AU730324B2 - Use of protein gax for treating cancer - Google Patents

Description

WO 97/16459 1 PCT/FR96/01690 APPLICATION OF THE GAX PROTEIN TO THE TREATMENT OF CANCER The present invention relates to a new method for the treatment of cancer. More particularly, it relates to a method for the treatment of cancer by blocking cell proliferation which may be deregulated in tumour cells expressing an oncogene such as mutated ras or in cells deficient for a tumour suppressor gene such as p53. It also relates to the use of vectors for gene therapy which make it possible to regulate the latter, as well as to 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 eukaryotic organisms where they have been examined. Some specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic. Thus, a large number of human tumours has been associated with the presence of modified ras genes. Likewise, an overexpression of these p21 proteins may lead to a deregulation of cell proliferation.

In a normal cellular context, this proliferation of oncogenes is probably checked, at least in part, by the generation of so-called tumour suppressor genes such as p53 and Rb. It is thus known that the p53 protein, at least in its wild-type form, is a transcription factor which negatively regulates growth and cell division and which, in some situations, is capable of inducing apoptosis (Yonish-Rouach et al., Nature, 352, 345-347, 1991). Given that these properties manifest themselves in a situation of stress where the integrity of the cellular DNA is threatened, p53 has been suggested to be a "guardian of the genome". However, certain phenomena may come to disrupt this mechanism of cellular self-regulation and thereby promote the development of a neoplastic state. One of these events consists in mutations in the tumour suppressor genes. Accordingly, inactivated forms of the Rb gene have been implicated in various tumours, and especially in retinoblastomas or in mesenchymatous cancers such as osteosarcomas and the presence of mutated p53 has been noted in about 40 of human tumours of all types (for revues, see Montenarh, Oncogene, 7, 1673-1680, 1992; Oren, FASEB 6, 3169-3176, 1992; Zambetti and Levine, FASEB 7, 855-865, 1993).

Consequently, the identification of biological compounds capable of interfering with this type of deregulation of cell proliferation and/or of compensating for this type of deficiency in tumour suppressor genes is consequently of a major interest in the therapeutic approach to carcinogenesis.

The present invention results precisely, in part, from the demonstration that the GAX protein constitutes a potential inhibitor of the cell proliferation induced by the ras proteins. It results, in particular, from the demonstration that cell proliferation, which is deregulated in tumour cells, can be reestablished by expressing the GAX protein therein.

The GAX protein is a protein of 303 amino acids. Its sequence has been characterized and its cDNA cloned (Gorski et al., Mol.Cell.Biol. 1993, 6, 3722-3733). The gax (growth arrest specific homeobox) gene belongs to the family of homeotic genes. These genes encode transcriptional factors which contain consensus sequences (or homeodomains) which recognize specific regions of the DNA (or homeoboxes) (revue: Gehring et al. Cell, 78: 211-223, 1994). The homeodomain of the rat GAX protein is situated between amino acids 185 and 245. This gene possesses certain properties which are similar to the gas and Gadd genes since it also appears to control the GO/G1 transition of the cell cycle. Thus, the gax mRNA levels are reduced in rat CMLV by a factor of 10 after two hours of exposure to PDGF (Gorski et al., Mol. Cell. Biol.

1993, 6, 3722-3733). The expression of the gax gene is therefore repressed during the mitogenic response of the CMLVs. These observations have their corollary in vivo since in rats, intimal hyperplasia caused by abrasion of the carotid is associated with a high reduction in the expression of gax (Weir et al. J.

Biol. Chem. 1995, 270, 5457-5461).

P:\OPER\TDO\74982-96 dla.doc-22/12 -4- The expression of the gax gene has been demonstrated in the cardiovascular system, especially the heart and the aorta, and until now the use of this gene in gene therapy was therefore limited to these cells which express it naturally.

Unexpectedly, the applicant has discovered that it was possible advantageously to promote an inhibitory activity of the GAX protein towards cell proliferation in tumour cells, that is to say cells not expressing the gax gene endogenously.

The present invention thus offers a particularly efficient new approach for the treatment of tumours associated with a deregulation of cell proliferation such as non-small cell lung tumours, pancreatic and colic carcinomas, osteosarcomas and the like.

The present invention also describes particularly efficient systems which allow the in vivo delivery, directly into the tumours, or such compounds and thereby to combat the development of cancer.

S*ee A first subject of the invention therefore provides use of the gax protein or of a variant thereof which is capable of inhibiting cell proliferation induced by the ras protein, for the preparation of a pharmaceutical composition for the treatment of cancer.

More specifically, it relates to the use of at least one nucleic sequence encoding either the GAX protein or a variant as defined above for the preparation of a pharmaceutical composition intended for the treatment of cancer.

As indicated above, this may be a gene encoding all or part of the GAX protein or a variant thereof. For the purposes of the present invention, the term variant designates any mutant, fragment or peptide having at least one biological property of GAX, as well as any homologue of GAX obtained from other species.

These fragments and variants may be obtained by any technique known to persons skilled in the art, and especially by genetic and/or chemical and/or enzymatic modifications, or alternatively by hybridization or by expression cloning, allowing the selection of variants according to their biological activity. The genetic modifications include suppressions, deletions, mutations and the like.

The nucleic sequence used may encode all or part of the rat GAX protein or one of its variants.

The gene used for the purposes of the invention is preferably the gene encoding the rat GAX protein or its human homologue. This may be more preferably a cDNA or a gDNA.

The claimed nucleic sequence may be injected as such at the level of the site to be treated, or incubated directly with the cells to be destroyed or to be treated. It has indeed been described that naked nucleic acids can penetrate into cells without a specific vector. However, it is preferable within the framework of the present invention to use an 6 administration vector, making it possible to enhance the efficiency of cell penetration, (ii) the targeting, (iii) the extra- and intracellular stability.

Various types of vectors can be used. They may be viral or nonviral vectors.

The vector according to the invention may thus be a nonviral agent capable of promoting the transfer and the expression of the nucleic sequence in prokaryotic or eukaryotic cells. The chemical or biochemical vectors represent an advantageous alternative to the natural viruses, in particular for reasons of convenience, security and also because of the absence of a theoretical limit as regards the size of DNA to be transfected.

These synthetic vectors have two main functions, to compact the nucleic acid to be transfected and to promote its cellular attachment as well as its passage across the plasma membrane and, where appropriate, both nuclear membranes. To make up for the polyanionic nature of the nucleic acids, the nonviral vectors all possess polycationic charges.

Among the synthetic vectors developed, the cationic polymers of the polylysine, (LKLK)n, (LKKL)n, polyethyleneimine and DEAE-dextran types or alternatively cationic lipids or lipofectants are most advantageous. They possess the property of condensing the DNA and of promoting its association with the cell membrane. Among these, there may be mentioned lipopolyamines (lipofectamine, transfectam, and the like) and various cationic or neutral lipids (DOTMA, DOGS, DOPE, and the like) or liposomes. More recently, the concept of targeted transfection, mediated by a receptor, was developed, which exploits the principle of condensing the DNA by means of the cationic polymer while directing the attachment of the complex to the membrane by means of a chemical coupling between the cationic polymer and the ligand of a membrane receptor, present at the surface of the cell type which it is desired to graft. The targeting of the receptor for transferrin or for insulin, or of the receptor for the asialoglycoproteins of the hepatocytes, has thus been described.

The nucleic sequence used in the present invention may be formulated for administration via the topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular or transdermal route and the like. Preferably, it is used in an injectable form. It can therefore be mixed with any pharmaceutically acceptable vehicle for an injectable formulation, especially for a direct injection at the level of the site to be treated. This may be in particular isotonic sterile solutions or dry, especially freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or of physiological saline, allow the preparation of injectable solutions. A direct injection of the nucleic sequence into the patient's tumour is advantageous because it makes it possible to concentrate the therapeutic effect at the level of the affected tissues. The nucleic sequence doses used may be adjusted according to various parameters, and especially according to the vector, the mode of administration used, the relevant pathology or the desired duration of treatment.

To carry out the present invention, it is most particularly advantageous to use a defective recombinant virus.

It is thus possible to use adenoviruses, herpes viruses, retroviruses and more recently adenoassociated viruses. These viruses have been found to be particularly efficient from the transfection point of view.

The second subject of the present invention is therefore the use of a defective recombinant virus containing at least one inserted gene encoding all or part of the GAX protein or a variant thereof. The invention also consists in the use of such a virus for the treatment of cancer. In the vectors of the invention, the gene inserted and/or present may be a complementary DNA (cDNA) or genomic DNA (gDNA) fragment, or a hybrid construct consisting, for example, of a cDNA into which one or more introns would be inserted. It may also be synthetic or semisynthetic sequences.

Generally, the inserted gene also comprises sequences permitting its expression in the infected cell. These may be sequences which are naturally responsible for the expression of the said gene when these sequences are capable of functioning in the infected cell. They may also be sequences of different origin (which are responsible for the expression of other proteins, or even synthetic). In particular, they may be eukaryotic or viral gene sequences or derived sequences, stimulating or repressing the transcription of a gene in a specific manner or otherwise and in an inducible manner or otherwise. By way of example, they may be promoter sequences obtained from the genome of the cell which it is desired to infect, or from the genome of a virus, and especially the promoters of the adenovirus genes ElA and MLP, the CMV and RSV-LTR promoter, and the like. Among the eukaryotic promoters, there may also be mentioned the ubiquitous promoters (HPRT, vimentin, actin, tubulin and the like), the promoters for the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (MDR, CFTR and factor VIII types, and the like), tissue-specific promoters (promoter for the actin of the smooth muscle cells), the promoters preferentially activated in dividing cells, or alternatively the promoters responding to a stimulus (receptor for the steroid hormones, receptor for retinoic acid, and the like). In addition, these expression sequences can be modified by the addition of activating or regulatory sequences, and the like.

Moreover, when the inserted gene does not contain expression sequences, it can be inserted into the genome of the defective virus downstream of such a sequence.

Moreover, the inserted gene generally comprises, upstream of the coding sequence, a signal sequence directing the synthesized polypeptide in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of GAX, but it may also be any other functional signal sequence (that of the thymidine kinase gene, for example) or an artificial signal sequence.

The viruses according to the present invention are defective, that is to say are incapable of autonomously replicating in the target cell.

Generally, the genome of the defective viruses used within the framework of the present invention therefore lacks at least the sequences necessary for the replication of the said virus in the infected cell.

These regions may be either eliminated (completely or partially), or made nonfunctional, or substituted by other sequences and, especially, by the inserted gene.

Preferably, the defective virus retains, nevertheless, the sequences of its genome which are necessary for the encapsidation of the viral particles.

Various adenovirus serotypes exist whose structure and properties vary somewhat. Among these serotypes, the use of the type 2 or 5 human adenoviruses (Ad 2 or Ad 5) or of the adenoviruses of animal origin (see application W094/26914) is preferred within the framework of the present invention. Among the adenoviruses of animal origin which can be used within the framework of the present invention, there may be mentioned adenoviruses of canine, bovine, murine (example: MAV1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or alternatively simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, or more preferably a CAV2 adenovirus [Manhattan strain or A26/61 (ATCC VR- 800) for example]. Preferably, adenoviruses of human or canine or mixed origin are used within the framework of the invention.

Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence allowing the encapsidation and the nucleic acid of interest. Still more preferably, in the genome of the adenoviruses of the invention, at least the El region is nonfunctional.

The viral gene considered can be rendered nonfunctional by any technique known to persons skilled in the art, and especially by total suppression, by substitution or partial deletion, or by addition of one or more bases in the gene(s) considered. Such modifications can be obtained in vitro (on the isolated 12 DNA) or in situ, for example by means of genetic engineering techniques, or alternatively by treating with mutagenic agents. Other regions can also be modified and especially the E3 (W095/02697), E2 (W094/28938), E4 (W094/28152, W094/12649, W095/02697) and L5 (W095/02697). According to a preferred embodiment, the adenovirus according to the invention comprises a deletion in the El and E4 regions.

According to another preferred embodiment, it comprises a deletion in the El region at the level of which are inserted the E4 region and the sequence encoding GAX (cf FR94/13355). In the viruses of the invention, the deletion in the El region extends preferably from nucleotides 455 to 3329 on the sequence of the adenovirus The defective recombinant adenoviruses according to the invention can be prepared by any technique known to persons 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 of interest. The homologous recombination occurs after co-transfection of the said adenoviruses and plasmid into an appropriate cell line. The cell line used should preferably be transformable by the said elements, and (ii) contain the sequences capable of complementing the defective adenovirus genome part, preferably in integrated form in order to avoid risks of recombination. As an example of a cell line, there may be mentioned the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains especially, integrated in its genome, the left hand part of the genome of an Ad5 adenovirus (12 or lines capable of complementing the El and E4 functions as described especially in applications Nos. W094/26914 and W095/02697.

Next, the adenoviruses which have multiplied are recovered and purified according to conventional molecular biology techniques.

As regards the adeno-associated viruses (AAV), they are relatively small DNA viruses which become integrated into the genome of the cells which they infect, in a stable and site-specific manner. They are capable of infecting a broad spectrum of cells, without inducing any effect on cell growth, morphology or differentiation. Moreover, they do not seem to be involved in pathologies in man. The genome of the AAVs has been cloned, sequenced and characterized. It comprises about 4700 bases and contains, at each end, an inverted repeat region (ITR) of about 145 bases which serves as replication origin for the virus. The remainder of the genome is divided into 2 essential regions carrying the encapsidation functions: the left hand part of the genome, which contains the rep gene involved in the viral replication and the expression of 14 the viral genes; the right hand part of the genome, which contains the cap gene encoding the virus capsid proteins.

The use of vectors derived from AAVs for the transfer of genes in vitro and in vivo has been described in the literature (see especially 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, from which the rep and/or cap genes are deleted and replaced by a gene of interest, and their use for the transfer in vitro (on cells in culture) or in vivo (directly in an organism) of the said gene of interest. The defective recombinant AAVs according to the invention can be prepared by co-transfection, into a cell line infected by a human helper virus (for example an adenovirus), of a plasmid containing the nucleic sequence of interest bordered by two AAV inverted repeat regions (ITR), and of a plasmid carrying the AAV encapsidation genes (rep and cap genes). The recombinant AAVs produced are then purified by conventional techniques. The invention therefore relates to a recombinant virus derived from AAVs whose genome comprises a sequence encoding GAX bordered by AAV ITRs. The invention also relates to a plasmid comprising a sequence encoding GAX bordered by two ITRs from an AAV. Such a plasmid may be used as it is to transfer the GAX sequence, optionally incorporated into a liposomal vector (pseudo-virus).

As regards the retroviruses, the construction of recombinant vectors has been widely described in the literature: see especially EP 453 242, EP 178 220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like. In particular, the retroviruses are integrative viruses which infect the dividing cells. The genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In the recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted, completely or partly, and replaced by a heterologous nucleic acid sequence of interest. These vectors can be prepared from various types of retroviruses, such as especially MoMuLV (murine Moloney leukaemia virus, also called MoMLV), MSV (murine Moloney sarcoma virus), HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Rous sarcoma virus) or alternatively Friend's virus.

To construct recombinant retroviruses containing a sequence encoding GAX according to the invention, a plasmid containing especially the LTRs, the encapsidation sequence and the said coding sequence is generally constructed and then used to transfect a so-called encapsidation cell line capable of providing in trans the retroviral functions which are deficient in the plasmid. Generally, the encapsidation lines are therefore capable of expressing the gag, pol and env 16 genes. Such encapsidation lines have been described in the prior art, and especially the PA317 line (US 4,861,719), the PsiCRIP line (WO 90/02806) and the GP+envAm-12 line (WO 89/07150). Moreover, the recombinant retroviruses may contain modifications in the LTRs so as to suppress the transcriptional activity, as well as extended encapsidation sequences containing a portion of the gag gene (Bender et al., J.

Virol. 61 (1987) 1639). The recombinant retroviruses produced are then purified by conventional techniques.

For the treatment of cancer, it is most particularly advantageous to use a defective recombinant adenovirus. It is possible to use relatively low quantities of active ingredient (recombinant adenovirus), and this also allows an effective and very rapid action on the sites to be treated. The adenoviruses of the invention are also capable of expressing, at high levels, the gax gene introduced, which confers a very effective therapeutic action on them. Furthermore, because of their episomal character, the adenoviruses of the invention last for a limited period in proliferative cells and therefore have a transient effect which is perfectly adapted to the desired therapeutic effect.

The doses of virus used for the injection can be adapted according to various parameters, and especially according to the mode of administration used and the desired duration of the treatment. In general, the recombinant viruses according to the invention are formulated and administered in the form of doses of between 10 4 to 1014 pfu/ml. For the AAVs and the adenoviruses, doses of 106 to 101 0 pfu/ml can also be used. The term pfu ("plaque forming unit") corresponds to the infectivity of a suspension of virions, and is determined by infection of an appropriate cell culture, and measurement, generally after 48 hours, of the number of plaques of infected cells. The techniques for determining the pfu titre of a viral solution are well documented in the literature.

The present invention is advantageously used in vivo for the destruction of cells undergoing hyperproliferation (that is to say abnormal proliferation).

It is thus applicable to the destruction of tumour cells. It is most particularly appropriate for the treatment of cancers in which an activated oncogene is involved. As example, there may be mentioned: colon adenocarcinomasa, thyroid cancer, lung carcinomers, myeloid leukaemias, colorectal cancer, breast cancer, lung cancer, gastric cancer, oesophageal cancer, B lymphomas, ovarian cancer, cancer of the bladder, glioblastomas, and the like.

In addition, this treatment can be applied both to man and to any animal such as ovines, bovines, domestic animals (dogs, cats and the like), horses, fish and the like.

The present invention is more fully described with the aid of the following examples which should be considered as illustrative and nonlimiting.

LEGEND TO THE FIGURES Figure 1: Representation of the plasmid pCOl.

Figure 2: Representation of the plasmid pXL-CMV-GaxHA Figure 3: Representation of the effect of the expression of AdCMV gax on cell proliferation in human tumour cells H460.

Figure 4: Tumour cells H460 treated with the adenovirus AdCMV gax at MOI 1000 (Figure 4A), MOI 100 (Figure 4B) and MOI 1000 (Figure 4C).

Figure 5: Representation of the effect of AdCMV gax on cell proliferation in human tumour cells Saos.

Figure 6: Representation of the effect of the expression of AdCMV gax on cell proliferation in human tumour cells H358.

GENERAL MOLECULAR BIOLOGY TECHNIQUES The methods conventionally used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in caesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, phenol or phenol-chloroform extraction of proteins, ethanol or isopropanol precipitation of DNA in saline medium, transformation in Escherichia coli, and the like, are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley Sons, New York, 1987].

The pBR322 and pUC type plasmids and the phages 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 agarose or acrylamide gel electrophoresis, extracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the supplier.

The filling of the protruding 5' ends can be performed with the Klenow fragment of E. coli DNA polymerase I (Biolabs) according to the specifications of the supplier. The destruction of the protruding 3' ends is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5' ends is performed by a controlled treatment with S1 nuclease.

Site-directed mutagenesis in vitro by synthetic oligodeoxynucleotides can be performed 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 so-called PCR technique [Polymerase-catalysed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona Meth. Enzym.

155 (1987) 335-350] can be performed using a DNA thermal cycler (Perkin Elmer Cetus) according to the specifications of the manufacturer.

The verification of the nucleotide sequences can be performed by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463- 5467] using the kit distributed by Amersham.

EXAMPLE 1: Construction of the vector pXL-CMV-GaxHA carrying the gene encoding the rat GAX protein under the control of the CMV promoter.

This example describes the construction of a vector containing the cDNA encoding the GAX protein (species: rat) and adenoviral sequences allowing recombination. The influenza virus haemagglutinin epitope (HA1 epitope), comprising 18 amino acids, is added to the N-terminal end of the GAX protein (Field et al., Mol. Cell. Biol. 8: 2159-2165, 1988). This process of adding epitope makes it possible to monitor, especially by immunofluorescence techniques, the expression of gax with the aid of antibodies directed against the HA1 epitope. In addition to its sensitivity, this method makes it possible to get rid of the background noise corresponding to the expression of endogenous GAX proteins both in vitro and in vivo.

1.1. Construction of plasmid pCOl (Figure 1) A Construction of the plasmid pCE The EcoRI-XbaI fragment corresponding to the left end of the adenovirus Ad5 genome was first cloned between the EcoRI and XbaI sites of the vector pIC19H.

This generates the plasmid pCA. The plasmid pCA was then cut with HinfI, its protruding 5' ends were filed with the Klenow fragment of DNA polymerase I of E.coli, and then it was cut with EcoRI. The fragment thus generated from the plasmid pCA which contains the left end of the Ad5 adenovirus genome was then cloned between the EcoRI and SmaI sites of the vector (Marsh et al., Gene 32 (1984) 481). This generates the plasmid pCB. The plasmid pCB was then cut with EcoRI, its protruding 5' ends were filled with the Klenow fragment of DNA polymerase I of E. coli, and then it was cut with BamHI. The fragment thus generated from the plasmid pCB which contains the left end of the adenovirus genome was then cloned between the NruI and BglII sites of the vector pIC20H. This generates the plasmid pCE of which an advantageous characteristic is that it possesses the first 382 base pairs of the adenovirus followed by a multiple cloning site.

22 B Construction of the plasmid pCD' The Sau3A (3346) SstI (3645) fragment and the SstI (3645) NarI (5519) fragment of the adenovirus genome were first ligated and cloned between the Clal and BamHI sites of the vector generating the plasmid pPY53. The SalI-TaqI fragment of the plasmid pPY53, prepared from a dam- context, containing the part of the Ad5 adenovirus genome between the Sau3A (3346) and TaqI (5207) sites was then cloned between the SalI and the Clal sites of the vector pIC20H, generating the plasmid pCA'. The TaqI (5207) NarI (5519) fragment of the Ad5 adenovirus genome, prepared from a dam- context, and the SalI-TaqI fragment of the plasmid pCA' were then ligated and cloned between the SalI and NarI sites of the vector This generates the plasmid pCC'. The NarI (5519) NruI (6316) fragment of the Ad5 adenovirus genome, prepared from a dam- context, and the SalI-NarI fragment of the plasmid pCC' were then ligated and cloned between the SalI and NruI sites of the vector This generates the plasmid pCD'.

C Construction of the plasmid pC01 A partial digestion with XhoI and then a complete digestion with SalI of the plasmid pCD' generates a restriction fragment which contains the adenovirus sequence, from the Sau3A (3446) site to the NruI (6316) site. This fragment was cloned into the 23 SalI site of the plasmid pCE. This generates the plasmid pCO1 (Figure which contains the left part of the Ad5 adenovirus up to the HinfI (382) site, a multiple cloning site and the Sau3A (3446) NruI (6316) fragment from the Ad5 adenovirus.

1.2. Construction of the vector pXL-CMV-GaxHA (cf. Figure 2) The gax cDNA was cloned between the XbaI-BamHI sites of the vector pCGN (Tanaka and Herr, Cell 60 375-386, 1990). The resulting vector pGCN-Gax contains the early promoter and the enhancer sequence of the cytomegalovirus (CMV) (-522, +72; Boshart et al., Cell, 41 521-530, 1985), the leader sequence of herpes simplex virus thymidine kinase including the initiation codon AUG as well as the first three amino acids +104; Rusconi and Yamamoto, EMBO 6 1309-1315, 1987), encoding the HA1 epitope, the rat gax cDNA and finally the polyadenylation sequence of the rabbit P-globin gene (Pabo et al, cell, 35 445-453, 1983).

The vector pCGN-Gax was then cut with XmnI and SfiI and the fragment obtained, containing the promoter, cDNA and polyadenylation sequence, previously treated with Klenow, was introduced at the EcoRV site of the shuttle vector pCO1 containing the adenoviral sequences necessary for the recombination. The plasmid obtained was designated pXL-CMV-GaxHA (cf. Figure 2).

EXAMPLE 2: Construction of the recombinant adenovirus Ad-CMVqax The vector pXL-CMV-GaxHA prepared in Example 1 was then linearized and co-transfected for recombination with a deficient adenoviral vector, into the helper cells (line 293) providing in trans the functions encoded by the adenovirus El regions (E1A and E1B).

The adenovirus Ad-CMVgax was obtained by homologous recombination in vivo between the adenovirus Ad.RSVpgal (Stratford-Perricaudet et al., J. Clin.

Invest 90 (1992) 626) and the vector pXL-CMV-GaxHA according to the following procedure: the vector pXL-CMV-GaxHA, linearized with the enzyme XmnI, and the adenovirus Ad.RSV3gal, linearized with Clal, were cotransfected into the 293 line in the presence of calcium phosphate so as to allow homologous recombination. The recombinant adenoviruses thus generated were selected by plaque purification. After isolation, the recombinant adenovirus is amplified in the 293 cell line, giving a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of about 101 0 pfu/ml.

The viral particles are purified by centrifugation on a caesium chloride gradient according to known techniques (see especially Graham et al., Virology 52 (1973) 456). The adenovirus Ad-CMVgax is stored at -80 0 C in 10 glycerol.

EXAMPLE 3: Control of the expression of Ad-CMVqax in human tumour cells H460 The human tumour cells H460, derived from a non-small cell lung cancer, were transduced by the recombinant adenovirus Ad-CMV gax and by a control virus expressing P-galactosidase (Ad-RSV-pgal) at increasing multiplicities of infection (MOI) (Figure After incubating for 1 hour 30 min in the presence of the adenoviral solution, the culture medium comprising 10 foetal serum is changed and the cells incubated for a period of 48 hours. The cell viability being checked by the trypan blue exclusion test, the number of viable cells per culture well is then determined. Two batches of adenovirus AdCMV gax, Ad-gax and Ad-gax(2), were used and correspond to two independent productions in the cells 293. The activities of the two batches were found to be comparable and quite distinct from the control adenovirus AdRSV pgal.

The use of Ad-RSV Pgal made it possible, beforehand, to demonstrate the capacity of a recombinant adenovirus to transduce efficiently the H460 cells. Thus, the nuclear Pgal activity was detected in more than 75 of the cells treated with Ad-RSV-Pgal (MOI 1000). In parallel, the expression of the GAX protein was detected in the cells transduced by Ad-CMV gax by immunofluorescence. The efficiencies of transduction of the Ad-CMV gax and Ad-RSV-Pgal viruses were found to be comparable. The treatment of the H460 cells with Ad-CMV gax is associated with a reduction in cell proliferation but also with a massive cytotoxicity detected 48 hours after the transduction by the adenovirus (cf. Figures 4A, 4B and 4C). Such an effect is not observed with the control adenovirus. These data therefore demonstrate that the overexpression of the GAX protein is accompanied by a stoppage of the growth of the H460 cells.

Advantageously, the H460 cells express a mutated ras protein (Ki-ras). Our data demonstrate, surprisingly, that the expression of the GAX protein makes it possible to reestablish the control of cell proliferation which was initially deregulated by the modified ras protein. The dominant character of the gax factor in relation to oncogenic mutations of ras is not restricted to lung carcinomas. Similar results, namely a stoppage of growth by AdCMV gax, were obtained on the HCT116 cells derived from a colon carcinoma. A mutation of the ras oncogene (G13C) and of the DCC gene have been described in this cell line (Brattain et al., Cancer Research, 41:1751-1756, 1981).

EXAMPLE 5: Control of the expression of Ad-CMV-qax in human tumour cells with a null p53 environment The H358 cells, also derived from a non-small cell lung carcinoma, constitute a deficient model for the p53 protein. This cell line has, indeed, a homozygous deletion for the p53 gene (Maxwell and Roth, Oncogene 8 3421, 1993). Advantageously, the Ad-gax adenovirus effectively blocks the growth of the H358 cells and causes a massive cytotoxicity 48 hours after the adenoviral transduction (Figure This cell death is not detected in the presence of a control adenovirus Ad-RSV-pgal. These data demonstrate that the overexpression of gax blocks specifically and effectively the growth of tumour cells deficient for p53. Advantageously, these data are not limited to the cells derived from lung carcinomas. Indeed, the growth of the Saos human cells, derived from an osteosarcoma, is also blocked after treatment with Ad-CMV gax. The Saos cells constitute a cellular model with null p53 and pRb environment. Again, this growth arrest is dependent on the concentration of virus used and is not observed after transduction of the cancer cells by the control virus (Figure Thus, more than 90 of the Saos-2 cells are positive for the 3gal activity after induction by AdRSV pgal at a multiplicity of infection of 250. Under the same experimental conditions (multiplicity of infection 250), AdCMV gax causes a reduction in cell viability greater than 70 Gax having initially been described as a growth arrest gene, the applicant observed, surprisingly, that the growth arrest is followed by a cell death which resembles apoptosis.

The compensation in vivo for the cell death P:OPER\TDO\74982-96 ca.doc-22/1200 -28induced by the overexpression of gax may, advantageously, be associated with a therapeutic benefit in the cancer patient.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

eo

Claims (18)

1. Use of the GAX protein or of a variant thereof which is capable of inhibiting cell proliferation induced by the ras protein, for the preparation of a pharmaceutical composition for the treatment of cancer.

2. Use of at least one nucleic sequence encoding either the GAX protein or a variant as defined in claim 1, for the preparation of a pharmaceutical composition for the treatment of cancer.

3. Use according to claim 2, characterized in that the :nucleic sequence is used in a form complexed with DEAE- dextran, with receptor proteins, or with lipids or cationic polymers, in the form of liposomes or alternatively as it is.

4. Use according to claim 2, characterized in that the nucleic sequence is part of a recombinant viral vector.

5. Use according to claim 4, characterized in that the vector is chosen from adenoviruses, retroviruses and adeno-associated viruses.

6. Use according to claim 4 or 5, characterized in that an e. adenovirus is involved. oe

7. Use according to claim 6, wherein the adenovirus is of the Ad5 or Ad2 type.

8. Use according to claim 4, 5 or 6, characterized in that an adenovirus of animal origin is involved.

9. Use according to claim 8, wherein the adenovirus is of canine origin. Use according to any one of claims 2 to 9, characterized in that the nucleic sequence used encodes the rat GAX protein or a variant thereof.

P: aprtdo\74982-96 la.doc.28/12/U0

11. Use according to claim 10, characterized in that the nucleic sequence used encodes the rat GAX protein or its human homologue.

12. Use according to any one of claims 2 to 11, characterized in that the nucleic sequence used is a cDNA.

13. Use according to any one of claims 2 to 11, characterized in that the nucleic sequence used is a gDNA.

14. Use according to any one of claims 4 to 13, characterized in that the nucleic sequence used comprises sequences allowing its expression in the infected cell.

Use according to any one of claims 4 to 14, characterized in that the nucleic sequence used comprises a signal sequence which directs the synthesized polypeptide in the secretory pathways of the target cell.

16. Method of treating cancer comprising administering a GAX protein or variant as defined in claim 1 or a nucleic sequence as defined in any one of claims 2 to S.

17. Use according to claim 1 or 2 substantially as hereinbefore described in any one of the Examples.

18. Method according to claim 16 substantially as hereinbefore described in any one of the Examples. DATED this 27th day of December, 2000. RHONE-POULENC RORER S.A. A 5 by its Patent Attorneys DAVIES COLLISON CAVE