates to a defective recombinant virus comprising a DNA sequence coding for an ETS derivative under the control of a promoter allowing predominant expression in tumor cells.

Expression is considered to be predominant within the meaning of the invention when, even if residual expression is observed in other cell types, the expression levels are higher in tumor cells.

The present invention also relates to any pharmaceutical composition comprising one or more defective recombinant viruses as described above. These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, especially for direct injection into the patient's tumor. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. Direct injection into the patient's tumor is advantageous because it allows the therapeutic effect to be concentrated in the affected tissues.

The doses of defective recombinant virus used for injection can be adapted as a function of various parameters, and in particular as a function of the viral vector, of the mode of administration used, of the pathology concerned or also of the duration of the treatment sought. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ^ and 101 ⁴ pfu / ml, and preferably 10 ^ to 10-O pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of ranges of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature. With regard to retroviruses, the compositions according to the invention can directly comprise the producer cells, with a view to their implantation. The pharmaceutical compositions according to the invention can be used to modulate the activation of the p21 proteins and therefore to modulate the proliferation of certain cell types. More particularly, these pharmaceutical compositions are intended for the treatment of cancers having an activated ras oncogene. Many cancers have in fact been associated with the presence of ras oncogenic proteins. Among the cancers most often containing mutated ras genes, mention may be made in particular of adenocarcinomas of the pancreas, 90% of which have a Ki-ras oncogene mutated on the twelfth codon (Almoguera et al., Cell 53 (1988) 549), colon adenocarcinoma and thyroid cancer (50%), or lung carcinoma and myeloid leukemia (30%, Bos, JL Cancer Res. 49 (1989) 4682).

Another subject of the invention is the use of the molecules described above to modulate the activity of the p21 proteins. In particular, the invention relates to the use of these molecules to at least partially inhibit the activation of the p21 proteins.

Other advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.

The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and not limiting.

General molecular biology techniques

Classically used methods in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, the extraction of proteins with phenol or phenol-chloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation in Escherichia coli, etc. are well known. skilled in the art and are abundantly described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel FM et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].

The pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories). For the ligations, the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations.

The filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I from E. coli (Biolabs) according to the supplier's specifications. The destruction of the prominent 3 ′ ends is carried out in the presence of DNA Polymerase from phage T4 (Biolabs) used according to the manufacturer's recommendations. The destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI. Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. . (1985) 8749-8764] using the kit distributed by Amersham.

The enzymatic amplification of DNA fragments by the technique known as PCR [Polymerase-catalyzed Çhain Reaction, Saiki RK et al., Science 220 _. (1985) 1350-1354; Mullis KB and Faloona FA, Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications. The amplification of genomic DNA is carried out more particularly under the following conditions: 5 minutes at 100 ° C., 30 cycles of one minute at 95 ° C., 2 minutes at 58 ° C. then 3 minutes at 72 ° C. using appropriate probes. The amplification products are analyzed by gel electrophoresis.

Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5477] using the kit distributed by Amersham.

Free

Example 1. Materials and Methods

1 1 Constructions used

- The plasmid pBLCAT4 (4B) notably comprises the domain a of the enhancer of the polyoma virus, the promoter of thymidine kinase and a gene marker (the CAT gene). The construction of this plasmid has been described in Wasylyk et al., Genes Dev., 6, 965-974. The plasmid pBLCAT4 corresponds to the plasmid pBLCAT4 (4B), but lacking the α domain of the enhancer of the polyoma virus.

- The following constructions have been described in the literature: N70, NM (2), DELTA PU.1, [NN (P)], N69, N72 (Wasylyk et al., Genes Dev., 6, 965-974 and Cell Growth and Differ., 3, 617-625); N70-DELTAB (Wasylyk et al., Eur. J. Biochem., 21 1, 7-18 and Nucl. Acids Res., 1, 523-529); Gal4, Gal4-VP16, UAS-CAT (Webster et al., Cell, 52, 169-178); pSLJDELTA9Jun (Lloyd et al., Nature, 352, 635-638); pKJ-1 (Lohnes et al., Cell ,, 73, 381-393).

1.2. Cell cultures

The NIH3T3 cells transformed by Ras (DT line) and their non-transformed NIH3T3 mother cells (line Ci 1) are cultured on Dubelco medium enriched with antibiotics and 10% fetal calf serum. Unless specified, the cell transformations were carried out as follows: 5 90 mm plates containing DT cells are incubated for 20 hours in the presence of a calcium phosphate precipitate containing 5 μg of an expression vector of a Ets derivative, 0.5 μg of pKJ-1 and 15 μg of pEMBL18 per plate. The plates are then washed, again incubated for 24 hours with fresh medium, then with a medium containing 1 mg / ml of G418 (mixture of geneticin sulfate, GIBCO) until the clones appear. The clones are selected by adhesion enrichment (Lloyd et al., See above), cultivated in mass culture and frozen. The cell lines are then propagated in a medium containing 500 μg / ml then 300 μg / ml of G418. Six cell lines derived from DT cells, comprising an expression plasmid of a derivative of the PU.1 protein, have thus been established: ΔPU-R1 (49PU), ΔPU-R2 (481 1), ΔPU-R3 (4711) , ΔPU-R4 (51), ΔPU-R5 (461 1) and ΔPU-R6 (52).

Example 2 Inhibition of ras-induced transformation using ETS protein deletion derivatives

The inhibitory activity of the ETS protein deletion derivatives according to the invention was first demonstrated using the transient transfection technique in LMTK fibroblasts. These transfections contain a ras expression plasmid and a plasmid reporter, pBLCAT4 (4B), including the α domain of the enhancer of the polyome virus, the thymidine kinase promoter and a marker gene (the CAT gene) (cf. example 1.1.). In these cells, the expression of ras stimulates transcription by the α domain of the enhancer of the polyoma virus. The co-expression in these cells of derivatives of the ETS proteins according to the invention makes it possible to inhibit this activation by ras.

The LMTK ^" cells were transfected in the presence of calcium phosphate by the reporter plasmid, pBLCAT4 (4B), or a control plasmid devoid of flush response elements (pBLCAT4); and different quantities (total amount: 20 μg): - of a ras expression vector (pRCBx2)

a vector for expression of a protein derived from ETS according to the invention, and

- neutral plasmid pEMBLlδ.

The cells are incubated for 36 hours in a medium poor in serum (0.05% FCS) and the cell extracts are then analyzed for the expression of the CAT activity. The CAT activities observed were normalized with respect to the activity obtained in cells transformed by the plasmid pBLCAT4 (4B) only, arbitrarily fixed at 100. The results obtained are presented in table 1. They represent the average of 2 to 10 different transfection experiences.

The results obtained show that the sequence coding for ΔPU. l is capable of specifically inhibiting both the activation of the α domain by ras and the constitutive activity of ras. On the other hand, this sequence has no effect on transactivation by a heterologous factor consisting of the DNA binding site of GAL4 linked to the transactivating domain of VP16 (GAL4-VP16). These results demonstrate (i) a very high inhibition activity of the sequences according to the invention and (ii) that this inhibition activity is specific for the α domain and is not linked to a non-specific effect on the viability or cellular transcription.

The results obtained show that the same selective inhibition activity is observed using the derivatives of the ETS proteins N70, N70-ΔB and NM (2). On the other hand, the use of an ETS derivative modified at the level of the DNA domain at DNA (N69 comprising residues 382-485, and N72 comprising residues 383-485) has no effect on the activation by ras, which confirms the specific nature of the inhibition observed with the derivatives according to the invention. Example 3: The deletion derivatives of the ETS proteins inhibit the formation of agar colonies, growth in medium poor in serum, the saturation density and apoptosis.

The activity of the constructs according to the invention on the ras signaling pathway was confirmed by study of their effect on the formation of colonies, growth in medium poor in serum, saturation density and apoptosis. 1. Formation of colonies

For this, 10 ⁴ cells (Ci 1, DT cells, and established ΔPU lines) were seeded in 0.75% agar in Falcon multiwell plates, diameter of the wells: 3.5 cm), according to the technique described by Wharton. and Smith (In R. Baserga (ed), "Cell Growth and Division"; IRL Press, Oxford, 1989, pi 39). The number of colonies formed and the number of cells per colony (size of the colonies) were determined 8 days later. Each given value corresponds to an average of four duplicate experiments. The results obtained are presented in Table 2. The results show that the DT cells (transformed by ras) form much larger colonies than the cells Ci 1 (not transformed by ras). This table also shows that the ΔPU lines give only a few colonies of small sizes, which clearly shows that, in these lines, the transforming activity of ras is inhibited. 2. Growth in a low serum medium and saturation density

Furthermore, this experiment made it possible to show that Ci 1 cells (not transformed by ras) are more sensitive than DT cells at low serum concentrations: they grow more slowly and their growth stops at much lower saturation densities. due to contact inhibitions. This experiment made it possible to show that the cells of the ΔPU lines also grew much slower than the DT cells, and reached saturation at much lower densities. These results confirm that the ΔPU lines behave like the line not transformed by ras, and are therefore capable of reversing the effects of ras. 3. Apoptosis The effect of ras and the compounds of the invention on cell death by apoptosis was studied by culturing 10-5 cells on 90 mm plates under the conditions described in Example 1.2. At different times, one plate was used to count the number of viable cells and 2 plates to isolate the cellular DNA and analyze its profile according to the technique described by Debbas et al. (Genes & Dev. 7 (1993) 546). It has thus been observed that the Cl 1 cells (not transformed by ras) stop grow at confluence but remain viable, as long as the DT cells die by apoptosis. In particular, a degradation of the DNA of DT cells characteristic of apoptosis could be observed, detectable even before the decrease in the number of cells. This experiment also made it possible to show (i) that the DNA of the Ci 1 cells does not exhibit the degradation profile characteristic of apoptosis although, in some cases, a certain degradation is observed; (ii) that the DNA of the ΔPU-R1 and ΔPU-R2 cells shows no degradation of the type observed in apoptosis and (iii) some degradation is observed for the ΔPU-R3-6 lines, but much later than for DT cells. These results confirm that the ΔPU lines have the characteristics of the untransformed C 1 1 cell, both from the point of view of colony formation, growth in medium poor in serum, saturation density and apoptosis. , which clearly demonstrates that the in vivo expression of the ETS derivatives according to the invention is capable of reversing the transforming phenotype induced by ras.

Example 4: The ETS protein deletion derivatives inhibit the formation of tumors in "nude" mice.

This example demonstrates the inhibitory activity of the ETS proteins according to the invention on the formation of tumors in vivo. For this, 10 ^ cells (Ci 1 cells, DT, and established ΔPU lines) freshly thawed were cultured in Dulbecco medium supplemented with 10% S VF, trypsinized and then used for injection. The cells were thus injected (lO ^ / lOOμl PBS) under the skin, on each side of the back of 2 "nude" mice. The diameter of the tumors formed was then measured using calipers on days 3, 6 and 8. The results obtained are presented in Table 3. Each value represents the average obtained for each mouse.

The results obtained show that the DT cells (transformed by ras) form tumors in nude mice which are clearly detectable 3 days after the injection. On the contrary, the non-transformed cells (Ci l) do not induce any detectable tumor. These results also show that the ΔPU lines have, compared to DT cells, a tumorigenic effect markedly reduced, or even eliminated. Thus, the ΔPU-R1, ΔPU-R2 and ΔPU-R6 lines do not cause the formation of any tumor (on day 3). Furthermore, the ΔPU-R3, ΔPU-R4 and ΔPU-R5 lines induce the slower appearance of smaller tumors, indicating that in these cells also, the transforming activity of ras has been at least partially inhibited. These results therefore clearly show that the compounds according to the invention have significant anti-tumor properties in vivo.

Example 5 Inhibitory effects of deletion derivatives of the proteins c-ETS-1 and c-ETS-2.

This example shows that the activity demonstrated in the context of the present invention is not limited to PU derivatives, but also exists for derivatives of other proteins of the ETS family.

Six independent clones corresponding to each subsequent ETS derivative were established according to the protocol described in Example 1.2. : ΔPU, N70, N70-Δ and NM (2). These clones were then cultivated in mass culture, selected, and analyzed for different criteria for reversion of the transformed ras phenotype as described in examples 2 to 4: integrated DNA, DNA binding activity, levels of ras protein, formation. of colonies in soft agar, growth in medium poor in serum, levels of RNA of cathepsin L. The results obtained are presented in Table 4. The values given are the average of the experiments carried out. The results presented in Table 4 show that the different cell lines contain integrated DNA and express a protein capable of binding the ETS motifs. These lines form only a few soft agar colonies, have a reduced doubling time in a low serum medium, stop growing at a low saturation density, and have intact cathepsin levels. These results clearly confirm that the ras phenotype can be reversed according to the invention by ETS derivatives of different origins.

Example 6: Construction of a recombinant adenovirus expressing a deletion derivative of the ETS proteins.

This example describes the construction of a recombinant adenovirus comprising a nucleic acid sequence coding for a derivative of the ETS proteins according to the invention. This adenovirus is constructed by homologous recombination between the defective adenovirus Ad-dll324 and a plasmid carrying the sequence coding for said derivative plasmid pΔPU.l.

4.1. Construction of the plasmid pΔPU.l

The plasmid pΔPU.l comprises the sequence coding for the deletion derivative of the protein PU.l under the control of the LTR-RSV promoter, as well as regions of the adenovirus allowing homologous recombination. It is built by insertion of the sequence coding for ΔPU.l in the plasmid pAd.RSVβgal. The plasmid pAd.RSVβGal contains, in the orientation 5 '->3',

- the PvuII fragment corresponding to the left end of the Ad5 adenovirus comprising: the ITR sequence, the origin of replication, the packaging signals and the E1A amplifier;

- the gene coding for β-galactosidase under the control of the RSV promoter (from Rous sarcoma virus),

- A second fragment of the genome of the Ad5 adenovirus, which allows homologous recombination between the plasmid pAd.RSVβGal and the adenovirus dl324. The plasmid pAd.RSVβGal has been described by Stratford-Perricaudet et al. (J. Clin. Invest. 90 (1992) 626).

4.2. Construction of the recombinant adenovirus

The vector described in 4.1. is linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions encoded by the El regions (El A and E11B) of adenovirus.

More specifically, the recombinant adenovirus is obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector described in Example 4.1., According to the following protocol : the plasmid pΔPU.l and the adenovirus Ad-dll324, linearized by the enzyme Clal, are co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated are then selected by plaque purification. After isolation, the DNA of the recombinant adenovirus is amplified in the cell line 293, which leads to a culture supernatant containing the non-purified recombinant defective adenovirus having a titer of approximately 10 pfu / ml.

The viral particles are purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456). The adenovirus obtained can be stored at -80 ° C in 20% glycerol.

Example 7: Construction of a recombinant retrovirus expressing a deletion derivative of the ETS proteins.

This example describes the construction of a recombinant retrovirus comprising a nucleic acid sequence coding for a derivative of the ETS proteins according to the invention. This retrovirus further comprises a transcription blocking sequence making it possible to prevent the possible toxicity of the ETS deletion derivative on the growth of the packaging line used.

The recombinant retrovirus is constructed from the plasmid pnlsLacZ ^RT described by Tchénio et al (J. Virol. 66 (1992) 1571) by substitution of the nlsLacZ gene with the sequence coding for the deletion derivative of the protein PU.l. The plasmid thus obtained essentially comprises the following elements:

- The LTRs of the MoMLV retrovirus; - The packaging sequence including a part of the retrovirus gag gene

MoMLV; and,

- An expression cassette for the PU.l derivative comprising the coding sequence under the control of the thymidine kinase gene promoter from the HSV virus separated by a transcription blocking sequence (polyadenylation site of the SV40 virus) flanked by an acceptor site and a sleeper splicing site.

This plasmid is then used to generate the corresponding recombinant retroviruses by transfection, in the presence of calcium phosphate, in the GP + envAm-12 packaging cells. The recombinant retroviruses thus produced are then isolated from the culture medium, purified and then stored for use. It is also possible to directly use the transfected packaging lines (producing lines) in the form of implants.

35 Table 1

35 Table 2

30

35 Table 3 Table 4

Claims

1 Use of a compound capable of antagonizing the activity of ETS proteins for the preparation of a pharmaceutical composition intended for the treatment of cancers

2 Use according to claim 1 of a compound capable of at least partially inhibiting the attachment to DNA of ETS proteins

3 Use according to claim 2 characterized in that the compound is a derivative of ETS proteins

4 Use according to claim 2 or 3 characterized in that the compound is a derivative of ETS proteins capable of binding to the site of attachment on DNA, but devoid of transactivating function

5 Use according to claim 3 or 4 characterized in that the compound is derived from ETS proteins by mutation and / or deletion

6 Use according to claim 5 characterized in that the compound is derived from ETS proteins by mutation and / or deletion in the transactivating region

7 Use according to one of claims 3 to 6 characterized in that the compound is chosen from ΔPU 1, N70, N70-DB, and NM (2)

8 Use of a nucleic sequence coding for a derivative of ETS proteins capable of antagonizing the activity of ETS proteins for the preparation of a pharmaceutical composition intended for the treatment of cancers

9 Use according to claim 8 characterized in that the nucleic acid is used in complexed form with DEAE-dextran, with nuclear proteins, or with cationic lipids or polymers, in the form of liposomes or as such

10 Use according to claim 8 or 9 characterized in that the nucleic acid is part of a vector 1 1 Use according to claim 10 characterized in that the nucleic acid is part of a viral vector, chosen from adenoviruses, retroviruses and adeno-associated viruses

12 Viral vector comprising a nucleic acid sequence coding for a compound capable of at least partially inhibiting attachment to DNA of proteins

ETS

13 Viral vector according to claim 12 characterized in that it is chosen from adenoviruses, retroviruses and adeno-associated viruses

14 Viral vector according to one of claims 12 or 13 characterized in that it comprises a sequence coding for a derivative of ETS proteins capable of binding to the site of attachment on DNA, but devoid of transactivating function

15 Defective recombinant virus comprising a cDNA sequence coding for a derivative of the ETS proteins capable of binding to the binding site on DNA, but lacking a transactivating function, under the control of a viral promoter

16 Defective recombinant virus comprising a cDNA sequence coding for a derivative of the ETS proteins capable of binding to the binding site on the DNA, but lacking a transactivating function, under the control of a promoter allowing predominant expression in the cells tumor

17 defective recombinant virus according to claims 15 or 16 characterized in that it is an adenovirus or a retrovirus

18 Pharmaceutical composition comprising a nucleic acid sequence coding for an ETS protein derivative capable of binding to the site of attachment to DNA, but lacking a transactivating function

19 Pharmaceutical composition according to claim 18 characterized in that it comprises a viral vector according to one of claims 12 to 17.

20 Composition according to claim 17 formulated for intra-tumor administration