AU774391B2 - Composition designed for implementing an antitumoral or antiviral treatment in a mammal - Google Patents

Description

Composition designed for implementing an antitumoral or antiviral treatment in a mammal The present invention relates to a cytotoxic composition comprising a first nucleic acid sequence encoding all or part of the p 5 3 protein and a second nucleic acid sequence encoding all or part of a polypeptide having at least a cytotoxic, in particular antitumor or antiviral, activity. The present invention is particularly useful in the context of carrying out a gene therapy treatment of proliferative or infectious diseases.

In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item S 10 of knowledge or any combination thereof was at the priority date: S part of common general knowledge; or known to be relevant to an attempt to solve any problem with which this specification is concerned.

p53 is a nuclear phosphoprotein involved in particular in controlling the expression of the proteins involved in the cell cycle (Ozbun et al., 1995, Adv. Cancer Res.

66, 71-141 Selter et al., 1994, Int. J. Biochem. 26, 145-154) and participating in numerous cellular processes linked to the stability of the genome and to cellular apoptosis (Harris et al., 1996, J. Natl. Cancer Inst. 88, 1442-1445; Kastan et al., 1991, Cancer Res. 51, 6304-6311; Kuerbitz et al., 1992, PNAS, 89, 7491-7495).

S 20 The p 5 3 gene has been identified and sequenced. The sequence of the cDNA is described in Matlashewski et al., 1984, EMBO 3, 3257-3262 and that of the protein in Lamp, 1986, Mol. Cell Biol., 6, 1379-1385. Likewise, natural and functional polymorphic variants have been identified for which certain amino acids are replaced by others without, however, affecting the p53 function. Moreover, numerous mutations have been described in the literature relating to cancer which can result in a loss of the function of this protein (Holstein et al., 1991, Science, 253, 49-53; Levine et al., 1991, Nature, 351, 453-456). For example, Baker et al. (1989, Science, 244, 217) have observed that in more than 70% of colorectal tumors, the function of this p53 gene is lost.

2 Moreover, several in vitro studies have shown that the restoration of the p53 activity in cells deficient for this activity makes it possible to suppress cell growth or to induce apoptosis of the cells (Baker et al., 1990, Science, 249, 912-915; Shaw et al., 1992, PNAS, 89, 4495-4499). Similarly, several studies have confirmed that it is possible to suppress in vivo the growth of tumor cells by applying a therapy aimed at restoring the activity of the defective p53 gene (Fujiwara et al., 1994, J. Natl. Cancer Inst. 86, 1458-1462; Wills et al., 1994, Hum. Gene Therapy, 1079-1088; Hamada et al., 1996, Cancer Res. 56, 3047- 3054) In addition, following the work by Lowe et al.

(1993, Cell 74, 957-967, 1994, Science 266, 807-810) demonstrating an increased resistance of tumor cells not expressing the p53 gene toward various types of cytotoxic agents and radiation, the use of the functional p53 protein, or of its gene, was envisaged in order to develop a method for sensitizing tumor cells to said agents. More particularly, it has been shown that the transfection of a human colon tumor cell whose p53 gene has been made inactive by mutation with a plasmid expressing the wild-type p53 gene makes it possible in vitro to sensitize this cell to (5-FU) (Yang et al., 1996, Clin. Cancer Res. 2, 1649-1657) We have now identified novel cytotoxic compositions in which the various constituents are chosen so as to obtain a synergistic effect of their respective activities and improved properties of said constituents. More particularly, such compositions make it possible to inhibit or to slow down cell proliferation by inducing the specific death of the tumor cells, a better presentation of the antigens and/or a stimulation of the immune cells of the host organism. The present invention offers an advantageous and effective alternative to the prior art techniques, in particular for treating cancer in humans or animals.

3 The invention relates, in the first instance, to a composition intended for carrying out an antitumor or antiviral treatment, or any applications requiring cell death, in a mammal comprising: a nucleic acid sequence encoding all or part of the p53 polypeptide, (ii) at least one nucleic acid sequence encoding all or part of a polypeptide having at least a cytotoxic activity, said nucleic acid sequences being placed under the control of the elements necessary for their expression in a host cell of said mammal.

In the context of the present invention, it is possible to use in the entire nucleic acid sequence encoding the p53 polypeptide or only part of this polypeptide, or a derived or mutated polypeptide, in so far as the p53 function is conserved. Such sequences are well known to persons skilled in the art and reference may be made for example to Matlashewski et al., 1984, EMBO 3, 3257-3262, Prives et al., 1994, Cell, 78, 543-546 or Chen et al., 1996, Gene et Deve., 2438-2451, whose contents are incorporated into the present application.

The expression "polypeptide having at least a cytotoxic activity" is understood to mean any peptide substance capable of inducing or of activating an immune response directed specifically against a tumor cell (the cytotoxic activity is then called antitumor activity) or a cell infected by a virus (the cytotoxic activity is then called antiviral activity) or of inhibiting the growth and/or the division of such a tumor or infected cell. According to a preferred case, said cytotoxic activity results in the death of said cell.

Given the properties of the p53 polypeptide as transcriptional transactivator (Farmer et al., 1992, Nature, 358, 83-86) or as a polypeptide capable of interacting with other proteins (Harris, 1996, Carcinogenesis, 17, 1187-1198), the p53 activity can be 4 measured by analyzing the arrest of the cell cycle in the Gl/S and G2/M phase, the induction of apoptosis, the suppression of cell transformation induced by oncogenes or the inhibition of angiogenesis.

The cytotoxic activity of a given polypeptide, in particular an antitumor activity, may be evaluated in vitro by measuring cell survival either by tests of short term viability (such as for example the tryptan blue or MTT test), or by tests of clonogenic survival (formation of colonies) (Brown and Wouters, 1999, Cancer Research, 59, 1391-1399) or in vivo by measuring the growth of tumors (size and/or volume) in an animal model (Ovejera and Houchens, 1981, Semin. Oncol., 8, 386-393).

According to a first variant, the invention relates to a composition, characterized in that said polypeptide having a cytotoxic activity is chosen from cytokines, proteins encoded by a gene called "suicide gene" and antiangiogenic protein factors.

More particularly, when said polypeptide in (ii) is a cytokine, it is preferably a cytokine chosen from interferons a, P and y, interleukins, and in particular IL-2, IL-4, IL-6, IL-10 or IL-12, tumor necrosis factors (TNF) and colony stimulating factors (GM-CSF, C-CSF, M-CSF and the like).

According to a preferred embodiment, said cytokine is selected from interleukin-2 (IL-2) and interferon-gamma (IFN-y). Interleukin-2 is in particular responsible for the proliferation of the activated T lymphocytes, the multiplication and the activation of the cells of the immune system (for the nucleic acid sequence see in particular FR 85 09480).

IFN-y activates the phagocytic cells and increases the expression of the class I and II surface antigens of the major histocompatibility complex (for the nucleic acid sequence see in particular FR 85 09225).

According to a second variant, the invention also relates to such a composition, characterized in that said polypeptide in (ii) exhibits at least an 5 enzymatic activity selected from thymidine kinase activity, purine nucleoside phosphorylase activity, guanine or uracil or orotate phosphoribosyl transferase activity and cytosine deaminase activity.

Several studies have made it possible to identify polypeptides which are not toxic as such but which exhibit enzymatic catalytic properties capable of converting an inactive substance (prodrug), for example a nucleoside or a nucleoside analog, to a substance which is highly toxic for the cell, for example a modified nucleoside which may be incorporated into the DNA or RNA genes undergoing extension, with, as a consequence, in particular the inhibition of cell division or cellular dysfunctions leading to the death of the cell containing such polypeptides. The genes encoding such polypeptides are termed "suicide genes".

Numerous suicide gene/prodrug pairs are currently available. There may be mentioned more particularly the pairs: herpes simplex virus type I thymidine kinase (HSV-1 TK) and acyclovir or ganciclovir (GCV) (Caruso et al., 1993, Proc. Natl. Acad. Sci. USA 90, 7024-7028; Culver et al., 1992, Science 256, 1550-1552; Ram et al., 1997, Nat. Med. 3, 1354-1361); rat cytochrome p450 and cyclophosphophamide (Wei et al., 1994, Human Gene Therapy 5, 969-978); purine nucleoside phosphorylase from Escherichia coli coli) and 6-methylpurine deoxyribonucleoside (Sorscher et al., 1994, Gene Therapy 1, 233-238); guanine phosphoribosyl transferase from E. coli and 6-thioxanthine (Mzoz and Moolten, 1993, Human Gene Therapy 4, 589-595) and cytosine deaminase (CDase) and cytosine More particularly, CDase is an enzyme which is involved in the pyrimidine metabolic pathway through which exogenous cytosine is converted to uracil by means of a hydrolytic deamination. CDase activities 6 have been demonstrated in prokaryotes and lower eukaryotes (Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615; Beck et al., 1972, J. Bacteriol. 110, 219-228; De Haan et al., 1972, Antonie van Leeuwenhoek 38, 257- 263; Hoeprich et al., 1974, J. Inf. Dis. 130, 112-118; Esders and Lynn, 1985, J. Biol. Chem. 260, 3915-3922) but they are absent from mammals (Koechlin et al., 1966, Biochem Pharmacol. 15, 435-446; Polak et al., 1976, Chemotherapy 22, 137-153). The Saccharomyces cerevisiae cerevisiae) FCY1 and E. coli codA genes encoding respectively the CDase of these two organisms are known and their sequences have been published (EP 402 108; Erbs et al., 1997, Curr. Genet. 31, 1-6; WO93/01281).

CDase also deaminates a cytosine analog, to 5-fluorouracil (5-FU) which is a highly cytotoxic compound in particular when it is converted to 5-fluoro-UMP (5-FUMP). Cells lacking CDase activity, either because of an inactivating mutation of the gene encoding the enzyme, or because of their natural deficiency in this enzyme (for example mammalian cells) are resistant to 5-FC (Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615; Kilstrup et al., 1989, J. Bacteriol. 1989 171, 2124-2127). On the other hand, it has been shown that it is possible to transmit the sensitivity to 5-FC to mammalian cells to which the sequence encoding a CDase activity has been transferred (Huber et al., 1993, Cancer Res. 53, 4619- 4626; Mullen et al., 1992, Proc. Natl. Acad. Sci. USA 89, 33-37; WO 93/01281). Furthermore, in this case, the nontransformed neighboring cells also become sensitive to 5-FC (Huber et al., 1994, Proc. Natl. Acad. Sci. USA 91, 8302-8306). This phenomenon, called bystander effect, is due to the excretion, by the cells expressing the CDase activity, of 5-FU which intoxicates the neighboring cells by mere diffusion across the cell membrane. This property of passive diffusion of 5-FU constitutes an advantage in relation to the tk/GCV reference system for which the bystander 7 effect requires contact with the cells which express tk (Mesnil et al., 1996, Proc. Natl. Acad. Sci. USA 93, 1831-1835). This effect consequently constitutes an additional advantage for the use of CDase in the context of gene therapy, in particular anticancer therapy.

However, the sensitivity to 5-FC varies a lot according to the cell lines. A low sensitivity is observed for example in human tumor lines PANC-1 (pancreas carcinoma) and SK-BR-3 (breast adenocarcinoma) transduced with a retrovirus expressing the E. coli codA gene (Harris et al., 1994, Gene Therapy 1, 170-175). This undesirable phenomenon could be explained by the absence or the low endogenous conversion of the 5-FU formed by the enzymatic action of CDase to cytotoxic 5-FUMP. This step, normally performed in mammalian cells by orotate phosphoribosyl transferase (Peters et al., 1991, Cancer 68, 1903- 1909), may be absent from certain tumors and thus make gene therapy, based on CDase, ineffective.

In prokaryotes and lower eukaryotes, uracil is converted to UMP by the action of uracil phosphoribosyl transferase (consequently exhibiting a UPRTase activity). This enzyme also converts 5-FU to Thus, furl mutants of the yeast S. cerevisiae are resistant to high concentrations of 5-FU (10 mM) and of (10 mM) because in the absence of UPRTase activity, the 5-FU, resulting from the deamination of by CDase, is not converted to cytotoxic (Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615) The upp and FUR1 genes encoding the UPRTase of E. coli and of S. cerevisiae respectively have been cloned and sequenced (Andersen et al., 1992, Eur. J. Biochem. 204, 51-56; Kern et al., 1990, Gene 88, 149-157).

For the purposes of the present invention, a polypeptide having a UPRTase activity designates a polypeptide capable of converting uracil or one of its derivatives to a monophosphate-containing analog and, in particular, 5-FU to 5-FUMP. The expression 8 "mutation" should be understood to mean the addition, deletion and/or substitution of one or more residues at any site of said polypeptide.

The native UPRTase in question in the present invention may be of any origin, in particular of prokaryotic, fungal or yeast origin. By way of illustration, the nucleic acid sequences encoding the UPRTases from E. coli (Anderson et al., 1992, Eur. J.

Biochem 204, 51-56), from Lactococcus lactis (Martinussen and Hammer, 1994, J. Bacteriol. 176, 6457- 6463), from Mycobacterium bovis (Kim et al., 1997, Biochem Mol. Biol. Int 41, 1117-1124) and from Bacillus subtilis (Martinussen et al., 1995, J. Bacteriol. 177, 271-274) may be used in the context of the invention.

However, it is most particularly preferred to use a yeast UPRTase and in particular that encoded by the S. cerevisiae FUR1 gene whose sequence disclosed in Kern et al. (1990, Gene 88, 149-157) is introduced here by way of reference. As a guide, the sequences of the genes and those of the corresponding UPRTases may be found in the literature and the specialist databanks (SWISSPROT, EMBL, Genbank, Medline and the like).

Moreover, application PCT/FR99/00904 describes an FUR1 gene lacking 105 nucleotides in 5' of the coding part allowing the synthesis of a UPRTase from which the 35 first residues have been deleted at the N-terminal position and starting with the methionine at position 36 in the native protein. The product of expression of the mutant gene, designated FUR1A105, is capable of complementing an furl mutant of S. cerevisiae. In addition, the truncated mutant exhibits a higher UPRTase activity than that of the native enzyme. Thus, according to a particularly advantageous embodiment, the polypeptide encoded according to the invention is a deletion mutant of a native UPRTase. The deletion is preferably located in the N-terminal region of the original UPRTase. It may be complete (affecting all the residues of said N-terminal region) or partial (affecting one or more 9 continuous or discontinuous residues in the primary structure). In general, a polypeptide consists of N-terminal, central and C-terminal parts, each representing about a third of the molecule. For example, since the S. cerevisiae UPRTase has 251 amino acids, its N-terminal part consists of the first 83 residues starting with the so-called initiator methionine situated at the first position of the native form. As for the E. coli UPRTase, its N-terminal part covers positions 1 to 69.

Thus, most preferably, the polypeptide according to PCT/FR99/00904 is derived from a native UPRTase at least by deletion of all or part of the N-terminal region upstream of the second ATG codon of said native UPRTase. The complete deletion of the abovementioned region is preferred. For example, the UPRTase encoded by the FUR1 gene comprises a first ATG codon (initiator ATG codon) at position +1 followed by a second at position +36. Thus, the deletion of residues +1 to 35 may be envisaged in the context of the present invention, giving a polypeptide starting at the methionine normally found at position +36 of the native form.

A preferred polypeptide according to PCT/FR99/00904 comprises an amino acid sequence substantially as represented in the sequence identifier SID NO: 1, starting with the Met residue at position 1 and ending with the Val residue at position 216. The term "substantially" refers to a degree of identity with said sequence SID NO: 1 greater than advantageously greater than 80%, preferably greater than 90%, and most preferably greater than 95%. More preferably still, it comprises the amino acid sequence represented in the sequence identifier SID NO: 1. As mentioned above, it may comprise additional mutations.

There may be mentioned in particular the substitution of the serine residue at position 2 (position 37 in the native UPRTase) by an alanine residue.

10 In addition, patent applications W096/16183 and PCT/FR99/00904 describe the use of a fusion protein encoding an enzyme with two domains having the CDase and UPRTase activities and demonstrate that the transfer of a hybrid gene codA::upp or FCY1::FUR1 or FCY1::FUR1A105 carried by an expression plasmid increases the sensitivity of the transfected B16 cells to 5-FC. The protein and nucleic sequences described in these two applications are incorporated into the description of the present application.

According to another embodiment, the polypeptide according to PCT/FR99/00904 is a fusion polypeptide in which it is fused in phase with at least a second polypeptide. Although the fusion can take place at any site of the first polypeptide, the N- or C-terminal ends are preferred and in particular the N-terminal end. Advantageously, the fusion in phase uses a second polypeptide having a cytosine deaminase (CDase) activity and which is derived from a native cytosine deaminase, such that the fusion polypeptide according to the invention exhibits the CDase and UPRTase activities. An FCY1::FUR1 fusion is preferred.

Such a bifunctional polypeptide makes it possible to improve the sensitivity of the target cells to 5-FC and to 5-FU. Preferably, the second polypeptide according to the invention is capable of metabolizing 5-FC to According to PCT/FR99/00904, a CDase of prokaryotic or lower eukaryotic origin is used. More preferably still, it is a yeast CDase, and in particular that encoded by the Saccharomyces cerevisiae FCY1 gene. The cloning and the sequencing of the genes encoding CDases from various sources are available in the literature and specialist databanks. It should be mentioned that the sequence of the FCY1 gene is disclosed in Erbs et al. (1997, Curr. Genet. 31, 1-6).

It is of course possible to use a mutant of CDase having a conversion capacity which is comparable to or greater than that of the native enzyme. Persons skilled 11 in the art are capable of cloning the CDase sequences from the published data and of carrying out possible mutations, of testing the enzymatic activity of the mutant forms in an acellular or cellular system according to the prior art technology or based on the protocol indicated below, and of fusing in phase the polypeptides with CDase and UPRTase activity.

A preferred example is a polypeptide comprising an amino acid sequence substantially as represented in the sequence identifier SID NO: 2, starting with the Met residue at position 1 and ending with the Val residue at position 373. The term "substantially" has the definition given above. A polypeptide comprising the amino acid sequence as represented in the sequence identifier SID NO: 2 is most particularly suitable for carrying out the invention.

A fusion of the CDase and UPRTase activities makes it possible to improve the sensitivity of the target cells to 5-FC and to Persons skilled in the art are capable of cloning the CDase or UPRTase sequences from the published data and of carrying out possible mutations, of testing the enzymatic activity of the mutant forms in an acellular or cellular system according to the prior art technology or based on the protocol indicated in application PCT/FR99/00904, and of fusing, in particular in phase, the polypeptides with CDase and UPRTase activity, and consequently all or part of the corresponding genes.

In general, a polypeptide according to the invention may be produced by conventional methods of chemical synthesis or by recombinant DNA techniques (see for example Maniatis et al., 1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY). Thus, according to the method of preparation described in PCT/FR99/00904, a nucleotide sequence encoding said polypeptide is introduced into a cell in order to generate a transformed cell, said transformed cell is cultured under appropriate 12 conditions in order to allow the production of said polypeptide, and said polypeptide is harvested from the cell culture. The producing cell may be of any origin and, without limitation, a bacterium, a yeast or a mammalian cell, insofar as the nucleotide sequence considered is either integrated into its genome or integrated into an appropriate expression vector capable of replicating. Of course, the nucleotide sequence is placed under the control of transcriptional and translational signals allowing its expression in the producing cell. Expression vectors and control signals are known to persons skilled in the art. As for the polypeptide, it may be recovered from the medium or from the cells (after lysis thereof) and subjected to conventional purification steps (by chromatography, electrophoresis, filtration, immunopurification and the like) PCT/FR99/0904 [sic] also describes a nucleotide sequence encoding, a said polypeptide which may be a cDNA or genomic sequence or a sequence of the mixed type. It may optionally contain one or more introns, the latter being of native origin, heterologous origin (for example the intron of the rabbit P-globin gene and the like) or synthetic origin in order to increase expression in the host cells. As already indicated, said sequence may encode a polypeptide derived from the native enzyme or a mutant exhibiting a comparable or improved activity. The sequences used may be obtained by conventional molecular biology techniques, for example by screening a library using specific probes, by immunoscreening an expression library, by PCR using suitable primers or by chemical synthesis. The mutants may be generated from native sequences by substitution, deletion and/or addition of one or more nucleotides using the techniques of site-directed mutagenesis, PCR, digestion with restriction enzymes and ligation or by chemical synthesis. The functionality of the mutants and of the constructs may be verified.by assaying the 13 enzymatic activity or by measuring the sensitivity of target cells to 5-FC and/or F-FU.

Consequently, according to a specific case, the composition of the invention is characterized in that the nucleic acid sequence (ii) is selected from the nucleic sequences of the CodA, upp, FUR1, FCY1 and FUR1A105 genes, or using a combination of all or part of said sequences.

The invention relates more particularly to said composition, characterized in that said polypeptide in (ii) exhibits at least a CDase activity and a UPRTase activity.

The expression "combination of nucleic acid sequences" is intended to designate both distinct sequences which encode at least two distinct polypeptides and fused sequences which encode fusion polypeptides, it being understood that the production of such polypeptides may be carried out under the control of the same regulatory elements (polycistronic cassette) or of independent elements, which are identical or different, homologous or heterologous with respect to the vector containing them, constitutive or inducible.

According to a particular embodiment, the composition of the invention comprises at least one nucleic acid sequence (ii) encoding a fusion polypeptide in which a first polypeptide exhibiting a UPRTase or CDase activity is fused in phase with at least a second polypeptide, said second polypeptide exhibiting a CDase or UPRTase activity, respectively.

More particularly, such a polypeptide is characterized in that the fusion with the second polypeptide is carried out at the N-terminal end of said first polypeptide.

According to a preferred case, said composition is characterized in that the nucleic acid sequence encoding said fusion polypeptide is a hybrid sequence comprising: 14 a first nucleic acid sequence encoding a first polypeptide exhibiting a UPRTase or CDase activity, a second nucleic acid sequence encoding a second polypeptide exhibiting a CDase or UPRTase activity, respectively.

Such a hybrid nucleic acid sequence encoding said fusion polypeptide may in addition contain an IRES-type sequence.

The invention relates in particular to such a composition for which the first nucleic acid sequence is selected from upp, FUR1 and FURlA105, and in that the second nucleic acid sequence is selected from CodA and FCY1, and vice versa. Most preferably, such a hybrid nucleic acid sequence is chosen from the hybrid sequences described in patent applications W096/16183 and PCT/FR99/00904.

According to a third variant, the composition according to the present invention is characterized in that said polypeptide having a cytotoxic activity (ii) is an antiangiogenic protein factor. Angiogenesis is the process responsible for the formation of new capillaries from the vascular network which already exists. This complex process is finely regulated in healthy tissues by the balance of the effects of numerous angiogenic and antiangiogenic factors.

However, in certain pathological conditions, and in particular during the formation of a tumor, this process is deregulated: the angiogenic factors become dominant over the antiangiogenic factors, which allows considerable vascularization of the tumors and, consequently, their rapid development and/or the appearance of metastases. Accordingly, in the context of the present invention, an antiangiogenic factor is considered to be a cytotoxic, in particular antitumor, agent. Among the different antiangiogenic factors known to date, there may be mentioned in particular angiostatin, endostatin, platelet factor PF4, thrombospondin-l, PRP (for Proliferin Related Protein), VEGI (for Vascular Endothelial Growth Inhibitor) and urokinase.

The nucleic acid sequences or (ii) may be easily obtained by cloning, by PCR or by chemical synthesis according to the conventional techniques in use. They may be native genes or genes derived from the latter by mutation, deletion, substitution and/or addition of one or more nucleotides. Moreover, their sequences are widely described in the literature which can be consulted by persons skilled in the art.

The present invention also relates to a composition as presented above, characterized in that said nucleic acid sequences and (ii) are inserted into a recombinant vector of plasmid or viral origin, and to such a recombinant vector carrying such nucleotide sequences placed under the control of the elements necessary for their expression in a host cell.

More particularly, the compositions of the invention may comprise said nucleic acid sequences (i) and (ii) inserted into the same recombinant vector or into distinct recombinant vectors.

The expression "recombinant vector" according to the invention is understood to mean a vector of plasmid or viral origin, and optionally such a vector combined with one or more substances improving the transfectional efficiency and/or the stability of said vector and/or the protection of said vector in vivo toward the immune system of the host organism. These substances are widely documented in the literature which is accessible to persons skilled in the art (see for example Felgner et al., 1987, Proc. West.

Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry 5, 647-654). By way of illustration but without limitation, they may be polymers, lipids, in particular cationic lipids, liposomes, nuclear or viral proteins or neutral lipids.

These substances may be used alone or in combination.

Examples of such compounds are in particular available 16 in patent applications WO 98/08489, WO 98/17693, WO 98/34910, WO 98/37916, WO 98/53853, EP 890362 or WO 99/05183. A combination which may be envisaged is a plasmid recombinant vector combined with cationic lipids (DOGS, DC-CHOL, spermine-chol, spermidine-chol and the like) and neutral lipids (DOPE).

The choice of the plasmids which can be used in the context of the present invention is vast. They may be cloning and/or expression vectors. In general, they are known to a person skilled in the art and a number of them are commercially available, but it is also possible to construct them or to modify them by genetic engineering techniques. There may be mentioned, by way of examples, the plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene) or p Poly (Lathe et al., 1987, Gene 57, 193-201). Preferably, a plasmid used in the context of the present invention contains a replication origin ensuring the initiation of replication in a producing cell and/or a host cell (for example, the ColE1 origin may be selected for a plasmid intended to be produced in E. coli and the oriP/EBNAl system may be selected if it is desired for it to be self-replicating in a mammalian host cell, Lupton and Levine, 1985, Mol.

Cell. Biol. 5, 2533-2542; Yates et al., Nature 313, 812-815). It may in addition comprise a selection gene which makes it possible to select or identify the transfected cells (complementation of an auxotrophic mutation, gene encoding resistance to an antibiotic and the like). Of course, it may comprise additional elements improving its maintenance and/or its stability in a given cell (cer sequence which promotes the monomeric maintenance of a plasmid (Summers and Sherrat, 1984, Cell 36, 1097-1103, sequences for integration into the cell genome).

As regards a viral vector, it is possible to envisage a vector derived from a poxvirus (vaccinia virus, in particular MVA, canarypox and the like), from an adenovirus, from a retrovirus, from a herpesvirus, 17 from an alphavirus, from a foamy virus or from an adeno-associated virus. A nonreplicative and nonintegrative vector will preferably be used. In this regard, the adenoviral vectors are most particularly suitable for carrying out the present invention.

However, it should be noted here that, in the context of carrying out the present invention, the nature of the vector is of little importance.

Retroviruses have the property of infecting and of becoming predominantly integrated into dividing cells and, in this regard, are particularly appropriate for cancer application. A recombinant retrovirus according to the invention generally comprises the LTR sequences, an encapsidation region and the nucleotide sequence according to the invention placed under the control of the retroviral LTR or of an internal promoter such as those described below. It may be derived from a retrovirus of any origin (murine, primate, feline, human and the like) and in particular from MoMuLV (Moloney murine leukemia virus), MVS (Murine sarcoma virus) or Friend murine retrovirus (Fb29). It is propagated in an encapsidation line capable of providing en trans the viral polypeptides gag, pol and/or env which are necessary for the constitution of a viral particle. Such lines are described in the literature (PA317, Psi CRIP GP AM-12 and the like). The retroviral vector according to the invention may comprise modifications, in particular in the LTRs (replacement of the promoter region with a eukaryotic promoter) or in the encapsidation region (replacement with a heterologous encapsidation region, for example of the VL30 type) (see French applications 94 08300 and 97 05203).

It will also be possible to use an adenoviral vector which is replication-defective, that is to say which lacks all or part of at least one region essential for replication which is selected from the El, E2, E4 and regions. A deletion of the El region is preferred. However, it may be combined with 18 other modification(s)/deletion(s) affecting in particular all or part of the E2, E4 and/or regions insofar as the defective essential functions are complemented en trans by means of a complementation line and/or of a helper virus in order to ensure the production of the viral particles of interest. In this regard, the second generation of vectors of the state of the art may be used (see for example international applications WO 94/28152 and WO 97/04119). By way of illustration, the deletion of the majority of the El region and of the E4 transcription unit is most particularly advantageous. With the aim of increasing cloning capacities, the adenoviral vector may also lack all or part of the nonessential E3 region. According to another alternative, it is possible to use a minimal adenoviral vector retaining the sequences essential for encapsidation, namely the 5' and 3' ITRs (Inverted Terminal Repeat) and the encapsidation region.

Moreover, the origin of the adenoviral vector according to the invention may be varied both from the point of view of the species and of the serotype. It may be derived from the genome of an adenovirus of human origin or of animal (canine, avian, bovine, murine, ovine, porcine or simian and the like) origin or from a hybrid comprising fragments of adenoviral genome of at least two different origins. There may be mentioned more particularly the adenoviruses CAV-1 or CAV-2 of canine origin, DAV of avian origin or Bad type 3 of bovine origin (Zakharchuk et al., Arch. Virol., 1993, 128; 171-176; Spibey and Cavanagh, J. Gen. Virol., 1989, 70: 165-172; Jouvenne et al., Gene, 1987, 21-28; Mittal et al., J. Gen. Virol., 1995, 76: 93-102). However, an adenoviral vector of human origin which is preferably derived from an adenovirus of serotype C, in particular of type 2 or 5, will be preferred. An adenoviral vector according to the present invention may be generated in vitro in Escherichia coli coli) by ligation or homologous recombination (see for example international 19 application WO 96/17070) or by recombination in a complementation line. The various adenoviral vectors and the techniques for their preparation are known (see for example Graham and Prevect, 1991, in Methods in Molecular Biology, Vol. 7, p. 109-128; Ed: E.J. Murey, The Human Press Inc).

The elements necessary for the expression consist of the set of elements allowing the transcription of the nucleotide sequence to RNA and the translation of the mRNA to a polypeptide, in particular the promoter sequences and/or regulatory sequences which are effective in said cell, and optionally the sequences required to allow the excretion or the expression at the surface of the target cells for said polypeptide. These elements may be regulatable or constitutive. Of course, the promoter is adapted to the vector selected and to the host cell. There may be mentioned, by way of example, the eukaryotic promoters of the genes PGK (Phospho Glycerate Kinase), MT (metallothionein; McIvor et al., 1987, Mol. Cell Biol.

7, 838-848), a-1 antitrypsin, CFTR, the promoters of the gene encoding muscle creatine kinase, actin pulmonary surfactant, immunoglobulin or 3-actin (Tabin et al., 1982, Mol. Cell Biol. 2, 416-436), SRa (Takebe et al., 1988, Mol. Cell. 8, 466-472), the SV40 virus (Simian Virus) early promoter, the RSV (Rous Sarcoma Virus) LTR, the MPSV promoter, the TK-HSV-1 promoter, the CMV virus (Cytomegalovirus) early promoter, the vaccinia virus promoters p7.5K pH5R, pK1L, p28, pll and the adenoviral promoters E1A and MLP or a combination of said promoters. It may also be a promoter which stimulates expression in a tumor or cancer cell. There may be mentioned in particular the promoters of the MUC-1 genes which is overexpressed in breast and prostate cancers (Chen et al., 1995, J. Clin. Invest.

96, 2775-2782), CEA (for carcinoma embryonic antigen) which is overexpressed in colon cancers (Schrewe et al., 1990, Mol. Cell. Biol. 10, 2738-2748), tyrosinose which is overexpressed in melanomas (Vile et al., 1993, 20 Cancer Res. 53, 3860-3864), ERB-2 which is overexpressed in breast and pancreatic cancers (Harris et al., 1994, Gene Therapy 1, 170-175) and -fetoprotein which is overexpressed in liver cancers (Kanai et al., 1997, Cancer Res. 57, 461-465). The Cytomegalovirus (CMV) early promoter is most particularly preferred. It is also possible to use a tissue-specific promoter region, in particular when the tumor to be treated is derived from a particular cell type, or is activable under defined conditions. The literature provides a large amount of information relating to such promoter sequences.

The elements necessary may, in addition, include additional elements which improve the expression of the nucleotide sequence according to the invention or its maintenance in the host cell. There may be mentioned in particular the intron sequences (WO 94/29471), secretion signal sequences, nuclear localization sequences, internal sites for reinitiation of translation of the IRES type, poly A sequences for termination of transcription.

According to a preferred embodiment, the invention relates more particularly to a recombinant vector, in particular a replication-defective adenoviral vector, comprising: a nucleic acid sequence encoding all or part of the p53 polypeptide, (ii) at least one nucleic acid sequence encoding all or part of a polypeptide having at least a cytotoxic activity, said nucleic acid sequences being placed under the control of the elements necessary for their expression in a host cell and being defined as indicated above.

The subject of the present invention is also a viral, in particular adenoviral, particle comprising a recombinant viral vector according to the invention.

Such a viral particle may be generated from a viral vector according to any conventional technique in the 21 field of the art. Its propagation is carried out in particular in a complementation cell suitable for the deficiencies of the vector. As regards an adenoviral vector, use may be made for example of a complementation line as described in application WO 94/28152, of the 293 line established from human embryonic kidney cells, which effectively complements the El function (Graham et al., 1977, J. Gen. Virol.

36, 59-72), the A549-El line (Imler et al., 1996, Gene Therapy 3, 75-84) or a line allowing double complementation (Yeh et al., 1996, J. Virol. 70, 559- 565; Krougliak and Graham, 1995, Human Gene Therapy 6, 1575-1586; Wang et al., 1995 Gene Therapy 2, 775-783; international application WO 97/04119). It is also possible to use helper viruses to at least partially complement the defection functions. The expression complementation cell is understood to mean a cell capable of providing en trans the early and/or late factors necessary for the encapsidation of the viral genome into a viral capsid in order to generate a viral particle containing the recombinant vector. Said cell may not, on its own, complement all the defective functions of the vector and, in this case, may be transfected/transduced with a helper virus/vector providing the complementary functions.

The invention also relates to a method for preparing a viral particle, according to which: a recombinant vector according to the invention is introduced into a cell, in particular a complementation cell capable of complementing en trans said vector, so as to obtain a so-called transfected cell, (ii) said transfected cell is cultured under appropriate conditions to allow the production of said viral particle, and (iii) said viral particle is recovered from the cell culture.

Of course, the viral particle may be recovered from the culture supernatant but also from the cells.

22 One of the methods commonly used consists in lysing the cells by successive freeze/thaw cycles in order to recover the virions from the lysis supernatant. These may be amplified and purified according to prior art techniques (chromatographic method, ultracentrifugation in particular through a cesium chloride gradient and the like).

The invention also relates to a eukaryotic host cell comprising the DNA fragments present in the composition according to the invention. Said host cell is advantageously a mammalian cell and, preferably, a human cell. It will preferably be a 293, LCA4 or PERC6 cell. Such a cell is in particular useful for producing the viral particles in a high titer, without generating replication-competent particles. The invention also relates to a host cell comprising a nucleotide sequence, a recombinant vector according to the invention or infected with a viral particle according to the invention. For the purposes of the present invention, a host cell consists of any cell which can be transfected with a recombinant vector or which can be infected with a viral particle, as defined above. A mammalian, and in particular human, cell is most particularly suitable. It may comprise said vector in a form integrated into the genome or otherwise (episome).

It may be a primary or tumor cell of any origin, in particular of hematopoietic (totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage and the like), muscle (satellite cell, myocyte, myoblast, smooth muscle cell and the like), cardiac, pulmonary, tracheal, hepatic, epithelial or fibroblast origin.

The invention also relates to a composition intended for carrying out an antitumor or antiviral treatment, or any application requiring cell death, in a mammal comprising: all or part of the P53 polypeptide, (ii) all or part of a polypeptide having at least a cytotoxic activity, 23 said polypeptides being defined as indicated above.

Another subject according to the invention consists in a formulation intended for carrying out an antitumor or antiviral treatment in a mammal, characterized in that it comprises a composition (based on nucleic acid or polypeptide as described above), an adenoviral vector or a viral particle according to the invention, as well as a pharmaceutically acceptable carrier. Such a carrier is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as for example a sucrose solution.

Moreover, such a carrier may contain any solvent, or aqueous or partially aqueous liquid such as nonpyrogenic sterile water. The pH of the formulation is, in addition, adjusted and buffered so as to meet the requirements of use in vivo. The formulation may also include a pharmaceutically acceptable diluent, adjuvant or excipient, as well as solubilizing, stabilizing and preserving agents. For injectable administration, a formulation in aqueous, nonaqueous or isotonic solution is preferred. It may be provided in a single dose or in a multidose in liquid or dry (powder, lyophilisate and the like) form which can be reconstituted at the time of use with an appropriate diluent.

According to a particular embodiment of the invention, said formulation comprises, in addition, pharmaceutically acceptable quantities of a prodrug capable of being converted to a cytotoxic molecule by a polypeptide having at least a cytotoxic activity.

Such a prodrug will be in particular selected from the group consisting of acyclovir or ganciclovir (GCV), cyclophosphophamide, 6-methylpurine deoxyribonucleoside, 6-thioxanthine, cytosine or one of its derivatives or uracil or one of its derivatives. Most preferably, said prodrug is 5-fluorocytosine (5FC) or 24 Moreover, in particular in the context of formulations containing a composition according to the second variant mentioned above, it should be noted that said formulation may also comprise one or more substances which potentiate the cytotoxic effect of There may be mentioned in particular the drugs inhibiting the enzymes of the pathway of the biosynthesis de novo of pyrimidines (for example those cited below), the drugs such as Leucovorin (Waxman et al., 1982, Eur. J. Cancer Clin. Oncol. 18, 685-692) which, in the presence of the product of the metabolism of 5-FU (5-FdUMP), increases the inhibition of thymidylate synthase which causes a decrease in the dTMP pool necessary for replication, and finally the drugs such as methotrexate (Cadman et al., 1979, Science 250, 1135-1137) which, by inhibiting dihydrofolate reductase and by raising the pool for incorporation of PRPP (phosphoribosyl pyrophosphate), causes an increase in 5-FU in the cellular RNA.

A formulation according to the invention is more particularly intended for the preventive or curative treatment of diseases by gene therapy and is intended more particularly for proliferative diseases (cancers, tumors, restenosis and the like) and for diseases of infectious origin, in particular of viral origin, for which it is necessary to limit the proliferation of the cells infected (induced by hepatitis B or C viruses, HIV, herpes, retroviruses and the like).

A formulation according to the invention may be manufactured conventionally for administration by the local, parenteral or digestive route. The routes of administration which may be envisaged are many. There may be mentioned, for example, the intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumor, intranasal, intrapulmonary or intratracheal route. For the latter three embodiments, administration by aerosol or instillation is advantageous. The administration may be made as a 25 single dose or repeated once or several times after a certain time interval. The appropriate route of administration and dosage vary as a function of various parameters, for example, of the individual, of the disease to be treated or of the gene(s) of interest to be transferred. The viral particle-based preparations according to the invention may be formulated in the form of doses of between 104 and 1014 pfu (plaqueforming units), advantageously 105 and 1013 pfu and, preferably, 106 and 1012 pfu. As regards the recombinant vector according to the invention, doses comprising from 0.01 to 100 mg of DNA, preferably 0.05 to 10 mg and, most preferably, 0.5 to 5 mg may be envisaged. A composition based on polypeptides preferably comprises from 0.05 to 10 g and, most preferably, from 0.5 to 5 g of said polypeptide. Of course, the doses may be adjusted by the clinician.

The present invention also relates to the therapeutic or prophylactic use of a composition, of a recombinant vector or of a viral particle according to the invention for the preparation of a medicament intended for the treatment of the human or animal body by gene therapy, in particular for the preparation of an antitumor or antiviral medicament intended to inhibit growth or to cause the rejection of a tumor or the death of an infected cell. According to a first possibility, the medicament may be administered directly in vivo (for example by intravenous injection, into an accessible tumor or at its periphery, into the lungs by aerosol, into the vascular system by means of an appropriate probe and the like). It is also possible to adopt the ex vivo approach which consists in collecting cells from the patient (bone marrow stem cells, peripheral blood lymphocytes, muscle cells and the like), transfecting or infecting them in vitro according to prior art techniques and readministering them to the patient. A preferred use consists in treating or preventing cancers, tumors and diseases resulting from an undesirable cell proliferation. Among 26 the applications which may be envisaged, there may be mentioned cancers of the breast, of the uterus (in particular those induced by papilloma viruses), of the prostate, of the lungs, of the bladder, of the liver, of the colon, of the pancreas, of the stomach, of the esophagus, of the larynx, of the central nervous system and of the blood (lymphomas, leukemia and the like). It is also useful in the context of cardiovascular diseases, for example for inhibiting or slowing down the proliferation of the smooth muscle cells of the vascular wall (restenosis). Finally, as regards infectious diseases, the application to AIDS may be envisaged.

It is moreover possible to envisage, where appropriate and without departing from the scope of the present invention, carrying out simultaneous or successive administrations, by different routes, of the various components contained in the pharmaceutical formulation or composition according to the invention.

The invention also extends to a method for the treatment of diseases by gene therapy, characterized in that a nucleotide sequence, a recombinant vector, a viral particle or a host cell according to the invention is administered to an organism or a host cell requiring such a treatment.

When the method of treatment uses a nucleotide sequence, a recombinant vector or a viral particle allowing the expression of a polypeptide according to the invention having a UPRTase activity, it may be advantageous to further administer a second nucleotide sequence encoding a second polypeptide exhibiting a CDase activity, said second nucleotide sequence being carried by said recombinant vector or viral particle or by a vector or an independent viral particle. In the latter case, the administration of the UPRTase and CDase sequences may be simultaneous or consecutive, the order of administration being unimportant.

According to an advantageous embodiment, the therapeutic use or the method of treatment also 27 comprises an additional step according to which pharmaceutically acceptable quantities of a prodrug, advantageously of a cytosine analog and, in particular, of 5-FC, are administered to the organism or the host cell. By way of illustration, a dose of 50 to 500 mg/kg/day may be used with a preference for 200 mg/kg/day. In the context of the present invention, the prodrug is administered according to standard practices, prior to, concomitantly with or subsequent to that of the therapeutic agent according to the invention. The oral route is preferred. It is possible to administer a single dose of prodrug or doses repeated over a sufficiently long period to allow the production of the toxic metabolite in the organism or host cell.

According to an advantageous embodiment of the invention, the therapeutic use or the method of treatment is combined with a second treatment of the patient by surgery (partial or complete removal of the tumor), by radiotherapy or chemotherapy. In this particular case, the treatment according to the invention is applied prior to, concomitantly with or subsequent to said second treatment. Preferably, this treatment will be applied subsequent to said second treatment.

The examples which follow are intended to illustrate the various subjects of the present invention and are consequently not limiting in character.

Figure 1 shows the in vitro antiproliferative effect of a buffer (Mock), of an empty adenovirus (Ad-null), of an adenovirus expressing FCU1 (AD-FCU1) or p53 (Ad-p53) or p53 and FCU1 (Ad-p53FCU1) on SW480 cells at an MOI of 1 in the absence of prodrug (100% corresponds to the viability of the noninfected cells).

Figure 2 shows the in vitro antiproliferative effect of Mock, of an empty adenovirus, of an adenovirus expressing FCU1 or p53 or p53 and FCU1 on B16FO cells at an MOI of 100 in the absence of prodrug 28 (100% corresponds to the viability of the noninfected cells).

Figure 3 shows the in vitro antiproliferative effect of Mock, of an empty adenovirus, of an adenovirus expressing FCU1 or p53 or p53 and FCU1 on LoVo cells at an MOI of 1 in the absence of prodrug (100% corresponds to the viability of the noninfected cells).

Figure 4 shows the sensitization, in the presence of various concentrations of 5FU, of the SW480 cells infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

Figure 5 shows the sensitization, in the presence of various concentrations of 5FU, of the B16FO cells infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

Figure 6 shows the sensitization, in the presence of various concentrations of 5FU, of the LoVo cells infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

Figure 7 shows the sensitization, in the presence of various concentrations of 5FC, of the [sic] cells infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

Figure 8 shows the sensitization, in the presence of various concentrations of 5FC, of the B16FO cells infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

29 Figure 9 shows the sensitization, in the presence of various concentrations of 5FC, of the LoVo cells infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

Figure 10 represents the survival rate for B6D2 mice into which B16FO tumor cells treated with various adenoviral compositions have been implanted.

Figure 11 shows the sensitization, in the presence of various concentrations of 5FC, of the SK-BR-3 (ATCC HTB-22) cells infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

Figure 12 shows the sensitization, in the presence of various concentrations of 5FC, of the T47-D cells (ATCC HTB-133) infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

Figure 13 shows the sensitization, in the presence of various concentrations of 5FC, of the WiDr cells (ATCC CCL-218) infected with Mock, an empty adenovirus, an adenovirus expressing FCU1 or p53 or p53 and FCU1 (100% corresponds to the viability of the cells infected in the absence of prodrug).

EXAMPLES:

The present invention is illustrated, without being limited as a result, by the following examples.

The constructs described below are produced according to the general genetic engineering and molecular cloning techniques detailed in Maniatis et al. (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY) or according to the manufacturer's recommendations when a commercial kit is used. The homologous recombination steps are preferably carried out in the E. coli BJ 5183 strain 30 (Hanahan, 1983, J. Mol. Biol. 166, 557-580). As regards the repair of the restriction sites, the technique used consists in filling in the protruding 5' ends with the large fragment of E. coli DNA polymerase I (Klenow).

Moreover, the fragments of adenoviral genome used in the various constructs described below are indicated specifically according to their position in the nucleotide sequence of the Ad5 genome as disclosed in the GeneBank databank under the reference M73260.

As regards the cell biology, the cells are transfected or transduced and cultured according to standard techniques well known to persons skilled in the art.

EXAMPLE 1: Construction of an adenovirus expressing p53 (Adp53).

The coding region of p53 was amplified by PCR from the plasmid pC53-SN3 (Baker et al., 1990, Science 249, 912-915) used as template and the following primers: At the 5' terminus: 3 (SEQ ID NO :3) At the 3' terminus 'ggctgtcagtggggatctagaagtggag 3 (SEQ ID NO :4) The EcoRI and XbaI sites were introduced in and in respectively, of the coding sequence of p53.

The PCR fragment of p53 was cut with EcoRI and XbaI and then inserted into the plasmid pCI-neo (Promega Corp) to give the plasmid pCI-neop53. The XhoI-XbaI fragment of pCI-neop53 containing the p53 gene is isolated and introduced into the vector pTG6600 (Lathe et al., 1987, Gene 57, 193-201) cleaved with these same enzymes, to give the transfer vector pTG6600p53. The adenoviral vector Adp53 is reconstituted by homologous recombination in the E. coli BJ5183 strain between the PacI-BstEII fragment of pTG6600p53 and the vector pTG6624 linearized with Clal. The final construct Adp53 contains the genome of Ad5 from which there have been deleted most of the El (nucleotides 459 to 3328) and E3 31 (nucleotides 28249 to 30758) regions and in place of El, a cassette for expressing the p53 gene placed under the control of the CMV early promoter and the P-globin/Ig hybrid splicing sequences. The viral particles are generated by transfection into a 293 cell line (ATCC CRL1573) which complements the El function.

EXAMPLE 2: Construction of an adenovirus expressing a bicistronic unit p53-IRES-FCU1 (Adp53FCU1).

The NcoI-SalI fragment of the plasmid pCI-neoFCUl described in French patent application No. 98 05054 containing the fusion gene FCU1 is isolated and introduced into the vector pTG4369 linearized with NcoI-SalI. The plasmid pTG4369FCUl thus obtained contains the FCU1 gene downstream of the IRES (for Internal Ribosome Entry Site) sequence of EMCV (encephalomyocarditis virus). The SacI-NotI fragment of pCI-neop53 is isolated and then inserted into the vector pTG4369FCU1 linearized with SacI-NotI to give the vector pTG4369p53FCUl into which the p53 gene is placed upstream of the IRES sequence. The NheI-MluI fragment of pTG4369p53FCUl containing the p53IRESFCU1 sequence is inserted into the vector pTG6600 cleaved with these same enzymes to give the transfer vector pTG6600p53IRESFCU1. The adenoviral vector Adp53FCU1 is reconstituted by homologous recombination in the E. coli BJ5183 strain between the PacI-BstEII fragment of pTG6600p53IRESFCUl and the vector pTG6624 linearized with Clal. The final construct Adp53FCUl contains the genome of Ad5 from which there have been deleted most of the El (nucleotides 459 to 3328) and E3 (nucleotides 28249 to 30758) regions and in place of El, a cassette for expressing the bicistron p53-FCUl placed under the control of the CMV early promoter and the 0-globin/Ig hybrid splicing sequences. The adenoviral particles are generated by transfection into a 293 cell line (ATCC CRL1573) which complements the El function.

32 EXAMPLE 3: Construction of the adenovirus expressing the FCU1 fusion gene (AdFCU1).

The XhoI-MluI fragment of pCI-neoFCUl (described in French patent application No. 98 05054) containing the FCU1 fusion gene is isolated and introduced into the vector pTG6600 linearized with XhoI-MhuI to give the transfer vector pTG6600FCUl. As described above, homologous recombination between the PacI-BstEII fragment carrying FCU1 and isolated from pTG6600FCUl and the vector pTG6624 linearized with Clal makes it possible to generate the adenoviral vector pTG6624FCUl from which the El and E3 regions have been deleted and comprising in place of El the FCU1 gene placed under the control of the CMV promoter and the P-globin/Ig hybrid splicing sequences. The adenoviral particles are generated by transfection into a 293 cell line (ATCC CRL1573) which complements the El function.

EXAMPLE 4: Infection with the adenoviruses AdFCU1, Adp53 and Adp53FCU1.

4.1 Results in vitro The adenoviral constructs of the preceding examples are used to infect in vitro 3 tumor cell lines: the human tumor line SW480 (colon adenocarcinoma/ATCC CCL-228) whose p53 gene is not functional, the human tumor cell line LoVo (colon adenocarcinoma/ATCC CCL-229) whose p53 gene is functional and the mouse melanoma line B16(FO) (ATCC CRL-6322).

The cells are infected (MOI of 1 for SW480 and LoVo and MOI of 100 for B16(FO)) and then cultured in the presence or in the absence of a prodrug (5-fluorocytosine (5FC) or 5-fluorouracil (5FU)) at various concentrations. After trypsinization (at for SW480 and at D+8 for B16(FO)), the viability of the cells is evaluated with trypan blue. The values 33 reported correspond to the mean values obtained after 4 counts.

In the absence of a prodrug (Figures 1 to 3), there is observed an antiproliferative effect of the cells infected with an adenovirus expressing p53 (Adp53 and Adp53FCUl) in mainly the SW480 lines and more weakly in the B16FO lines. However, in spite of the expression of p53 which induces apoptosis or the suppression of cell growth, no antiproliferative effect is observed in the LoVo line; this indicates that the sole administration of p53, in particular into tumor cells whose p53 gene is functional, is insufficient, or even ineffective, to allow the use of an effective antitumor therapy protocol.

By contrast, it is observed that, in the presence of various concentrations of prodrug (Figures 4 to the cells infected with Adp53 are sensitized compared with the noninfected cells or the cells infected with a nonrecombinant adenovirus, thus illustrating the known action of p53 in the toxicity of However, in accordance with the present invention, it is also observed that, surprisingly, the presence of FCU1 very substantially increases the toxicity of in the cells- (100% corresponds to the viability of the cells infected in the absence of 5FU), thus indicating a clearly synergistic effect of the two types of agent.

Figures 7 to 9 illustrate the analogous results obtained using 5FC as prodrug.

These results demonstrate that there is synergy between the products of expression of FCU1 and of p53 for the induction of mortality when 5FC or 5FU are used as prodrugs.

4.2 Results in vivo B16F(0) cells (3x10 5 cells) are injected by the subcutaneous route into 4 groups of 8 immunocompetent B6D2 mice at DO. As soon as the tumors become palpable (about the adenoviral constructs comprising the p53 gene (Adp53) or the p53 and FCU1 genes (Adp53FCUl) or a nonrecombinant adenoviral construct (Ad null) are 34 injected at a dose of 5x10 8 IU on three consecutive days D+10 and D+11). From D+9, 1 ml of a saline solution at 0.9% or 1 ml of a 5-FC solution at 1% are injected by the intraperitoneal route, twice per day.

The results presented in Figure 10 demonstrate an increase in the rate of survival of the mice into which Adp53FCU1 had been injected and which had been treated with EXAMPLE 5: Infection of cells expressing a nonfunctional p53 protein with AdFCU1, Adp53 and Adp53FCU1 The cancer cells present in patients to be treated generally express a nonfunctional form of p53.

Consequently, to test the compositions of the invention under conditions comparable with pathological situations, the adenoviral constructs of the preceding examples were used to infect in vitro 3 types of tumor cell line: The tumor line SK-BR-3 (ATCC HTB-22) whose p53 gene is mutated and encodes a nonfunctional p53 protein (Arg 175 ->His 175 (Kovach et al., 1991, J. Nat. Cancer Inst., 83, 1004-1009) The tumor line T47-D (ATCC HTB-133) whose p53 gene is mutated and encodes a nonfunctional p53 protein (Leu194->His 194 (Nigro et al., 1989, Nature, 342, 705-708) The tumor line WiDr (ATCC CCL-218) whose p53 gene is mutated and encodes a nonfunctional p53 protein (Arg 27 3->HiS 273 (Li et al., 1995, Int. J. Cancer, 64, 383-387) The cells are infected (MOI of 1) and then cultured in the presence or in the absence of various concentrations of prodrug (5-fluorocytosine according to the conditions described in Example 4.

After trypsinization, the viability of the cells is evaluated with trypan blue. The values reported in Figures 11 to 13 correspond to the mean values obtained after 4 counts.

For each of these lines, an antiproliferative effect is observed in the absence of prodrug in the cases where the infection is produced with an adenovirus expressing the p 5 3 polypeptide (Adp53 and Adp53FCU1).

The results obtained (Figures 11 to 13) are comparable to those observed in Example 4. More particularly, it is observed that the coexpression of p53 and of FCU1 in the cells cultured in the presence of 5-FC very substantially increases in toxicity of this prodrug toward the cells, thus confirming the synergistic effect of these two products of expression.

The word 'comprising' or forms of the word 'comprising' as used in this description and in the claims do not limit the invention claimed to exclude any variants or •additions.

°o 0* 0 *0 e0 S0 *5

S

EDITORIAL NOTE APPLICATION NUMBER 49314/00 The following Sequence Listing pages 1 to 3 are part of the description.

The -claims pages follow on pages "36" to "38".

SEQUENCE LISTING <110> TRANSGENE <120> Composition designed for implementing an antitumoral or antiviral treatment in a mammal <130> Polypeptide p53 <140> <141> <150> FR9906892 <151> 1999-05-25 <160> 4 <170> Patentln Ver. 2.1 <201> 1 <211> 216 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Ala Ser Giu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gin Thr Asn 1 5 10 Gin Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr Arg 25 Pro Asp Phe Ilie Phe Tyr Ser Asp Arg Ilie Ilie Arg Leu Leu Val Giu 40 Giu Gly Let2 Asn His Leu Pro Val Gin Lys Gin Ile Val G'iu Thr Asp 55 Thr Asn Giu Asn Phe Giu Gly Val Ser Phe Met Gly Lys Ile Cys Gly 70 75 Val Ser Ile Val Arg Ala Gly Glu Ser Met Giu Gin Gly Leu Arg Asp 90 Cys Cys Arg Ser Val Arg Ile Gly Lys Ilie Leu Ile Gin Arg Asp Glu 100 105 110 Giu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Giu Asp Ile 115 120 125 Ser Giu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly Gly 130 135 140 Ser Ala Ilie Met Ala Thr Glu Vai Leu Ile Lys Ary Gly Val Lys Pro 145 150 155 160 Giu Arg Ile Tyr Phe Leu Asn Lieu Ile Cys Ser Lys Glu Gly Ile Glu 165 170 175 Lys Tyr Asp Ary Phe Gly 210 His Ala Ala 180 Gly Leu Asp 195 Asp Arg Tyr Glu Val Arg Ile Val Thr Gly Ala Leu 1.85 190 Lys TyT: Leu Val Pro Gly Leu Gly Asp 200 205 ValI <~210> 2 <211> 373 <22 PRT <213> Saccliaromyces cerevisiae <400> 2 met Val

I

Ile Ala Ile Gly Gly His Ilie Ser Asp Thr Ala Ile Asn Phe Val Val 130 Ile Asp 145 Glu Pro Gly Leu Ile Phe Thr Gly Gly Met Ala Ser Gly Met 1s Gly Val Leu Giy His Gly Val Tyr Cys Thr Giu Asn 110 Gly His Lys Gin Giu Ala Gln Leu 175 Pro Asp 190 Glu Gly 195 Asn His Leu Pro Val Gin 210 Gln Ile Val Glii Thr Asp Thr Asn Giu 220 Asn Phe Glu Gly Val Ser Phe met Gly Lys Ile Cys Gly Val Ser Ile 225 230 235 240 Val Arg Ala Gly Glu Ser Met Glu Gin Gly Leu Arg Asp Cys Cys Arg 245 250 255 Ser Val Arg Ile Gly Lys Ile Leu Ile Gin Arg Asp Glu Giu Thr Ala 260 265 270 Leu Pro Lys Leu Phe Tyr Giu Lys Leu Pro Glu Asp Ile Ser Giu Arg 275 280 285 Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly Gly Ser Ala Ile 290 295 300 Met Ala Thr Giu Val Leu Ile Lys Arg Gly Val Lys Pro Giu Arg Ile 305 310 315 320 Tyr Phe Leu Asn Leu Ile Cys Ser Lys Giu Gly Ile Giu Lys Tyr His 325 330 335 Aia Ala Phe Pro Giu Val Arg Ile Vai Thr Gly Ala Leu Asp Arg Gly 340 345 350 Leu Asp Giu Asn Lys Tyr Leu Vai Pro Giy Leu Gly Asp Phe Gly Asp 355 360 365 Arg Tyr Tyr Cys Vai 370 <210> 3 <211> 26 <212> DNA <213> Aitificial sequence <220> <223> Description of the 'artificial sequence: primer <400> 3 9gcagccaga attccttccg ggtcac 26 <210> 4 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: primer <400> 4 ggctgtcagt ggggatctag aagtggag 28

Claims (9)

1. 6.MAY.2004 17:03 MRLLESONS 96435999 N0.257 P.9 36 CLAIMS 1. Composition used to carry out an antitumor or antiviral treatment in a mammal comprising: a nucleic acid sequence encoding the p53 polypeptide, (ii) at least one nucleic acid sequence encoding all or part of a polypeptide having at least a cytotoxic activity selected from thymidine kinase activity, purine nucleoside phosphorylase activity, guanine phosphoribosyl transferase activity and cytosine deaminase activity, said nucleic acid sequences being placed under the o: control of the elements necessary for their expression in a host cell of said mammal. 2. Composition according to claim 1, characterized in 15 that said polypeptide exhibits at least a CDase activity and a UPRTase activity. 3. Composition according to claim 1, characterized in that the nucleic acid sequence (ii) is selected from CodA, upp, FUR1, FCY1 and FUR1A105. 20 4. Composition according to claim 2, characterized in that said polypeptide is a fusion polypeptide in which a first polypeptide exhibiting a UPRTase or CDase activity is fused in phase with at least a second polypeptide, said second polypeptide exhibiting a CDase or UPRTase activity, 25 respectively. 5. Composition according to claim 4, characterized in that the fusion with the second polypeptide is carried out at the N-terminal end of said first polypeptide. 6. Composition according to claim 4 or characterized in that the nucleic acid sequence encoding said fusion polypeptide is a hybrid sequence comprising: a first nucleic acid sequence encoding a first polypeptide exhibiting a UPRTase activity, a second nucleic acid sequence encoding a second polypeptide exhibiting a CDase activity. COMS ID No: SMBI-00737505 Received by IP Australia: Time 16:55 Date 2004-05-06 6.MAY.2004 17:04 MALLESONS 96435999 NO.257 37
2. 7. Composition according to claim 6, characterized in that the first nucleic acid sequence is selected from upp, FUR1 and FUR1A105, and in that the second nucleic acid sequence is selected from CodA and FCY1.
3. 8. Composition according to claim 1, characterized in that said nucleic acid sequences and (ii) are inserted into a recombinant vector of plasmid or viral origin.
4. 9. Composition according to claim 8, characterized in that said nucleic acid sequences and (ii) are inserted into the same recombinant vector. 0 10. Composition according to claim 8, characterized in that said nucleic acid sequences and (ii) are inserted into distinct recombinant vectors.
5. 11. Vector comprising: 15 a nucleic acid sequence encoding the p53 1polypeptide, (ii) at least one nucleic acid sequence encoding all or part of a polypeptide having at least a cytotoxic activity selected from thymidine kinase activity, purine 20 nucleoside phosphorylase activity, guanine phosphoribosyl transferase activity and cytosine deaminase activity, said nucleic acid sequences being placed under the control of the elements necessary for their expression in a host cell. 25 12. Vector according to claim 11, characterized in that it is a viral vector.
6. 13. Viral particle comprising a vector according to claim 12.
7. 14. Method for preparing a viral particle according to claim 13, according to which: a viral vector according to claim 12 is introduced into a cell capable of producing said vector, so as to obtain a transfected cell, (ii) said transfected cell is cultured under appropriate conditions to allow the production of said viral particle, and COMS ID No: SMBI-00737505 Received by IP Australia: Time 16:55 Date 2004-05-06 6.MRY.2004 17:04 MALLESONS 96435999 NO.257 P.11 38 (iii) said viral particle is recovered from the cell culture. Composition used to carry out an antitumor or antiviral treatment in a mammal comprising: the p53 polypeptide, (ii) all or part of a polypeptide having at least a cytotoxic activity, according to which said polypeptide having at least a cytotoxic activity is as defined in claims 1 to 7.
8. 16. Formulation used to carry out an antitumor or antiviral treatment in a mammal, characterized in that it comprises a composition according to one of claims 1 to 10, a vector according to claims 11 or 12, a viral particle according to claim 13, or a composition according 15 to claim 15, as well as a pharmaceutically acceptable *carrier. 4 17. Formulation according to claim 16, characterized in that it comprises pharmaceutically acceptable quantities of a prodrug capable of being converted to a 20 cytotoxic molecule by a polypeptide having at least a cytotoxic activity.
9. 18. Formulation according to claim 17, characterized in that said prodrug is selected from and 5-fluorocytosine 25 19. Use of a composition according to claims 1 to of a vector according to claims 11 or 12, of a viral particle according to claim 13, or of a composition according to claim 15 for the,preparation of an antitumor or antiviral medicament. TRANSGENE 5 May 2004 COMS ID No: SMBI-00737505 Received by IP Australia: Time 16:55 Date 2004-05-06