CA2339900A1 - Composition for implementing a cytotoxic, in particular an antitumoral or antiviral, treatment in a mammal - Google Patents

Abstract

The invention concerns a composition for implementing a cytotoxic treatment in a mammal comprising: (i) a nucleic acid sequence coding for all or part of an MIP chemokine; (ii) at least a nucleic acid sequence coding for all or part of a polypeptide having at least a cytotoxic activity, said nucleic acid sequences being placed under the control of elements required for their expression in said mammal's host cell.

Description

Composition intended for the implementation of a treatment cytotoxic, in particular antitumor or antiviral, in a mammal The present invention relates to a cytotoxic composition comprising a first nucleic acid sequence coding for all or part of a MIP chemokine and a second nucleic acid sequence encoding all or part of a polypeptide having at least one activity cytotoxic, in particular antitumor or antiviral. The present invention is particularly useful in the context of the implementation of a treatment by gene therapy for proliferative or infectious diseases.
To date, the most encouraging results obtained within the framework of anti-tumor treatments relate to combined treatments combining a treatment with chemical compounds (chemotherapy) and treatment based on the use of radiation (radiotherapy). Besides the significant inconvenience caused by the patient with this type of treatment, it is found in a large number of cases that tumor cells, of the type metastatic or not, persist in the subject treated, which may cause relapse and therefore not allowing complete remission.
Recent work in the field of cancer has proposed to adapt gene therapy protocols to tumor therapy. In this regard, we can for example cite the work of Meneguzzi et al., 1991, Virology, 181, 61-69 relating to immunization against tumor cells using a recombinant vaccine vector expressing the E6 and E7 genes of the Human papillomavirus type 16. Mention may also be made of the content of the French patent FR 92/03120 relating to the use of an adenovirus recombinant expressing a cytokine as part of gene therapy antitumor.
Cytokines are molecules naturally produced as a result antigenic stimulation or inflammatory reaction (Gillis and Williams, 1998, Curr. Opin. Immunol., 10, 501-503) whose utility in framework for the treatment of certain cancers has been shown in particular by Oettger

2 (Curr. Opin. Immunol., 1991, 3, 699-705). Thus, Leroy et al. (1998, Res.Immunol. 149 (7-8): 681-684) have shown that the production of cytokines at tumor sites after infra-tumor administration of vectors recombinant virals allow the induction of an immune response associated with inhibition of tumor growth. However, this answer ar ~ titumoral although encouraging does not allow the final disappearance of tumor cells, and therefore the implementation of a treatment satisfactory antitumor.
The chemokines for their part constitute a subclass of the family cytokines. They are distinguished from other cytokines by their property chemo-attractive, especially during natural chemotaxis processes, including attraction of immune system cells to tissue in which sits (inflammation or (infection, as well as by their properties anti-angiogenic.
Chemokines are low molecular weight proteins (between 8 and 10 kd), of small size (from 70 to 80 amino acids) whose sequences in amino acids have a low omology rate (varying from 10 to 70 according to the chemokines considered) allowing to define to date approximately 50 different chemokines. These chemokines can nevertheless be subdivided in 4 main families relating to the position of the cysteine residues they contain. A families whose N-terminal end includes 2 cysteines separated by a single amino acid (chemokines of t3rpe IL-8, NAP-2, GCP-2) and (3 whose N-terminal end comprises 2 adjacent cysteines (RANTES, MIP1, MCP1 type chemokines) are best characterized (Horuk, R., 1994, Trends Pharmacol. Sci., 15, pages 159-165; Murphy, PM, 1994, Annu. Rev. Immunol., 12, pages 593-633).
Furthermore, the group of Dilloo et al. (1996, Nature Medicine, vol. 2, Number 10, 1090-1095) has shown that co-expression, after administration recombinants in mice of fibroblasts modified ex vivo using retroviral vectors, of a particular chemokine, lymphotactin (Lptn), and finterleukin-2 (IL2), stimulates the anti-tumor immune response of the treated animal. However, this effect is limited in time, and does not allows

3 a transient control of the tumor volume and no remission che2 les animals treated.
It is therefore desirable to have new compositions allowing in particular the implementation of anti-tumor treatments effective, easy to set up, i.e. allowing control extended tumor volume and increased survival rate of treated patients.
We have now identified new cytotoxic compositions whose different constituents are chosen so as to obtain an effect synergistic of their respective activities and the improved properties of said lo constituents. More particularly, such compositions allow inhibit or delay cell proliferation by inducing death specific to cells, especially tumor cells, better presentation of antigens and / or stimulation of immune cells in the host organism. The present invention provides an advantageous and effective alternative to techniques i5 of the prior art, in particular for treating cancer of man or the animal.
The invention relates firstly to a composition intended for the using cytotoxic treatment, for example antitumor or antiviral treatment, or any application requiring cell death, in a mammalian including (I) a nucleic acid sequence encoding all or part a MIP chemokine, (üj at least one nucleic acid sequence coding for all or part of a polypeptide having at least one cytotoxic activity, said nucleic acid sequences being placed under the control of 25 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 (i) the entire nucleic acid sequence encoding the chemokine MIP
(for Macrophage Inflammatory Protein, in English) or only part 3o of this polypeptide, or a derived or mutated polypeptide, insofar as the function and properties of the MIP chemokine are retained. In the sense of

4 present invention means by mutation, a deletion and / or a substitution and / or addition of one or more nucleotides. Likewise, it it is possible to use a sequence coding for a hybrid chemokine originating from the fusion of the sequence coding for a chemokine of the type MIP and of the sequence coding for at least one chemokine of another type (MANTES, MCP 1, ...).
In the context of the present invention, the preferred MIP chemokine is MIP 1 type chemokine, and more particularly selected from the group consisting of the chemokines MIPla and MIP1 (i whose properties have have been highlighted by Wolpe et al, 1988, J. Exp. Med, 167, 570-581.
MIPla, whose nucleic acid and peptide sequences are described in Obaru et al. 1986, J. Biochem. 99, 885-894, the content of which is incorporated by reference in this application, is produced by T cells and monocytes. It allows the chemo-attraction of eosinophils and T cells during pathway infections respiratory; monocytes and neutrophils in arthritis rheumatic, inflammations of the digestive system or meningitis of origin bacterial. In addition, it inhibits the proliferation of precursors hematopoietic.
2o MIP1 (i, whose nucleic acid and peptide sequences are described in Brown et al. 1989, J. Immunol. 142, 679-68, the content of which is incorporated by reference in this application, is also produced by T cells and monocytes. It exerts its chemo-attractive to monocytes and neutrophils in arthritis cases bone and bacterial meningitis. Like MIPla, it inhibits proliferation of hematopoietic precursors.
There are natural variants of said proteins MIPla and MIP1 (3 which are known to those skilled in the art and which carry, for example, the names GOS 19, LD78, pAT464, TY5 (mouse) or SIS a (mouse) for MIPla or pAT744, Act-2, G-26, H-400 (mouse) or hSIS y (mouse) for MIP1 (3.
particular of MIP1 (3, we will choose for example the sequence corresponding to Act-2 (Lipes et al., 1988, PNAS, 85, 9704-9708, the content of which is incorporated here for reference).
by “polypeptide having at least one cytotoxic activity u, is meant designate any peptide substance capable of inducing or activating a

5 immune response directed specifically against a tumor cell (the activity cytotoxic is then called anti-tumor activity) or an infected cell through a virus (cytotoxic activity is then called antiviral activity) or to inhibit the growth and / or division of such a cell, in particular a tumor or infected. According to a preferred case, said cytotoxic activity results in the dead 10 of said cell. Depending on a particular case, it would also be possible to use compositions according to the present invention in pathoiagic cases associated with cell proliferation, such as for example phenomena restenosis.
The chemo-attraction activity of a given polypeptide, in particular derived from the chemokine MIP, on cells involved in the reactions immune systems (such as eosinophils, T lymphocytes, monocytes or neutrophils can be assessed by a chemotaxis (Maghazachi, 1993, Nature Immunity, 12, 57). Likewise, this type of chemokine inhibiting the proliferation of precursors 2o hematopoietic, it is possible to evaluate such a property in vitro according to Graham et al., 1992, Growth Factors, 7, I51.
The cytotoxic activity of a given polypeptide, in particular an activity antitumor, can be evaluated in vitro by measuring cell survival either by short-term viability tests (such as for example the blue tryptan at MTT), either by clonogenic survival tests (formation of colonies) (Brown and Wouters, 1999, Cancer Research, 59, 1391-1399) or in in vivo by measuring tumor growth (size and / or volume) in a 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 cytotoxic activity is chosen from cytokines, proteins encoded by a gene called "gene suicide ~ and antiangiogenic protein factors.

6 More particularly, when said polypeptide in (ü) is a cytokine, it is preferably a cytokine chosen from interferons a, (3 and y, interleukins, and in particular fIL-2, IL-4, IL-6, IL-10 or fIL-12, the tumor necrosis factors (TNF) and colony stimulating factors (GM-CSF, C-CSF, M-CSF ...).
According to a preferred embodiment, said cytokine is selected among interleukin-2 (IL-2) and gamma interferon (IFN-y). Interleukin-2 East in particular responsible for the proliferation of activated T lymphocytes, multiplication and activation of immune system cells (for the nucleic acid sequence see in particular FR 85 09480). IFN-y activates phagocytic cells and increases the expression of surface antigens class I and II of the major istocompatibility complex (for the sequence in nucleic acid see in particular FR 85 09225). Said acid sequences nucleic acid are incorporated by reference in the present application.
According to another embodiment, the composition according to the invention is characterized in that it comprises in (ü) at least two acid sequences nucleic acid encoding all or part of interleukin-2 (IL-2) and, all or part of gamma interferon (IFN-y).
According to a second variant, the invention also relates to such a composition characterized in that said polypeptide in (ü) has at least an enzymatic activity selected from thymidine kinase activity, purine nucleoside phosphorylase activity, guanine or uracil activity or orotate phosphoribosyl transferase and cytosine deaminase activity.
Several studies have identified polypeptides that are not toxic as such but with enzymatic properties catalytic capable of transforming an inactive substance (predrogue), by example a nucleoside or a nucleoside analog, in substance highly toxic to the cell, for example a modified nucleoside which can be incorporated into elongated DNA or RNA chains, with for consequence, in particular, of the inhibition of cell division or cellular dysfunctions leading to the death of the cell containing

7 such polypeptides. The 1 genes coding for such polypeptides are said to be "N suicide genes. Many suicide / predrole gene couples are currently available. More particularly, the couples - herpes simplex virus type 1 thymidine kinase (TK HSV-1) 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 (E. Colt) 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 5-fluorocytosine (5FC).
More particularly, CDase is an enzyme which intervenes in the metabolic pathway for pyrimidines by which the exogenous cytosine is transformed by hydrolytic deamination into uracil. Of e CDase activities 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. Say. 130, 112-118; Esders and Lynn, 1985, J. Biol. Chem. 260, 3915-3922) but they are absent in mammals (Koechlin et al., 1966, Biochem Pharmacol. I5, 435-446; Polak et al., 1976, Chemotherapy 22, 137-153). The FCYI genes of Saccharomyces cerevisiae (S. cerevisiae) and codA from E. coli coding respectively for the CDase of these two organisms are known and their published sequences (EP 402 108; Erbs et al., 1997, Curr. Genet. 31, 1-6;
W093 / 01281).

8 CDase also deaminates a cytosine analog, 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) which is a highly cytotoxic especially when it is converted into 5-fluoro-UMP (5-FUMP). The cells lacking CDase activity, either due to a mutation inactive of the gene encoding the enzyme, or their natural deficiency for this enzyme (e.g. mammalian cells) are resistant to 5-FC (Jund & Lacroute, 1970, J. Bacteriol. 102, 607-615; Kilstrup et al., 1989, J.
Bacteriol. 1989 171, 2124-2127). However, it has been shown to be possible to transmit sensitivity to 5-FC to mammalian cells in which the sequence encoding 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). In addition, in this case, the cells nearby unprocessed also become sensitive to 5-FC
(Huber et aL, 1994, Proc. Natl. Acad. Sci. USA 91, 8302-8306). This phenomenon, called neighborhood effect (bystander in English), is due to excretion by cells expressing CDase activity, 5-FU which intoxicates cells neighbors by simple diffusion through the cell membrane. This property of passive diffusion of 5-FU is an advantage compared to the reference tk / GCV for which the neighborhood effect requires contact with the cells which express tk (Mesnil et al., 1996, Proc. Natl. Acad. Sci. USA 93, 1831-1835). This effect therefore constitutes an additional advantage of the use of CDase in the context of gene therapy, in particular anticancer.
However, the sensitivity to 5-FC varies a lot according to the lines.
cellular. Low sensitivity is observed for example in lines human tumor PANC-1 (pancreatic carcinoma) and SK-BR-3 (breast adenocarcinoma) transduced by a retrovirus expressing the gene E coda. Coli (Harris et al., 1994, Gene Therapy 1, 170-175). This phenomenon undesirable could be explained by the absence or the low conversion endogenous 5-FU formed by the enzymatic action of CDase into cytotoxic 5-FUMP.
This stage, normally carried out in mammalian cells by orotate phosphorybosyl transferase (Peters et al., 1991, Cancer 68, 1903-1909), can WO 00/7462

9 PCT / FR00 / 01559 be absent in some tumors and thus make gene therapy, based on CDase, inoperative.
In prokaryotes and lower eukaryotes, uracil is transformed in UMP by the action of uracil phosphoribosyl transferase (presenting by 5 therefore UPRTase activity). This enzyme also converts 5-FU
in 5-FUMP. Thus furl mutants of the yeast S. cerevisiae are resistant to high concentrations of 5-FU (10 mM) and 5-FC (10 mM) because in the absence of UPRTase activity, 5-FU, resulting from the deamination of 5-FC by the CDase, is not transformed into cytotoxic 5-FUMP (Jund and Lacroute, 1970,

J. Bacteriol. 102, 607-615). The upp and FURI 'genes encoding UPRTase respectively from E. coli and S. cerevisiae have been cloned and sequenced (Andersen et al., 1992, Eur. J. Biochem. 204, 51-56; Kern et al., 1990, Gene 88, 149-157).
Within the meaning of the present invention, a polypeptide having an activity 15 UPRTase denotes a polypeptide capable of converting uracil or one of its derivatives into a monophosphate analog and, in particular Ia 5-FU into 5-FUMP.
By "mutation ~" is meant the addition, deletion and / or substitution one or more residues at any location of said polypeptide.
The native UPRTase discussed in the present invention can 20 be of any origin, including prokaryotic, fungal or yeast. By way of illustration, the nucleic acid sequences coding for the UPRTases of E. coli (Anderson et al., 1992, Eur. J. Biochem 204, 51-56), de La.ctococcus lactis (Martinussen and Hammer, 1994, J. Bacteriol. 176, 6457-6463), from Mycobacterium bonis (Kim et al., 1997, Biochem Mol. Biol. Int 41, 25 1117-1124) and Bacillus subtilis (Martinussen et al., 1995, J. Bacteriol.
177, 271-274) can be used in the context of the invention. But we prefer especially to use a yeast UPRTase and in particular that encoded by the FUR1 gene of S. cerevisiae, the sequence of which is disclosed in Kern et al. (1990, Gene 88, 149-157) is introduced here by reference. As 30 indicative, the sequences of the genes and those of the corresponding UPRTases can be found in literature and databases specialized (SWISSPROT, EMBL, Genbank, Medline ...).

Furthermore, application PCT / FR99 / 00904 describes a FURI gene devoid of 105 nucleotides in 5 ′ of the coding part allowing the synthesis a UPRTase deleted from the first 35 residues in the N-terminal position and starting with methionine at position 36 in the native protein. The product 5 expression of the mutant gene, designated FUR1d105, is capable of complement a furl mutant of S. cerer ~ isiae. In addition, the truncated mutant has a higher UPRTase activity than that of the native enzyme. So, according to a particularly advantageous embodiment, the polypeptide encoded according to the invention is a deletion mutant of a native UPRTase. The deletion 10 is preferably located in the N-terminal region of UPRTase of origin.
It can be total (concern all the residues of said region N-final) or partial (concern one or more residues, continuous or not in the primary structure). Generally, a polypeptide is consisting of N-terminal, central and C-terminal parts, each representing about a third of the molecule. For example, the tJPRTase from S.
cerevisiae having 251 amino acids, its N-terminal part consists of 83 first residues starting with the so-called initiator methionine located in first position of the native form. As for UPRTase by E. coli, its part N-terminal covers positions 1 to 69.
2o In addition, patent applications W096 / 16183 and PCT / FR99 / 00904 describe the use of a fusion protein coding for an enzyme two domains with CDase and UPRTase activities and demonstrate that transfer of a hybrid gene codA :: upp or FCYl :: FURl or FCY1 :: FUR1d105 carried by a expression plasmid increases sensitivity to 5-FC of B cells 16 transfected. The protein and nucleic acid sequences described in these two requests are incorporated into the description of this request. According to this embodiment, the polypeptide is a polypeptide fused in phase with at least one second polypeptide. Although the merger may take place at anywhere in the first polypeptide, the N or C ends terminals are preferred and in particular the N-terminal end. A merger CDase and UPRTase activities improve cell sensitivity targets at 5-FC and 5-FU.

11 Those skilled in the art are capable of cloning the CDase sequences or UPRTase from published data, to carry out possible mutations, to test the enzymatic activities of the mutant forms in a system acellular or cellular according to art technology or by following the protocol indicated in application PCT / FR99 / 00904 and to merge, in particular by phase, the CDase and UPRTase activity polypeptides, and therefore all or part of the corresponding genes.
Consequently, according to a specific case, the composition of the invention is characterized in that the nucleic acid sequence (ü) is selected among the nucleic acid sequences of the CodA, upp, FUR1 genes; FCY1 and FUR1. ~ 105, or by a combination of all or part of said sequences.
The invention relates more particularly to a said composition characterized in that said polypeptide in (ü) exhibits at least one activity CDase and UPRTase activity.
By “combination of nucleic acid sequences” is meant to denote as well as separate sequences that code for at least two distinct polypeptides as fused sequences that code for fusion polypeptides, it being understood that the production of such polypeptides can be carried out under the control of the same regulatory elements (cassette polycistronic) or independent elements, 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 (ü) coding for a fusion polypeptide in which a first polypeptide having a UPRTase or CDase activity is merged in phase with at least one second polypeptide, said second polypeptide exhibiting CDase activity or UPRTase, 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.

12 According to a preferred case, said composition is characterized in that the nucleic acid sequence encoding said fusion polypeptide is a hybrid sequence including a first nucleic acid sequence coding for a first polypeptide exhibiting UPRTase or CDase activity, - a second nucleic acid sequence coding for a second polypeptide exhibiting Cdase or UPRTase activity, respectively.
Such a hybrid nucleic acid sequence coding for said the fusion polypeptide may further contain an IRES-like sequence.
The invention relates in particular to such a composition for which the first nucleic acid sequence is selected from upp, FUR1 and FUR10105, and in that the second nucleic acid sequence is selected from CodA and FCY1, and vice versa. So completely preferred, such a hybrid nucleic acid sequence is chosen from 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 cytotoxic activity (ü) is an anti-angiogenic protein factor. Angio ~ enesis is the process responsible for training new capillaries from the network already existing. This complex process is finely regulated in the healthy tissue by the balance of the effects of many angiogenic factors and anti-angiogenic. However, in certain pathologies, and in particular during of tumor formation, this process is deregulated: the factors angiogenic drugs take precedence over anti-angiogenic factors which allows an important vascularisation of the tumors and by way of consequence their rapid development and / or the appearance of metastases.
This is why, in the context of the present invention, an anti-angiogenic is considered to be a cytotoxic agent, in particular antitumor. Among the various anti-angiogenic factors known at the time current we can notably cite angiostatin, endostatin, factor

13 platelet PF4, thrombospondin-1, PRP (for Proliferin Related Protein), VEGI (for Vascular Endothelial Growth Inhibitor) metalloproteases and urokinase.
The nucleic acid sequences (i) or (ü) ~ can be easily obtained by cloning, by PCR or by chemical synthesis according to techniques conventional in use. They may be native genes or derived from these last by mutation, deletion, substitution and / or addition of one or more nucleotides. Furthermore, their sequences are widely described in the literature consultable by a person skilled in the art.
The present invention also relates to a composition such as presented above characterized in that said acid sequences nucleic acid (i) and (ü) are inserted into an original recombinant vector plasmid or viral, as well as to such a recombinant vector carrying such nucleotide sequences placed under the control of the elements necessary for their expression in a host cell. The nucleic acid sequences {i) and (ü) may be present in one or more copies on the same vector.
More particularly, the compositions of the invention can comprise said nucleic acid sequences (i) and (ü) inserted into a same recombinant vector or in separate recombinant vectors.
By N recombinant vector "according to the invention, bn means a H vector of plasmid or viral origin, and optionally such a vector associated with one or more efTicacity-enhancing substances transfectional and / or the stability of said vector and / or the protection of said vector in viUO â
the immune system of the host organism. These substances are widely documented in literature accessible to those 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 not limiting, they may be polymers, in particular cationic lipids, liposomes, of nuclear or viral proteins or neutral lipids. These substances can be used alone or in combination. Examples of such compounds are available in particular in WO patent applications

14 98/08489, WO 98/17693, WO 98/34910, WO 98/37916, WO 98/53853, EP
890362 or WO 99/05183. A possible combination is a vector recombinant plasmid associated with cationic lipids (DOGS, DC-CHOL, spermine-chol, spermidine-chol etc ...) and neutral lipids (DOPE).
The choice of plasmids usable in the context of this invention is vast. They may be cloning and / or expression vectors.
In general, they are known to those skilled in the art and many of them they are commercially available but it is also possible to construct or modify them by genetic manipulation techniques. We as examples, the plasmids derived from pBR322 (Gibco BRL) can be cited, pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene) or still p Poly (Lathe et al., 1987, Gene 57, 193-201). Preferably, a plasmid used in the context of the present invention contains a origin of replication ensuring the initiation of replication in a cell producer and / or a host cell (for example, the ColEl origin will be retained for a plasmid intended to be produced in E. coli and the oriP / EBNAl system if you want it to be self-replicating in a mammalian host cell, Lupton & Levine, 1985, Mol. Cell. Biol. 5, 2533-2542; Yates et al., Nature 313, 812-815). It can also comprise a selection gene making it possible to select or identify the transfected cells (complementing a auxotrophy mutation, gene coding for antibiotic resistance ...).
Of course, it can include 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, integration sequences in the cell genome).
Being a viral vector, we can consider a drifting vector a poxvirus (vaccinia virus, in particular MVA, canaripox, etc.), a adenovirus, retrovirus, herpes virus, alphavirus, foamyvirus or a virus associated with adenovirus. We will preferably use of a non-replicative and non-integrative vector. In this regard, the vectors adenovirals are particularly suitable for the implementation of present invention. However, it should be noted here that in the context of the implementation of the present invention, the nature of the vector takes little of importance.
Retroviruses have the property of infecting and integrating mainly in dividing cells and in this regard are particularly suitable 5 for cancer application. A recombinant retrovirus according to the invention generally includes LTR sequences, an encapsidation region and the nucleotide sequence according to the invention placed under the control of the LTR
retroviral or an internal promoter such as those described below. he can derived from a retrovirus of any origin (murine, primate, feline, 10 human etc ...) and in particular MoMuLV (Moloney; purine leukemia virus), MVS (Mucine sarcoma virus) or Friend mucin retrovirus (Fb29). It has spread in an packaging line capable of providing the polypeptides in trans gag, pol and / or env virals necessary for the constitution of a particle viral.
Such lines are described in the literature (PA317, Psi CRIP GP + Am-

15 etc ...). The retroviral vector according to the invention may include changes especially at the LTR level (replacement of the promoter region by a eukaryotic promoter) or of the encapsidation region (replacement with a heterologous packaging region, for example of the V30 type) (see the French requests 94 08 300 and 97 05 203).
A defective adenoviral vector may also be used to replication, i.e. devoid of all or part of at least one region essential for the replication selected from regions E1, E2, E4 and. A
deletion of the E1 region is preferred. But it can be combined with others modification (s) / deletion (s) affecting in particular all or part of the E2 regions, E4 and / or L1-L5, insofar as the defective essential functions are complemented in trans using a complementation line and / or an auxiliary virus to ensure the production of viral particles of interest. In this regard, we can use second vectors generation of the state of the art (see for example requests W094 / 28152 and W097 / 04119). By way of illustration, the deletion of the majority of the E 1 region and the E4 transcription unit is all particularly advantageous. In order to increase the capacities of

16 cloning, the adenoviral vector can also be devoid of all or part of the non-essential E3 region. According to another alternative, one can set uses a minimal adenoviral vector retaining the essential sequences for fencapsidation, namely the 5 'and 3' ITRs (Inverted Terminal Repeat) and the region packaging. Furthermore, the origin of the adenoviral vector according to the invention, can be varied both from the point of view of the species and of the serotype. he can derive from the genome of an adenovirus of human or animal origin (canine, avian, bovine, murine, ovine, porcine, simian ...) or a hybrid comprising adenoviral genome fragments of at least two origins l0 different. Mention may more particularly be made of the CAV-1 adenoviruses or CAV-2 of canine origin, DAV of avian origin or Bad of type 3 of origin bovine (Zakharchuk et al., Arch. Virol., 1993, 128: 171-176; Spibey and Cavanagh, J. Gen. Virol., 1989, 70: 165-172; Jouvenne et al., Gene, 198 ?, 60:
21-28; Mittal et al., J. Gen. Virol., 1995, 76: 93-102). However, we prefer to an adenoviral vector of human origin preferably derived from a adenovirus serotype C, especially type 2 or 5. An adenoviral vector according to the present invention can be generated in vitro in Escherichia coli (E.
coli) by ligation or homologous recombination (see for example the application W096 / 17070) or by recombination in a line of complementation. The different adenoviral vectors and their preparation techniques are known (see for example Graham and Prevect, 1991, in Methods in Molecular Biology, vol 7, p 109-128; Ed: EJ Murey, The Human Press Inc).
The elements necessary for expression consist of all of the elements allowing the transcription of the nucleodic sequence into RNA and the translation of mRNA into polypeptide, in particular the promoter sequences and / or efficient regulatory sequences in said cell, and possibly the sequences required to allow excretion or expression on the surface of target cells of said polypeptide. These elements 3U can be adjustable or constitutive. Of course, the promoter is adapted to the selected vector and to the host cell Mention may be made, by way of examples, of eukaryotic promoters of the PGK (Phospho Glycerate Kinase), MT genes (metallothioneine; Mc Ivor et al., 1987, Mol. Cell Biol. 7, 838-848), a-1

17 antitrypsin, CFTR, promoters of the gene encoding creatine kinase muscular, for actin, for pulmonary surfactant, immunoglobulin, / 3-actin (Tabin et al., 1982, Mol. Cell Biol. 2, 426-436), SRa (Takebe et al., 1988, Mol. Cell. Biol. 8, 466-472), the early promoter of the SV40 virus (Simian Virus), the RSV (Rous Sarcoma Virus) LTR, the MPSV promoter, the TK-HSV-1 promoter, the early promoter of the CMV virus (Cytomegalovirus), promoters of vaccinia virus p7.5K pHSR, pKl L, p28, p 11 and the EIA and MLP adenoviral promoters or a combination of said promoters.
It can also be a promoter stimulating expression in a tumor or cancer cell. Mention may in particular be made of the promoters of MUC-1 genes overexpressed in breast and prostate cancer (Chen and al., 1995, J. Clin. Invest. 96, 2775-2782), CEA (for carcinoma embryonic antigen) overexpressed in colon cancer (Schrewe et al., 1990, Mol.
Cell.
Biol. 10, 2738-2748), tyrosinosis overexpressed in melanomas (Vile et al., 1993, Cancer Res. 53, 3860-3864), ERB-2 overexpressed in cancer breast and pancreas (Harns et al., 1994, Gene Therapy 1, 170-I75) and a-overexpressed fetoprotein in liver cancer (Kanai et al., 199 ?, Cancer Res. 57, 461-465). The early promoter of Cytomegalovirus (CMV), or that RSV is particularly preferred. It is also possible to use a tissue-specific promoter region, especially when the tumor to be treated comes from a particular cell type, or can be activated under conditions defined. The literature provides a great deal of information relating to such promoter sequences.
The necessary elements may, in addition, include elements additions improving the expression of the nucleotide sequence according to the invention or its maintenance in the host cell. We can cite in particular the intronic sequences (WO 94/29471), secretion signal sequences, nuclear localization sequences, internal sites for re-initiation of IRES type translation, poly A transcription termination sequences.
According to a preferred embodiment, the invention relates more particularly a recombinant vector, in particular a viral vector, and more specifically a defective adenoviral vector for replication, comprising

18 (i) a nucleic acid sequence coding for all or part a MIP chemokine, (ü) at least one nucleic acid sequence coding for all or part of a polypeptide having at least one cytotoxic activity, _ said nucleic acid sequences being placed under the control of elements necessary for their expression in a host cell and being defined as shown above.
The present invention also relates to a viral particle, in particular adenoviral, comprising a recombinant viral vector according to the invention. Such a viral particle can be generated from a vector viral according to any conventional technique in the field of art. Her propagation is carried out in particular in a complementation cell adapted to the deficiencies of said vector. Being an adenoviral vector, we will for example use a complementaion line as described in application WO 94/28152, to line 293 established from cells of human embryonic kidney, which effectively complements the E1 function (Graham et al., 1977, J. Gen. Virol. 36, 59-72), the A549-El line (Imler and al., 1996, Gene Therapy 3, 75-84) or a line allowing a 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). We can also employing auxiliary viruses to complement at least part the defective functions. By complementation cell, we mean a cell capable of providing early and / or late factors in trans necessary for the packaging of the viral genome in a viral capsid for generate a viral particle containing the recombinant vector. Said cell alone may not complement all the defective functions of the vector and in this case can be transfected / transduced by a vector / virus auxiliary providing additional functions.
The invention also relates to a method for preparing a viral particle, according to which

19 (i) a recombinant vector according to the invention is introduced into a cell, in particular a complementation cell capable of complement in trans said vector, so as to obtain a so-called transfected cell, (ü) said transfected cell is cultured under conditions suitable for allowing the production of said viral particle, and (iii) recovering said viral particle in cell culture.
Of course, the viral particle can be recovered from the supernatant of culture but also from cells. One of the commonly used methods employed consists in lysing the cells by consecutive cycles of freeze / thaw to collect virions in the supernatant of lysis. These can be amplified and purified according to the techniques of the art (chromatographic process, ultracentrifugation in particular through a cesium chloride gradient ...).
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 cell, LCA4 or PERC6. Such a cell is particularly useful for producing the particles viral in high titer, without generating particles competent for the replication. 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 is constituted by any cell transfectable by a vector recombinant or infectable by a viral particle, as defined above.
A mammalian and in particular human cell is suitable for everything particularly. It can include said vector in integrated form in the genome or not (episome). It can be a primary or tumor cell of any origin, including hematopoietic (stem cell totipotent, leukocyte, lymphocyte, monocyte or macrophage ...), muscular (satellite cell, myocyte, myoblast, smooth muscle ...), cardiac, pulmonary, tracheal, hepatic, epithelial or fibroblast.
The invention also relates to a composition intended for anti-tumor or antiviral treatment, or any applications 5 requiring cell death, in a mammal comprising (i) all or part of the MIP polypeptide, (ü) all or part of a poiypeptide having at least one cytotoxic activity, said polypeptides being defined as indicated above.
Another object according to the invention consists of a formulation intended for the implementation of a cytotoxic treatment, in particular anti-tumor or antiviral, in a mammal characterized in that it comprises a composition (based on nucleic acids or polypeptides as described previously), an adenoviral vector or a viral particle according to 15 invention, as well as a support acceptable from a point of view pharmaceutical.
Such a support is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as by example a sucrose solution. Furthermore, such a support may contain any solvent, or aqueous or partially aqueous liquid such as water sterile

20 non-pyrogenic. The pH of the formulation is further adjusted and buffered to of meet the requirements for use in viao. The formulation can also include a diluent, adjuvant or excipient acceptable from a point of view pharmaceutical, as well as solubilizing and stabilizing agents, of preservation. For an injectable administration, a formulation in aqueous, non-aqueous or isotonic solution. She may be presented in single dose or multidose in liquid or dry form (powder, lyophilisate ...) capable of being reconstituted so extemporaneous with an appropriate thinner.
According to a particular embodiment of the invention, said formulation comprises in in addition to pharmaceutically acceptable amounts of a

21 prodrug capable of being transformed into a cytotoxic molecule by a polypeptide having at least one cytotoxic activity.
Such a prodrug will be selected in particular 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. So completely favorite, said prodrug is 5-fluorocytosine (5FC) or 5-fluorouracil (5-FU).
Furthermore, 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 include one or more substances potentiating the cytotoxic effect of 5-FU. We can cite in in particular, drugs that inhibit enzymes in the biosynthetic pathway novo pyrimidines (e.g. those listed below), drugs such as the 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 inhibition of thymidylate synthase which leads to a decrease in the pool of dTMP necessary for replication and finally drugs such as methotrexate (Cadman et al., 1979, Science 250, 1135-1137) which by inhibiting dihydrofolate reductase and raising the PRPP incorporation pool (phosphoribosyipyrophosphate) causes the increase of 5-FU in RNA
cellular.
A formulation according to the invention is more particularly intended for preventive or curative treatment of diseases by gene therapy and is intended more particularly to proliferative diseases (cancers, tumors, restenosis ... etc) and infectious diseases, especially viral for which it is necessary to limit the proliferation of cells infected (induced by hepatitis B or C viruses, HIV, herpes, retrovirus .... etc).
A formulation according to the invention can be manufactured so conventional for administration by local, parenteral or digestive. There are many possible routes of administration. We can

22 cite for example the intragastric, subcutaneous, intracardiac route, intramuscular, intravenous, intraperitoneal, intratumoral, intranasal, intrapulmonary or intratracheal. For these last three modes of embodiment, administration by aerosol or instillation is advantageous.
The administration can take place in single or repeated dose one or more times after a certain interval. The route of administration and dosage depending on various parameters, for example, the individual, the disease to be treated or the gene (s) of interest to to transfer. Preparations based on viral particles according to the invention can be formulated in doses between 104 and 1014 pfu (range forming units), advantageously 105 and 1013 pfu and, preference, 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 can be considered. A
composition based on polypeptides preferably comprises from 0.05 to 10 g and, most preferably 0.5 to 5 g of said polypeptide. Of course, doses can be adjusted by the clinician.
The present invention also relates to therapeutic use or prophylactic of a composition, a recombinant vector or a 2o viral particle according to the invention for the preparation of a medicament intended to the treatment of the human or animal body by gene therapy, in particular for the preparation of a cytotoxic, in particular antumoral or antiviral, intended to inhibit growth or cause rejection of a tumor or the death of an infected cell. According to a first possibility, the drug can be administered directly in vivo (e.g. by injection intravenously, in an accessible tumor or at its periphery, in the lungs by aerosol, in the vascular system by means of a probe appropriate ...). We can also adopt the ex vivo approach which consists of collect cells from the patient (stem cells from the bone marrow, per ~ ipher ~ ic blood cells, muscle cells ...), transfect or infect in vitro according to the techniques of the art and re-administer them to patient. A preferred use consists in treating or preventing cancers, tumors and diseases resulting from unwanted cell proliferation.
Among

23 possible applications include breast cancer, uterine cancer (especially those induced by papillomas virus), prostate, lung, bladder, liver, colon, pancreas, stomach, esophagus, larynx of the central nervous system and blood (lymphomas, leukemia etc ...).
It is also useful in the context of cardiovascular diseases, for example example to inhibit or delay the proliferation of muscle cells smooth of the vascular wall (restenosis). Finally with regard to diseases infectious, application to AIDS may be considered.
It is also possible, if necessary and without departing from the scope of the present invention, to carry out simultaneous or successive by different routes of the different components included in the pharmaceutical composition or formulation according to the invention.
The invention also extends to a method for the treatment of diseases by gene therapy, characterized in that one administers to a organism or host cell in need of such treatment a sequence nucleotide, a recombinant vector, a viral particle or a cell host according to the invention.
When the processing method implements a sequence nucleotide, a recombinant vector or a viral particle allowing the expression of a polypeptide according to the invention having ~ zne UPRTase activity, he may be advantageous to additionally administer a second sequence nucleotide encoding a second polypeptide having activity CDase, said second nucleotide sequence being carried by said vector recombinant or viral particle or by a vector or a viral particle independent. In the latter case, the administration of the UPRTase and CDase can be simultaneous or consecutive, the order of administration being unimportant.
According to an advantageous embodiment, the therapeutic use or the treatment method also includes an additional step whereby the host organism or cell is administered amounts pharmaceutically acceptable from a predrogue,

24 advantageously an analog of cytosine and, in particular of 5-FC. AT
For illustrative purposes, a dose of 50 to 500 mg / kg / day can be used with a preferably 200 mg / kg / day. In the context of the present invention, the predrogue is administered according to standard practices and this in a way 5 prior, concomitant or even later than that of the agent therapeutic according to the invention. The oral route is preferred. You can administer a dose single pre-drug or repeated doses for sufficient time long to allow the production of the toxic metabolite within the host organism or cell.
10 According to an advantageous embodiment of the invention, the therapeutic use or the treatment method is associated with a second treatment of the patient by surgery (including removal of the tumor partially or totally), by radiotherapy or chemotherapy. In this particular case, the treatment according to the invention is applied beforehand, concomitantly 15 or follows said second treatment. Preferably, this treatment will be applied following said second treatment.
The following examples are intended to illustrate the different objects of the present invention and therefore have no limiting character.
FIG. 1 represents the evolution of the tumor volume in B6D2 mice 2o implanted with B 16F0 tumor cells.
FIG. 2 represents the survival rate of these same mice. Of groups of 15 mice are treated using compositions comprising adenovirus expressing the following genes: huMIPa, huIL2, huMIPla + huIL2, Empty ad.

FIG. 3 represents the evolution of the tumor volume in mice B6D2 implanted with RENCA tumor cells.
FIG. 4 represents the survival rate of these same mice. Of groups of 15 mice are treated using compositions comprising adenovirus expressing the following genes: huMIP (3, huIL2, huMIP (3 + huIL2, 30 Ad empty.
FIG. 5 represents the evolution of the tumor volume in mice B6D2 implanted with P815 tumor cells.

FIG. 6 represents the survival rate of these same mice. Of groups of 15 mice are treated using compositions comprising adenovirus expressing the following genes: Tris buffer, huMIPa + huIL2, huMIPla + muIFNy, huIL2 + muIFNy.
5 Figure 7 represents the evolution of the tumor volume in mice B6D2 implanted with RENCA tumor cells. These mice are treated using compositions comprising adenoviruses expressing the MIP1 gene (i human (huMIP1 (i) in combination with adenoviruses expressing the gene murine IL12 (muILl2), or adenoviruses expressing the muILl2 gene, or 10 adenoviruses containing no transgene (Ad empty).
EXAMPLES
The constructions described below are carried out according to the general techniques of genetic engineering and molecular cloning, detailed 15 in Maniatis et aL, (19 $ 9, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY) or as recommended by manufacturer when using a commercial kit. The recombination steps homologous are preferably carried out in the strain E. soli BJ 5183 (Hanahan, 1983, J. Mol. Biol. 166, 557-580). Regarding the repair of 20 restriction sites, the technique used consists of filling in the protruding 5 'ends using the large fragment of DNA polymerase I
of. coli (Klenow). Furthermore, the adenoviral genome fragments used in the various constructions described below, are indicated precisely according to their position in the nucleotide sequence of the Ad5 genome such 25 as disclosed in the Genbank database under the reference M73260.
Regarding cell biology, cells are transfected or transduced and cultivated according to standard techniques well known from the skilled person.
Tumor models Three tumor cell models were chosen to assess the activity of the composition of the invention: P815 (mastocytoma H-2d, described in Dunn et al, 1957, J. Natl. Cancer Inst., 18, 587-590), B16F0 (melanoma

26 H-2b, described in Wu et al, 1996, Cancer Res., 56, 21-26) and RENCA
(H-2d renal cell carcinoma, described in Murphy et al, 1973, J. Natl. Cancer Inst., 50 (4), 1023-1025). The cells (3E + 5 for each tumor model) are implanted on D-7 / D-11 subcutaneously in the right flank of B6D2 mice 6 to 8 weeks old.
Administration of inventzon cytotoxic compositions A volume of 100 Nl of adenoviral vectors (5 x 108 units infectious) is injected directly into tumors when their volume is around 4 to 10 mm3 (OJ). This injection is repeated in the same conditions on D 1 and D 2.
The effectiveness of the composition of the invention administered is controlled by measuring the size of the tumors as well as by measuring the time of survival of mice treated with a status check if necessary immunology of the animal by ELISPOT, CTL test, ... Animals can in addition to then being subjected to a contra-lateral challenge during which a lethal dose of tumor cells is administered to the pre-treated animal.

Construction of pTG13010 (huMIPla).
Human MIPla cDNA (GenBank Accession Number X03754; sequence incorporated into the request by reference) has been assembled by synthetic oligonucleotides following the sequence described by Obaru, K. & al.
1986, J. Biochem. 99 (3), 885-894. This cDNA was introduced into a vector derived from pBluescript to give the vector pTG13006.
The NotI-Asp718 fragment of pTG13006 containing the MIPla gene is isolated and introduced into the vector pTG $ 347 cleaved by these same enzymes, for give the transfer vector pTG 13008. As an indication, pTG8347 is a vector p polyII (Lathe et al., 1987, Gene 57, 193-201) in which are inserted the Ad5 sequences 1 to 458, the RSV promoter, the sequences splice of rabbit beta-globin 1 intron 2, the sequences of polyadenylation of rabbit beta-globin 1 and the Ad5 sequences 3328-5788.
Such a construction is within the reach of those skilled in the art, especially on the

27 basis of French demand 97 06757. The adenoviral vector pTG 13010 is reconstituted by recombination in the E. coLi BJ 5183 strain between the PacI-BstEII fragment of pTG 13008 and the vector pTG6624 (described in French application 97 06757) linearized by CIaI. As an indication, pTG6624 corresponds to the plasmid p poly II carrying the genome Ad5 deleted from the E1 regions (nt 459 to 3327) and E3 (nt 28592 to 30470) ia MIP expression cassette being inserted in place of E 1.
The final construct pTG13010 contains the deleted Ad5 genome of most of the regions E1 (nt 459 to 3328) and E3 (nt 28249 to 30758) and instead 10 and place of E1, a cassette for the expression of the MIPIa gene placed under the control of the RSV promoter and of the intron 2 splicing sequences of the rabbit beta-globin 1. Adenoviral particles are generated by transfection into a line of complementation of the E1 function, by example line 293 (ATCC CRL1573) according to art techniques (Graham and Prevec, 1991, Methods in Molecular Biology Vol7, Gene Transfer and Expression Protocols; Ed EJ Murray, The Human Press Inc, Clinton, NJ).
Construction of pTG13023 (huMIP1 ~ i / variant Act2).
The MIP1 cDNA (3 human (GenBank Accession Number: J04130;
sequence incorporated into the request by reference) has been assembled by synthetic oligonucleotides following the sequence described by Lipes, MA and al.
1988, Proc. Natl. Acad. Sci. USA 85 (24), 9704-9708. This cDNA was introduced into a vector derived from M13TG130 (KIENY et al. 1983, Gene, 26, 91-99) to give the vector M 13TG 13013.
The NotI-Asp718 fragment of M13TG13013 containing the MIP1 gene (3 is isolated and introduced into the vector pTG8347 cleaved by these same enzymes, to give the transfer vector pTG13015. Such a construction is at the scope of a person skilled in the art, in particular on the basis of French demand 97 06757. The adenoviral vector pTG13023 is reconstituted by recombination in the E. coli BJ 5183 strain between the PacI-BstEII fragment of pTG 13015 and the vector pTG6624 (described in French application 97 0675?) linearized by CIaI.
The final construct pTG13023 contains the deleted Ad5 genome of most of the regions E 1 (nt 459 to 3328) and E3 (nt 28249 to 30758) and location

28 and place of E1, a cassette for the expression of the MIP1 gene (i placed under the control of the RSV promoter and of the intron 2 splicing sequences of (3 rabbit globin 1. Adenoviral particles are generated by transfection in an EI complementation line, for example the line 293 (ATCC CRL1573) according to art techniques (Graham and Prevec, 1991, Methods in Molecular Biology VoI7, Gene Transfer and Expression Protocols;
Ed EJ Murray, The Human Press Inc, Clinton, NJ).

In vivo experiments.
In order to evaluate the capacity of the compositions of the invention to inhibit the tumor growth in vivo, 3,105 B cells 16F0, RENCA or P815 are injected at (J = -10 / -?) in immunocompetent B6D2 mice. As soon as the tumors become palpable (D = 0) different compositions (see the legend of Figures 1 to 6) are injected three times (J0, J1, J2) by track infra-tumor at a dose of 5.108 infectious units.
The results show an increase in rates survival (Figures 1, 3 and 5) associated with a decrease in tumor volumes (Figures 2, 4 and 6) in mice treated with the compositions comprising a adenovirus expressing MIPla or MIP1 (3 associated with IL2 or l2FNy. These results confirm the interest of the compositions of the invention in the implementation artwork anti-tumor treatment.
The mice thus treated are then subjected to a counter challenge.
lateral consisting of administration under the conditions described previously from a lethal dose (3,105 cells) of tumor cells to J80 / J100. It was thus found that the results described above are in in addition to an immune status of the mouse such that no tumor is able to develop after this challenge stage.

In vivo experiments In order to evaluate the capacity of the compositions of the invention to inhibit the growth of tumors in vivo, 4,105 RENCA cells are injected at (D = 0) WO 00 / 74b29 PCTIFR00J01559

29 in B6D2 immunocompetent mice. As soon as the tumors become palpable (D - 6) different compositions containing either 2,108 units infectious (ui) adenovirus expressing the human MIPl gene (3 (huMIPl (3) in combination with 2.108 ui of an adenovirus expressing the mutated IL12 gene (muILl2), or 2.108 iu of an adenovirus expressing the muILl2 gene in combination with 2.108 ui of an adenovirus containing no transgene (Empty Ad), i.e. 4.108 of an empty Ad, are injected three times (J6, J7, J8) infra-tumor. For all these compositions, the adenoviruses are in a solution containing 100 mM Tris and 10 mM MgCla. He was by elsewhere verified that the mice treated under the same conditions by a composition comprising only adenoviruses expressing the MIP1 gene (3 had tumors of the same volume or even larger than those observed in mice treated with a composition comprising Ad empty.
The results obtained according to this example (Figure 7) highlight a decrease in tumor volumes in mice treated with compositions of the invention comprising an adenovirus expressing MIP1 (3 associated with an adenovirus expressing IL12, especially by comparison to the results observed when treating mice with muILl2 alone. These results confirm the interest of the compositions of the invention in the implementation of an anti-tumor treatment.

Claims (22)

1. Composition intended for the implementation of a treatment cytotoxic in a mammal comprising:
(i) a nucleic acid sequence encoding all or part a MIP chemokine, (ii) at least one nucleic acid sequence encoding any or part of a polypeptide having at least one cytotoxic activity, said nucleic acid sequences being placed under the control of elements necessary for their expression in a host cell of said mammal. 2. Composition according to claim 1, characterized in that said MIP chemokine is the MIP1 chemokine, and more particularly selected from the group consisting of the chemokines MIP1a and MIP1.beta.. 3. Composition according to one of claims 1 or 2, characterized in that said polypeptide having a cytotoxic activity is chosen from them cytokines, proteins encoded by suicide genes and factors protein anti-angiogenics. 4. Composition according to claim 3, characterized in that that said polypeptide having cytotoxic activity is a selected cytokine among the interferons .alpha., .beta. and .gamma., interleukins, necrotizing factors of tumors and colony stimulating factors. 5. Composition according to claim 4, characterized in that said polypeptide having cytotoxic activity is interleukin-2 (IL-2). 6. Composition according to claim 4, characterized in that said polypeptide having cytotoxic activity is interferon gamma (IFN-.gamma.). 7. Composition according to one of claims 1 to 6, characterized in what it comprises in (ii) at least two nucleic acid sequences encoding for all or part of interleukin-2 (IL-2) and all or part of interferon gamma (IFN-.gamma.). 8. Composition according to claim 4, characterized in that said polypeptide exhibits at least one cytotoxic activity selected from thymidine kinase activity, purine nucleoside phosphorylase activity, the activity guanine phosphoribosyl transferase and cytosine deaminase activity. 9. Composition according to claim 8, characterized in that said polypeptide has at least one CDase activity and one activity UPRTase. 10. Composition according to claim 4, characterized in that said polypeptide having cytotoxic activity is an anti-angiogenic selected from angiostatin, endostatin, factor platelet PF4, thrombospondin-1, PRP, VEGI, metalloproteases and urokinase. 11. Composition according to claim 1, characterized in that said nucleic acid sequences (i) and (ii) are inserted into a vector recombinant of plasmid or viral origin. 12. Composition according to claim 11, characterized in that said nucleic acid sequences (i) and (ii) are inserted into the same recombinant vector. 13. Composition according to claim 11, characterized in that said nucleic acid sequences (i) and (ii) are inserted into vectors distinct recombinants. 14. Vector including:
(i) a nucleic acid sequence encoding all or part of MIP chemokine, (ii) at least one nucleic acid sequence encoding any or part of a polypeptide having at least one cytotoxic activity, said nucleic acid sequences being placed under the control of elements necessary for their expression in a host cell. 15. Vector according to claim 14, characterized in that it is of a viral vector. 16. Viral particle comprising a vector according to claim 15. 17. Process for the preparation of a viral particle according to the claim 16, according to which:
(i) introducing a viral vector according to claim 15 into a cell capable of producing said vector, so as to obtain a cell transfected, (ii) said transfected cell is cultured under conditions appropriate to allow the production of said viral particle, and (iii) said viral particle is recovered in the cell culture. 18. Composition intended for the implementation of a treatment cytotoxic in a mammal comprising (i) all or part of a MIP polypeptide, (ii) all or part of a polypeptide having at least one activity cytotoxic, wherein said polypeptides (i) and (ii) are as defined in claims 1 to 10. 19. Formulation intended for the implementation of a treatment cytotoxic in a mammal characterized in that it comprises a composition according to one of claims 1 to 13, a vector according to claims 14 or 15, a virus particle according to claim 16, or a composition according to claim 18, as well as a support pharmaceutically acceptable. 20. Formulation according to claim 19, characterized in that it contains pharmaceutically acceptable amounts of a prodrug capable of being transformed into a cytotoxic molecule by a polypeptide having at least one cytotoxic activity. 21. Formulation according to claim 20, characterized in that said prodrug is selected from 5-fluorouracil (5-FU) and 5-fluorocytosine ((5-FC). 22. Use of a composition according to claims 1 to 13, of a vector according to claims 14 to 15, of a viral particle according to claim 16, or of a composition according to claim 18, for the preparation of a cytotoxic drug.