EP0763121A1

EP0763121A1 - Method of cancer treatment by p53 protein control

Info

Publication number: EP0763121A1
Application number: EP95920973A
Authority: EP
Inventors: Jean-Marie Blanchard; Serge Carillo; Marc Peichaczyk
Original assignee: Rhone Poulenc Rorer SA
Current assignee: Aventis Pharma SA
Priority date: 1994-05-31
Filing date: 1995-05-22
Publication date: 1997-03-19
Also published as: AU714209B2; JPH10500978A; WO1995033060A1; NO964772L; FR2720277A1; CA2190293C; FI120501B; NO321411B1; CA2190293A1; NO964772D0; FR2720277B1; AU2620695A; FI964783A; FI964783A0

Abstract

Method of cancer treatment by controlling cellular p53 protein levels. The invention concerns, in particular, the use of a compound capable of modulating calpaine activity.

Description

METHOD OF TREATING CANCER BY REGULATING THE

PROTEIN P53

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 cellular levels of the p53 protein. It also relates to gene therapy vectors making it possible to regulate the p53 protein, as well as the pharmaceutical compositions containing them.

For the past fifteen years, the molecular characterization of oncogenes and tumor suppressor genes has made it possible to take a new look at the process of carcinogenesis. Thus, the increasingly deep knowledge of the regulation of these genes and of the function of the corresponding proteins makes it possible to design new therapeutic approaches.

More particularly, the elucidation of the catabolism of oncogenic and anti-oncogenic proteins represents a major challenge in terms of anti-cancer control insofar as it suggests, in the case of oncogenic proteins, the possibility of accelerating their degradation and therefore to annihilate their action, in the case of tumor suppressors, to inhibit their degradation and therefore to increase their anti-proliferative or anti-tumor effect, in the case of mutated proteins, to potentiate their antigenic presentation by the molecules of the Major Histocompatibility Complex and thus stimulate a tumor-specific immune response, and, in cases where the strong expression of the oncogene or anti-oncogene is capable of inducing programmed cell death, the possibility of stabilizing these proteins to trigger the apoptotic process.

Originally, the p53 protein was classified as a nuclear oncogene since it could, in transfection experiments, lengthen the life of rodent cells in culture as well as cooperate with oncogenes activated as ras to transform cells into primary culture. In fact, the genes used in these first experiments were mutated and led to the expression of variant p53 proteins characterized by a gain in function. Without excluding functions which remain to be discovered, we now know that the p53 protein, at least in its wild form, is a transcription factor which negatively regulates growth and cell division and which, in certain situations, is capable of inducing apoptosis (Yonish-Rouach et al., Nature, 352, 345-347, 1991). These properties appear in stressful situations where the integrity of cellular DNA is threatened, p53 has been suggested be a "genome keeper". The presence of mutated p53 in approximately 40% of human tumors, all types combined, reinforces this hypothesis and underlines the probably crucial role played by mutations of this gene in tumor development (for reviews, see Montenarh, Oncogene, 7, 1673- 1680, 1992; Oren, FASEB J., 6, 3169-3176, 1992; Zambetti and Levine, FASEB I, 7, 855-865, 1993).

The wild-type p53 protein is subject to complex regulation which involves the control of its synthesis and catabolism as well as that of its intracellular localization and its post-translational modifications (see the journals cited above). The wild-type p53 protein is extremely unstable with a half-life of a few minutes. On the other hand, certain mutated proteins which accumulate at high level in tumors have a significantly extended half-life. Little has been clearly established regarding the degradation of p53. In fact, neither the intracellular degradation sites, nor the number and nature of the catabolic pathways used, nor the peptide motifs marking p53 for its degradation are known. To our knowledge, the only information available concerns the involvement of the enzyme E1 of the ubiquitin cycle under certain experimental conditions (Ciechanover et al., Proc. Natl. Acad. Sci. USA 88, 139-143, 1991; Chowdary et al., Molec. Cell. Biol. 14, 1997-2003, 1994). Furthermore, it has been shown that certain proteolytic products derived from p53 can be presented in an antigenic manner.

The present invention results in part from the demonstration that the p53 proteins are substrates for calcium-dependent proteases: calpains. It more particularly results from the demonstration that the p53 proteins are specifically degraded by m-calpain or μ-calpain. The present invention constitutes the first demonstration of a mechanism for regulating the cellular levels of the p53 proteins and thus offers a new particularly effective and specific approach for modulating the levels of this protein in pathological situations such as in particular certain cancers.

In particular, the present invention describes a new approach for the treatment of cancers, based on the use of compounds which modulate the activity of calpains on the p53 proteins, which make it possible either to activate the degradation of mutated p53 proteins, to block their tumorigenic effect and / or to increase the presentation of immunogenic peptides, ie to stabilize the wild-type p53 protein, to counterbalance the tumorigenic effect of mutated proteins expressed in tumors and / or to induce apoptosis of tumor cells.

A first object of the invention therefore resides in the use of a compound capable of modulating the activity of calpain for the preparation of a pharmaceutical composition intended for the treatment of cancers.

Calpains are ubiquitous enzymes found in most mammalian cells (for review, see Croall and deMartino, Physiol. Rev., 71, 813-847, 1991). They are essentially cytoplasmic but they can penetrate into the nucleus thanks to the destruction of the nuclear envelope during mitosis or following certain stimuli. As indicated above, the proteolytic activity of calpains is dependent on the presence of calcium.

The compounds capable of modulating the activity of calpain within the meaning of the present invention can be of several types.

They may be compounds capable of inhibiting the activity of calpain on the p53 proteins. These compounds are particularly advantageous since they can be used to inhibit, at least in part, the degradation of the wild-type p53 protein. These compounds therefore make it possible to intracellularly stabilize the wild-type p53 protein and to counterbalance the effect of the mutated forms. Among the inhibitor compounds which can be used in the context of the invention, there may be mentioned protease inhibitors (leupeptin, aprotinin, PMSF, etc.), calcium chelators (EGTA, EDTA, etc.), or more specific inhibitors such as calpastatin or any fragment or derivative thereof. Calpastatin is a known inhibitor of calpains. Its sequence has been described in the prior art (SEQ ID No. 1). A particularly advantageous embodiment of the present invention consists in transferring into the tumors a vector carrying all or part of the sequence coding for calpastatin. This approach is particularly suitable for the treatment of cancers always presenting a wild p53 allele, such as colonic or bronchial carcinomas for example. Different fragments or derivatives of calpastatin can be used in the context of the present invention. Such fragments or derivatives can be any molecule obtained from the sequence SEQ ID No. 1 by modification (s) of genetic and / or chemical nature, retaining the capacity to inhibit at least in part the activity of a calpain . By modification of a genetic and / or chemical nature is meant any mutation, deletion, substitution, addition, and / or modification of one or more nucleotides. Such modifications can be carried out for different purposes, and in particular that of preparing suitable sequences to expression in a particular type of vector or host, that of reducing the size of the sequence to facilitate their cell penetration, that of increasing the inhibition activity, or, in a particularly advantageous manner, of increasing the selectivity of the inhibitor with respect to the activity of calpains on the degradation of the wild-type p53 protein.

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 an inhibitor of the activity of calpains on the p53 proteins can be demonstrated in several ways, and in particular by bringing said inhibitor and the various forms into contact of p53 proteins, then by detecting the degradation products obtained (see examples 1 to 3). Any other technique known to those skilled in the art can obviously be used for this purpose.

In a particular embodiment of the present invention, all or part of calpastatin is used as an inhibitor, or a nucleic acid coding for all or part of calpastatin. Even more particularly, a peptide is used comprising all or part of the sequence SEQ ID No. 1 or a derivative thereof.

As regards the derivatives more specifically, mention may be made, by way of example, of the compound of sequence SEQ ID No. 2, which corresponds to a fragment of calpastatin. Advantageously, any derivative compound of sequence SEQ ID No. 1 or 2 capable of specifically or preferentially inhibiting the degradation of the wild-type p53 protein by calpain is used.

The compounds capable of modulating the activity of calpain on the p53 proteins within the meaning of the present invention may also be derivatives of calpain capable of specifically or preferentially degrading the mutated p53 proteins. Such derivatives are also very advantageous since they make it possible to activate the degradation of the mutated p53 proteins, to block their tumorigenic effect and / or to increase the presentation of immunogenic peptides, without significantly affecting the cellular levels of wild-type p53 protein. Such derivatives can be obtained from calpain, by structural modification (s) of genetic and / or chemical nature. The capacity of the derivatives thus obtained to specifically or preferentially degrade the mutated p453 proteins can then be demonstrated as described in Examples 1 to 3. Preferably, the modulators used in the context of the invention are proteins or polypeptides, or nucleic acid sequences coding for these polypeptides or proteins. Even more preferably, the modulator compounds are proteins or polypeptides specific for inhibiting the activity of calpain on the wild-type p53 protein or forms of calpains, modified or not, for specifically degrading the mutated p53 proteins.

In a particularly advantageous manner, the invention lies in the possibility of expressing, in cancer cells having both a wild-type p53 allele and a mutated p53 allele, nucleic sequences coding for calpain inhibitors, such as calpastatin or part calpastatin, or forms of calpains, modified or not, to specifically degrade mutated p53 proteins.

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 application WO 90/11092. 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. BioI. 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 present invention also relates to any recombinant virus comprising, inserted into its genome, a nucleic acid coding for a compound capable of modulating the activity of calpain. Preferably, 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 calpain modulator. 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 application 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 among others the DNA sequence coding for the calpain modulator. Homologous recombination 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 broad 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 virus. human helper (for example an adenovirus), of a plasmid containing the sequence coding for the modulator of calpains 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.

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 calpain modulator 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 modulator. 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 may in fact be particularly advantageous to use expression signals which are active specifically or mainly in tumor cells, so that the DNA sequence is not expressed and does not produce its effect until 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 a calpain modulator 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 a calpain modulator 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 10θ 4 pfu / ml, and preferably 10 à to 10 p 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 plaques of infected cells. pfu titer of a viral solution are well documented in the literature Concerning retroviruses, the compositions according to the invention can directly comprise the producer cells, with a view to their implantation.

The present invention is particularly suitable for the treatment of cancers in which mutated forms of p53 are observed. More specifically, the The present invention is particularly advantageous for the treatment of cancers in which the wild and mutated alleles of p53 are present. Such canccers are in particular colorectal cancers, breast cancers, lung cancers, gastric cancers, esophageal cancers, B lymphomas, ovarian cancers, bladder cancers, etc.

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

Legend of figures

Figure 1: Study of the regulation of the p53 protein by calpain. The reaction is carried out in a final volume of 30 μl, including 1 coming from the translation mixture, line 1: T0; line 2: 30 min in the presence of ImM Calcium + 20 μg ml Calpain; line 4: 30 min in the presence of ImM Calcium + 20 μg / ml Calpain + 0.5 mg / ml calpastatin; line 5: 30 min in the presence of ImM Calcium + 20 μg / ml Calpain + 10 mM EGTA; lane 6: PBS; lane 7: PBS + calcium; line 8: PBS + calpastatin.

General molecular biology techniques

Methods conventionally used 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 in the art. 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 F.M. 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 of E. coli (Biolabs) according to the supplier's specifications. The destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of 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. 13 _. (1985) 8749-8764] using the kit distributed by Amersham. The enzymatic amplification of DNA fragments by the technique known as

PCR [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications. 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.

Examples

Example 1

This example shows that the addition of m-calpain to the rabbit reticulocyte lysate induces the degradation of the wild-type p53 protein as well as that of certain mutated forms. This example also shows that inhibitors of calpains are capable of inhibiting the degradation of p53 and therefore of modulating the activity of this protein.

1.1. Demonstration of degradation: Wild p53 proteins from mice and humans as well as various mutated p53 proteins (human proteins C273, H273, H175, 1247) have been translated into the rabbit reticulocyte lysate. The proteins thus obtained are resistant to any degradation, even in the presence of high concentration of calcium (essential co-factor of calpains). The addition of beef m-calpain (Sigma) to the reticulocyte lysate in the presence of calcium led to the rapid disappearance of the neo-synthesized proteins and the appearance of proteolytic fragments which can be resolved by electrophoresis. Resistance to degradation of other proteins such as dihydrofolate reductase or glyceraldehyde-3-phosphate dehydrogenase under the same experimental conditions indicates the specificity of the reaction substrate.

1.2. Use of calpain inhibitors to modulate the levels of p53 proteins: In Example 1.1. above, the addition of m-calpain has been shown to induce degradation of the p53 proteins. In this example, in addition to m-calpain, various compounds were introduced into the medium to test their capacity to inhibit the activity of calpain. The results obtained show that the addition of a calcium chelator (EGTA) as well as a specific inhibitor peptide of calpains (derived from a physiological inhibitor, calpastatin; Maki et al., J. Biol. Chem ., 254, 18866-18869, 1989) are capable of inhibiting the degradation of p53 proteins induced by exogenous calpain.

Example 2

In the previous example, it was shown that the addition of exogenous calpain to a solution of p53 proteins caused their degradation. This example shows that the degradation of the wild-type p53 protein as well as that of certain mutated forms can be induced by endogenous calpains in cytoplasmic extracts. This example also shows that inhibitors of calpains are capable, in the presence of endogenous calpain, of inhibiting the degradation of p53 and therefore of modulating the activity of this protein.

2.1. Degradation by endogenous calpains: Wild p53 proteins from mice and humans, as well as certain mutated forms (cf. example 1) were translated into the reticulocyte lysate and then were incubated in the presence of cytoplasmic extracts of Daudi human lymphoblastoid cells. or Jurkat. The cytoplasmic extracts were prepared as follows: The cells (accessible to ATCC) were cultured in DMEM medium supplemented with 10% fetal calf serum. The cells were then harvested, washed in PBS buffer, then incubated for 5 min in hypotonic lysis buffer without detergent (HEPES 20 mM pH 7.5; KOAc 10 mM; MgOAc 1.5 mM; 2 ml for 5.10 ⁸ cells) . The lysis was completed using a Dounce homogenizer, then verified under a microscope. The nuclei were then removed by centrifugation at 2000 g for 5 min, and the supernatants were centrifuged at 10,000 g for 1 hour (Beckman SW60). The cytoplasmic extracts were then aliquoted at 5 to 12 mg / ml. When the reticulocute lysate was incubated in the presence of cytoplasmic extracts, in the absence of calcium, no degradation was observed. On the other hand, in the presence of calcium, a very rapid degradation of the p53 proteins was observed, with the appearance of a characteristic proteolytic product profile close to that obtained in Example 1. This experiment clearly shows that the p53 proteins are degraded by endogenous calpains.

2.2. Use of calpain inhibitors to modulate p53 protein levels: Calcium chelation by EGTA, as well as the use of a whole range of protease inhibitors (leupeptin, aprotinin, soybean trypsin inhibitor and PMSF) and above all the peptide calpastatin show that the degradation of these proteins is dependent on the calpains of the cytoplasmic extract, and that various compounds capable of modulating the activity of calpains can be used to regulate the levels of protein p53.

Example 3

This example demonstrates that wild mouse and human p53 proteins are direct substrates for calpains in cytoplasmic extracts.

Examples 1 and 2 show that calpains can induce the degradation of p53 in complex reaction mixtures. These experiments do not however exclude that under the conditions used the calpains activate secondary proteases which are those which really act on p53. In this example, the following experiment was carried out: (1) the wild-type p53 proteins of mice and of humans neo-synthesized in the rabbit reticulocyte lysate were incubated for 30 minutes in the presence of cytoplasmic extract of Daudi cells as well as in the presence of calcium to activate the calpains as in Example 2, (2) of the protein p53 was then added to the reaction mixture and the reaction was continued for 30 minutes under permissive conditions (same reaction conditions) or no (addition of either EGTA to chelate calcium, or of peptide calpastatin) for calpains. In the presence of calcium, the newly added p53 protein is completely degraded indicating that the protease activity is functional all the time of the experiment. When calpains are inhibited by the presence of EGTA or, more significantly, of the peptide calpastatin, the newly added p53 protein is no longer degraded. This last observation therefore excludes the possibility that in the first part of the experiment the calpains induced a second protease responsible for the degradation of p53 (FIG. 1).

Example 4

This example describes the construction of a recombinant adenovirus comprising a nucleic acid sequence coding for calpastatin. This adenovirus is constructed by homologous recombination between the defective adenovirus Ad-dll324 and a plasmid carrying the sequence SEQ ID No. 1 under the control of the RSV promoter.

4.1. Construction of the plasmid SEQ ID n ° 1

The plasmid SEQ ID No. 1 comprises the sequence coding for calpastatin under the control of the LTR-RSV promoter, as well as regions of the adenovirus allowing homologous recombination. It is constructed by inserting the sequence

SEQ ID No. 1 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 SEQ ID No. 1 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 purification on a plate. 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 × 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.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: RHONE-POULENC RORER S.A.

(B) STREET: 20, avenue Raymond ARON

(C) CITY: ANTONY (E) COUNTRY: FRANCE

(F) POSTAL CODE: 92165

(ii) TITLE OF THE INVENTION: Method for the treatment of cancers by regulation of the p53 protein.

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER-DEPENDABLE FORM: (A) TYPE OF MEDIA: Tape (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 2085 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(vi) ORIGIN: (A) ORGANISM: Homo sapiens

(ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 1..2085 (D) OTHER INFORMATION: / product = "Human Calpastatin"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG GAA GGA CCA CAT CTT CCT AAC AAG AAA AAA CAC AAA AAA CAG GCT 48 Met Glu Gly Pro His Leu Pro Asn Lys Lys Lys His Lys Lys Gin Ala 1 5 10 15

GTA AAA ACA GAA CCT GAG AAG AAG TC CAG TCA ACC AAG CTG TCT GTG 96 Val Lys Thr Glu Pro Glu Lys Lys Ser Gin Ser Thr Lys Leu Ser Val

20 25 30

GTT CAT GAG AAA AAA TCC CAA GAA GGA AAG CCA AAA GAA CAC ACA GAG 144 Val His Glu Lys Lys Ser Gin Glu Gly Lys Pro Lys Glu His Thr Glu 35 40 45

CCA AAA AGC CTA CCC AAG CAG GCA TCA GAT ACA GGA AGT AAC GAT GCT 192 Pro Lys Ser Leu Pro Lys Gin Ala Ser Asp Thr Gly Ser Asn Asp Ala 50 55 60

CAC AAT AAA AAA GCA GTT TCC AGA TCA GCT GAA CAG CAG CCA TCA GAG 240

His Asn Lys Lys Ala Val Ser Arg Ser Ala Glu Gin Gin Pro Ser Glu

65 70 75 80

AAA TCA ACA GAA CCA AAG ACT AAA CCA CAA GAC ATG ATT TCT GCT GGT 288

Lys Ser Thr Glu Pro Lys Thr Lys Pro Gin Asp Met Ile Ser Ala Gly

85 90 95

GGA GAG AGT GTT GCT GGT ATC ACT GCA ATA TCT GGC AAG CCG GGT GAC 336

Gly Glu Ser Val Ala Gly Ile Thr Ala Ile Ser Gly Lys Pro Gly Asp 100 105 110 AAG AAA AAA GAA AAG AAA TCA TTA ACC CCA GCT GTG CCA GTT GAA TCT 384 Lys Lys Lys Glu Lys Lys Ser Leu Thr Pro Ala Val Pro Val Glu Ser 115 120 125

AAA CCG GAT AAA CCA TCG GGA AAG TCA GGC ATG GAT GCT GCT TTG GAT 432 Lys Pro Asp Lys Pro Ser Gly Lys Ser Gly Met Asp Ala Ala Leu Asp 130 135 140

GAC TTA ATA GAT ACT TTA GGA GGA CCT GAA GAA ACT GAA GAA GAA AAT 480 Asp Leu Ile Asp Thr Leu Gly Gly Pro Glu Glu Thr Glu Glu Glu Asn 145 150 155 160

ACA ACG TAT ACT GGA CCA GAA GTT TCA GAT CCA ATG AGT TCC ACC TAC 528 Thr Thr Tyr Thr Gly Pro Glu Val Ser Asp Pro Met Ser Ser Thr Tyr 165 170 175

ATA GAG GAA TTG GGT AAA AGA GAA GTC ACA ATT CCT CCA AAA TAT AGG 576 Ile Glu Glu Leu Gly Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Arg 180 185 190 GAA CTA TTG GCT AAA AAG GAA GGG ATC ACA GGG CCT CCT GCA GAC TCT 624 Glu Leu Leu Ala Lys Lys Glu Gly Ile Thr Gly Pro Pro Ala Asp Ser 195 200 205

TCA AAA CCC ATA GGG CCA GAT GAT GCT ATA GAC GCC TTG TCA TCT GAC 672 Ser Lys Pro Ile Gly Pro Asp Asp Ala Ile Asp Ala Leu Ser Ser Asp 210 215 220

TTC ACC TGT GGG TCG CCT ACA GCT GCT GGA AAG AAA ACT GAA AAA GAG 720 Phe Thr Cys Gly Ser Pro Thr Ala Ala Gly Lys Lys Thr Glu Lys Glu 225 230 235 240

GAA TCT ACA GAA GTT TTA AAA GCT CAG TCA GCA GGG ACA GTC AGA AGT 768 Glu Ser Thr Glu Val Leu Lys Ala Gin Ser Ala Gly Thr Val Arg Ser 245 250 255

GCT GCT CCA CCC CAA GAG AAG AAA AGA AAG GTG GAG AAG GAT ACA ATG 816 Ala Ala Pro Pro Gin Glu Lys Lys Arg Lys Val Glu Lys Asp Thr Met 260 265 270 AGT GAT CAA GCA CTC GAG GCT CTG TCG GCT TCA CTG GGC ACC CGG CAA 864 Ser Asp Gin Ala Leu Glu Ala Leu Ser Ala Ser Leu Gly Thr Arg Gin 275 280 285

GCA GAA CCT GAG CTC GAC CTC CGC TCA ATT AAG GAA GTC GAT GAG GCA 912 Ala Glu Pro Glu Leu Asp Leu Arg Ser Ile Lys Glu Val Asp Glu Ala 290 295 300

AAA GCT AAA GAA GAA AAA CTA GAG AAG TGT GGT GAG GAT GAT GAA ACA 960 Lys Ala Lys Glu Glu Lys Leu Glu Lys Cys Gly Glu Asp Asp Glu Thr

305 310 315 320

ATC CCA TCT GAG TAC AGA TTA AAA CCA GCC ACG GAT AAA GAT GGA AAA 1008 Ile Pro Ser Glu Tyr Arg Leu Lys Pro Ala Thr Asp Lys Asp Gly Lys

325 330 335

CCA CTA TTG CCA GAG CCT GAA GAA AAA CCC AAG CCT CGG AGT GAA TCA 1056

Pro Leu Leu Pro Glu Pro Glu Glu Lys Pro Lys Pro Arg Ser Glu Ser 340 345 350

GAA CTC ATT GAT GAA CTT TCA GAA GAT TTT GAC CGG TCT GAA TGT AAA 1104

Glu Leu Ile Asp Glu Leu Ser Glu Asp Phe Asp Arg Ser Glu Cys Lys 355 360 365

GAG AAA CCA TCT AAG CCA ACT GAA AAG ACA GAA GAA TCT AAG GCC GCT 1152 Glu Lys Pro Ser Lys Pro Thr Glu Lys Thr Glu Glu Ser Lys Ala Ala 370 375 380 GCT CCA GCT CCT GTG TCG GAG GCT GTG TCT CGG ACC TCC ATG TGT AGT 1200

Ala Pro Ala Pro Val Ser Glu Ala Val Ser Arg Thr Ser Met Cys Ser

385 390 395 400

ATA CAG TCA GCA CCC CCT GAG CCG GCT ACC TTG AAG GGC ACA GTG CCA 1248 Ile Gin Ser Ala Pro Pro Glu Pro Ala Thr Leu Lys Gly Thr Val Pro

405 410 415

GAT GAT GCT GTA GAA GCC TTG GCT GAT AGC CTG GGG AAA AAG GAA GCA 1296

Asp Asp Ala Val Glu Ala Leu Ala Asp Ser Leu Gly Lys Lys Glu Ala 420 425 430

GAT CCA GAA GAT GGA AAA CCT GTG ATG GAT AAA GTC AAG GAG AAG GCC 1344

Asp Pro Glu Asp Gly Lys Pro Val Met Asp Lys Val Lys Glu Lys Ala 435 440 445

AAA GAA GAA GAC CGT GAA AAG CTT GGT GAA AAA GAA GAA ACA ATT CCT 1392 Lys Glu Glu Asp Arg Glu Lys Leu Gly Glu Lys Glu Glu Thr Ile Pro 450 455 460 CCT GAT TAT AGA TTA GAA GAG GTC AAG GAT AAA GAT GGA AAG CCA CTC 1440 Pro Asp Tyr Arg Leu Glu Glu Val Lys Asp Lys Asp Gly Lys Pro Leu 465 470 475 480

CTG CCA AAA GAG TCT AAG GAA CAG CTT CCA CCC ATG AGT GAA GAC TTC 1488 Leu Pro Lys Glu Ser Lys Glu Gin Leu Pro Pro Met Ser Glu Asp Phe

485 490 495

CTT CTG GAT GCT TTG TCT GAG GAC TTC TCT GGT CCA CAA AAT GCT TCA 1536 Leu Leu Asp Ala Leu Ser Glu Asp Phe Ser Gly Pro Gin Asn Ala Ser 500 505 510

TCT CTT AAA TTT GAA GAT GCT AAA CTT GCT GCT GCC ATC TCT GAA GTG 1584 Ser Leu Lys Phe Glu Asp Ala Lys Leu Ala Ala Ala Ile Ser Glu Val 515 520 525

GTT TCC CAA ACC CCA GCT TCA ACG ACC CAA GCT GGA GCC CCA CCC CGT 1632 Val Ser Gin Thr Pro Ala Ser Thr Thr Gin Ala Gly Ala Pro Pro Arg 530 535 540 GAT ACC TCG CAG AGT GAC AAA GAC CTC GAT GAT GCC TTG GAT AAA CTC 1680 Asp Thr Ser Gin Ser Asp Lys Asp Leu Asp Asp Ala Leu Asp Lys Leu 545 550 555 560 TCT GAC AGT CTA GGA CAA AGG CAG CCT GAC CCA GAT GAG AAC AAA CCA 1728 Ser Asp Ser Leu Gly Gin Arg Gin Pro Asp Pro Asp Glu Asn Lys Pro 565 570 575 ATG GGA GAT AAA GTA AAG GAA AAA GCT AAA GCT GAA CAT AGA GAC AAG 1776 Met Gly Asp Lys Val Lys Glu Lys Ala Lys Ala Glu His Arg Asp Lys 580 585 590

CTT GGA GAA AGA GAT GAC ACT ATC CCA CCT GAA TAC AGA CAT CTC CTG 1824 Leu Gly Glu Arg Asp Asp Thr Ile Pro Pro Glu Tyr Arg His Leu Leu 595 600 605

GAT GAT AAT GGA CAG GAC AAA CCA GTG AAG CCA CCT ACA AAG AAA TCA 1872 Asp Asp Asn Gly Gin Asp Lys Pro Val Lys Pro Pro Thr Lys Lys Ser 610 615 620

GAG GAT TCA AAG AAA CCT GCA GAT GAC CAA GAC CCC ATT GAT GCT CTC 1920 Glu Asp Ser Lys Lys Pro Ala Asp Asp Gin Asp Pro Ile Asp Ala Leu 625 630 635 640

TCA GGA GAT CTG GAC AGC TGT CCC TCC ACT ACA GAA ACC TCA CAG AAC 1968 Ser Gly Asp Leu Asp Ser Cys Pro Ser Thr Thr Glu Thr Ser Gin Asn 645 650 655 ACA GCA AAG GAT AAG TGC AAG AAG GCT GCT TCC AGC TCC AAA GCA CCT 2016 Thr Ala Lys Asp Lys Cys Lys Lys Ala Ala Ser Ser Ser Lys Ala Pro 660 665 670

AAG AAT GGA GGT AAA GCG AAG GAT TCA GCA AAG ACA ACA GAG GAA ACT 2064 Lys Asn Gly Gly Lys Ala Lys Asp Ser Ala Lys Thr Thr Glu Glu Thr 675 680 685

TCC AAG CCA AAA GAT GAC TAA 2085

Ser Lys Pro Lys Asp Asp * 690 695

(2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 399 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(vi) ORIGIN:

(A) ORGANISM: homo sapiens (ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 1..399

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

TCA GGC ATG GAT GCT GCT TTG GAT GAC TTA ATA GAT ACT TTA GGA GGA 48 Ser Gly Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu Gly Gly 700 705 71 0

CCT GAA GAA ACT GAA GAA GAA AAT ACA ACG TAT ACT GGA CCA GAA GTT 96

Pro Glu Glu Thr Glu Glu Glu Asn Thr Thr Tyr Thr Gly Pro Glu Val 715 720 725

TCA GAT CCA ATG AGT TCC ACC TAC ATA GAG GAA TTG GGT AAA AGA GAA 144

Ser Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly Lys Arg Glu 730 735 740

GTC ACA ATT CCT CCA AAA TAT AGG GAA CTA TTG GCT AAA AAG GAA GGG 192 Val Thr Ile Pro Pro Lys Tyr Arg Glu Leu Leu Ala Lys Lys Glu Gly 745 750 755 ATC ACA GGG CCT CCT GCA GAC TCT TCA AAA CCC ATA GGG CCA GAT GAT 240 Ile Thr Gly Pro Pro Ala Asp Ser Ser Lys Pro Ile Gly Pro Asp Asp 760 765 770 775

GCT ATA GAC GCC TTG TCA TCT GAC TTC ACC TGT GGG TCG CCT ACA GCT 288 Ala Ile Asp Ala Leu Ser Ser Asp Phe Thr Cys Gly Ser Pro Thr Ala

780 785 790

GCT GGA AAG AAA ACT GAA AAA GAG GAA TCT ACA GAA GTT TTA AAA GCT 336 Ala Gly Lys Lys Thr Glu Lys Glu Glu Ser Thr Glu Val Leu Lys Ala 795 800 805

CAG TCA GCA GGG ACA GTC AGA AGT GCT GCT CCA CCC CAA GAG AAG AAA 384 Gin Ser Ala Gly Thr Val Arg Ser Ala Ala Pro Pro Gin Glu Lys Lys 810 815 820

AGA AAG GTG GAG AAG 399 Arg Lys Val Glu Lys 825

Claims

1. Use of a compound capable of modulating the activity of calpain for the preparation of a pharmaceutical composition intended for the treatment of cancers.

2. Use according to claim 1 characterized in that the compound is a protein or a polypeptide inhibiting the activity of calpain, or a nucleic acid sequence coding for such a polypeptide or protein.

3. Use according to claim 2 characterized in that the compound is a protein or an inhibitory polypeptide specific for the activity of calpain on the wild-type p53 protein, or a nucleic acid sequence coding for such a polypeptide or protein.

4. Use according to claim 2 or 3 characterized in that the nucleic acid is part of a vector.

5. Use according to claim 4 characterized in that the nucleic acid is part of a viral vector, chosen from adenoviruses, retroviruses and adeno-associated viruses.

6. Use according to claim 4 characterized in that the nucleic acid is part of a liposomal, lipid vector

7. Use according to one of the preceding claims, characterized in that the compound is a nucleic acid coding for all or part of calpastatin.

8. Use according to claim 7 characterized in that the nucleic acid comprises all or part of the sequence SEQ ID No. 1 or a derivative thereof.

9. Use according to claim 8 characterized in that the nucleic acid is chosen from the sequences SEQ ID No. 1 and 2.

10. Use according to claim 8 characterized in that the nucleic acid is chosen from derivatives of sequences SEQ ϋ) n ° 1 or 2 coding for specific inhibitors of the degradation of the wild-type p53 protein.

11. Use according to one of claims 1 to 6 characterized in that the compound is a calpain derivative capable of specifically degrading the mutated p53 proteins.

12. Viral vector comprising a nucleic acid sequence coding for a protein or polypeptide inhibiting the activity of calpain.

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

14. Vector according to one of claims 12 or 13 characterized in that it comprises a sequence coding for all or part of the calpastatin.

15. Vector according to claim 12 characterized in that it comprises a sequence coding for a calpain derivative capable of specifically degrading the mutated p53 proteins.

16. Pharmaceutical composition comprising a nucleic acid sequence coding for all or part of calpastatin or for a calpain derivative capable of specifically degrading the mutated p53 proteins.

17. Composition according to claim 16 formulated for intra-tumor administration.