AU729908B2 - Antitumor cellular compositions expressing at least three transgenes - Google Patents

Description

WO 97/35995 PCT/FR97/00521 Antitumor cellular compositions expressing at least three transgenes The subject of the present invention is a cellular composition for the treatment or prevention of tumors in humans or animals. More particularly, it comprises a population of cells capable of expressing a combination of at least three therapeutic genes having an antitumor effect. The invention also relates to the therapeutic use of such a composition in the field of cancerology.

For the past ten years or so, numerous publications have mentioned the possibility of eliminating tumors or, failing this delaying their progression by the technique of gene transfer. The numerous clinical trials which have been put in place confirm the increasing importance of gene therapy for cancer. The majority of protocols uses retroviral vectors because of their capacity for integration into dividing cells to transfer an antitumorrelated gene. These vectors, as well as the encapsidation lines which make it possible to produce them, are described in numerous prior state of the art documents.

Several approaches have been considered, in particular the transfer of immunostimulatory genes (immunotherapy) capable of inducing or activating a cellular immune response toward the tumor, of cytotoxic genes which confer toxicity on the cells expressing them and of antioncogenes (tumor-suppressor genes or genes capable of inhibiting the activity of an oncogene). These various approaches are summarized in the prior state of the art (see for example Moolten, 1994, Cancer Gene Therapy 1, 279-287; Anderson, 1994, Human Gene Therapy 5, 1-2, Vile and Russel, 1994, Gene Therapy 1, 88-89; Culver and Blaese, 1994, Trends in Genetics 10, 174-178; Zier et al., 1996, Immunology Today, 17, 39-45).

Immunotherapy is based on the transfer of genes encoding 2 cytokines and costimulatory molecules with the aim of making the tumor cells more immunogenic and strengthening the host's antitumor immune response. The cytokines thus evaluated in murine models, and for which an antitumor property has been demonstrated, are interleukin (IL) 2, IL-4, IL-6, IL-7, IL-12, tumor necrosis factor (TNF) type alpha, GM-CSF (for Granulocyte-Macrophage Colony Stimulating Factor) and the interferons (IFN). Xenogenic cells producing IL-2 have thus demonstrated an antitumor effect (see European Application EP 0 579 791).

The cytotoxic approach consists in specifically increasing the sensitivity of the tumor cells to chemotherapy by transferring a so-called suicide gene whose product is capable of converting an inactive precursor to a highly cytotoxic product. The most widely used up until now is the Herpes Simplex virus type 1(HSV- 1) tk gene encoding the thymidine kinase (TK) enzyme which has the property of converting acyclovir and ganciclovir to phosphorylated analogs of nucleosides.

These abberant nucleotides are then incorporated, during cell division, into the newly formed chromosomal DNA chains, the consequence of which is an inhibition of genetic replication and death of dividing tumor cells (Moolten, 1986, Cancer Res. 46, 5276-5281). An additional advantage of this approach is conferred by the bystander effect which results from propagation of the cytotoxic properties to the neighboring cells, causing their destruction (Freeman et al., 1993, Cancer Res. 53, 5274- 5283). Thus, ;etroviruses carrying the tk gene have been used in the context of the treatment of malignant brain tumors with the aim of triggering cellular suicide after administration of ganciclovir (Izquierdo et al., 1996, Gene Therapy 3, 491-495). For the same purpose, Application WO 95/06486 proposes an injection of murine fibroblasts which produce a retroviral vector expressing the cytotoxic gene.

3 Finally, the antioncogene strategy is based on the introduction, into tumor cells, of a functional copy of a tumor-suppressor gene (for example the gene associated with retinoblastoma or p53) or the inhibition of the expression of the oncogenes by the transfer of genes encoding antisense polynucleotides or ribozymes capable of degrading the messenger RNAs of the oncogenes with the aim of reducing or abolishing the proliferation of the cancer cells.

A number of recent experimental anticancer protocols propose combining several therapeutic genes in order to obtain a synergistic antitumor effect (see for example Application PCT/FR94/00192; Rosenthal et al., 1994, Blood 83, 1289-1298; O'Malley et al., 1996, Cancer Res. 56, 1737-1741; Lollini et al., 1995, Human Gene Therapy 6, 743-752). However, given their large number and their diversity, all these studies do not currently allow the molecule or the combination of molecules best suited to the treatment of a given tumor to be clearly identified.

An antitumor composition has now been identified which is based on cells which are genetically modified in order to secrete IL-2 and to produce retroviral particles expressing the tk genes of the HSV-1 virus and IFNy. Such a composition makes it possible to inhibit or delay cell proliferation by inducing specific death of tumor cells, a better presentation of antigens and stimulation of the host's immune cells. The present invention offers an effective alternative to the prior art techniques for treating cancer in humans or animals.

Accordingly, the subject of the present invention is an antitumor composition comprising a cell population allowing the expression of at least three antitumor therapeutic genes.

"Antitumor therapeutic genes" are understood to mean 4 genes whose expression products have an antitumor effect, in particular for enhancing immunity directed specifically against the tumor and/or for at least partially inhibiting cell division. Preferably, they are genes encoding polypeptides immunostimulatory for the immune response and/or cytotoxic polypeptides.

"Immunostimulatory" is understood to mean any polypeptide capable of amplifying the production of antibodies directed against the tumor cells and antigens or of stimulating a cell-mediated immune response, by activating the T lymphocytes, so as to trigger a cytotoxic response or a significant retarded hypersensitivity-type response against tumor cells.

"Cytotoxic" is understood to mean any polypeptide capable of inducing cell death either directly or indirectly through a drug capable of being administered independently.

Among the immunostimulatory genes which can be used within the framework of the present invention, there may be considered the genes encoding cytokines and in particular interleukins colony stimulating factors (G-CSF, M-CSF and GM-CSF), interferons (IFN), tumor necrosis factors (TNF), costimulatory factors such as polypeptides B7.1 and B7.2 and factors activating the expression of class II histocompatibility antigens.

At present, 16 interleukins have been identified. It is particularly difficult to attribute a specific function to them because they exert pleiotropic effects. Within the framework of the present invention, all the interleukins are of interest, but there may be mentioned more particularly IL-2, IL-4, IL-6, IL-10 and IL-12. In this context, IL-2 is particularly preferred. It is responsible for the proliferation of the activated T lymphocytes and, in association with IFNy, stimulates the macrophages, the Natural Killer (NK) cells and the T cells. Furthermore, some studies appear to show that it 5 has a chemotactic role for the lymphocytes when it is produced at the level of a tumor.

Interferons possess antiviral and immunomodulatory properties. They can activate the phagocytic cells and increase the expression of the class I and II surface antigens of the major histocompatibility complex (MHC) and also stimulate the cytotoxicity of the NK cells and those activated by the lymphokines (LAK for Lymphokine Activated Killer) against tumor cells. Although the three principal classes of IFN, a, 3 and y respectively, are of interest within the framework of the present invention, IFNy would be most particularly preferred.

Colony stimulating factors are involved in the maturation of the hematopoietic stem cells and their differentiation into mature cells in the bloodstream. GM-CSF, G-CSF and M-CSF (for Granulocyte-Macrophage, Granulocyte and Macrophage respectively) can be distinguished according to the stage of maturation and the cell type on which these factors exert their influence.

As regards tumor necrosis factors, there may be distinguished TNFa produced by the macrophages and TNF3 produced by the T lymphocytes. Both are responsible for the antitumor cytotoxic activity of the macrophages and lymphocytes and for the local tissue impairment observed in the inflammatory reaction.

Among the cytotoxic genes, there may be mentioned those encoding a thymidine kinase and, in particular the thymidine kinase (TK) of the Herpes Simplex virus type 1 (HSV-1), cytosine deaminase, cytochrome P450 2B1, purine nucleoside phosphorylase encoded by the E.coli DeoD gene, nitroreductase and 3 -glucoronidase. In general, these toxic genes are described in the literature (see for example Moolten, 1994, Cancer Gene Therapy 1, 279-287).

6 According to a preferred embodiment, the three therapeutic genes used within the framework of the present invention encode the polypeptides HSVl-TK, IL-2 and IFNT. The origin of the immunostimulatory genes is chosen so that they can be functional in the host for which the antitumor composition according to the invention is intended. In the case of a human host, the coding sequences for human IL-2 and IFNy will be preferably selected.

The therapeutic genes may be obtained by cloning, by PCR or by chemical synthesis according to the conventional techniques commonly used. They may be native genes or genes derived from the latter by mutation, deletion, substitution and/or addition of one or more nucleotides.

Of course, they may comprise appropriate elements for regulation of transcription as well as signals for initiation and termination of translation allowing their expression. In general, use will be made of a promoter region which is functional in the cells of the host whom it is desired to treat, preferably in human cells. It may be the promoter region naturally controlling the expression of the said gene or a promoter region of a different origin, for example derived from eukaryotic or viral genes. Moreover, the promoter region may be modified so as to contain regulatory sequences, for example a transcription activating element (enhancer).

The promoter region selected may be consitutive or regulatable, and in the latter case, in response to certain cellular signals. It will be advantageous to use a tissue-specific promoter region when the tumor to be treated is derived from a specific cell type.

Alternatively, the use of a promoter responding to specifically tumoral signals (for example responding to factors which are overexpressed by tumor cells) may prove advantageous since the expression will be limited to the tumor cells.

7 Such promoters are generally known to a person skilled in the art. There may be mentioned in particular the (Simian Virus 40), PGK (Adra et al., 1987, Gene 60, 74), HMG (Hydroxy-Methyl-Glutaryl-coenzyme A) and TK (Thymidine Kinase) promoters, the promoters of the LTRs (Long Terminal Repeat) of RSV (Rous Sarcoma Virus), of Mo-MLV (Moloney Murine Leukemia Virus) and the promoters of the genes encoding the class I MHC antigens which are activated by IFNy. These examples are not limiting.

Within the framework of the present invention, the therapeutic genes may be placed under the control of elements allowing their expression independently or together. In other words, they may be expressed in a monocistronic or polycistronic manner. In the latter case, an element will be used which allows the reinitiation of translation at the level of the second cistron, for example an internal ribosome entry site (IRES) well known to a person skilled in the art. To date, a number of IRESs have been identified in the region of viral mRNAs and, in particular, from picornaviruses such as the poliomyelitis virus (Pelletier et al. 1988, Mol. Cell. Biol. 8, 1103-1112) and EMCV (Encephalomyocarditis virus; Jang et al., 1988, J. Virol.

62, 2636-2643). It is also possible to use the IRESs described in International Application WO 96/01324.

Moreover, it may be advantageous for the immunostimulatory polypeptides to be secreted outside the cells constituting the antitumor composition according to the invention. In this context, the corresponding genes may also include a signal sequence. This may be the natural signal sequence or a heterologous sequence as long as it is functional in the host cell.

Of course, the said cell population may derive from a primary or tumor cell, from a cell line, from an organ or may comprise a mixture of cells allowing the expression 8 of the various therapeutic genes. To illustrate this embodiment, use may be made of a mixture of cells, for example derived from the Vero line (available from ATCC), one portion expressing IL2 and another portion expressing IFNy, optionally combined with a retroviral encapsidation line allowing the production of virions or a virus population expressing the cytotoxic TK gene. Of course, other combinations may also be considered. For example, it is also possible to use a cell mixture comprising a line (for example Vero) allowing the expression of the IL2 gene and a retroviral encapsidation line allowing the production of infectious particles comprising a retroviral vector according to the invention as defined below.

According to a particularly advantageous embodiment, the cell population constituting the antitumor composition according to the invention comprises or consists of retroviral encapsidation cells allowing the production of infectious particles comprising a retroviral vector.

Generally, and for safety reasons, the retroviral vector is defective through deletion or mutation of the viral gag, pol and env genes and as a result cannot replicate autonomously. Its propagation requires the supply of the viral polypeptides for which it is deficient. An encapsidation cell is capable of providing in trans all the polypeptides which the retroviral vector cannot synthesize and which are necessary for the constitution of the infectious particles. Preferably, the encapsidation cells in use in the present invention are derived from a line of human origin, in particular from the 293 line. The latter can be generated by transfection of vectors allowing the expression of the gag/pol genes of the FMuLV virus (Friend Murine Leukemia Virus) of the FB29 strain and the env gene of the amphotropic virus 4070A. By way of example, there may be mentioned the E17 line described in international application PCT/IB96/00439 whose content is incorporated by way of 9 reference.

A preferred antitumor composition according to the invention comprises a cell population containing a vector allowing the expression of a gene encoding IL-2 and allowing the production of infectious particles which have incorporated a retroviral vector expressing a gene encoding thymidine kinase of the HSV-1 virus and a gene encoding IFNy.

According to an advantageous embodiment, the cell population in use within the framework of the present invention is sensitive to a drug allowing its elimination. In the case where it expresses the HSV-1 tk gene, it is possible to consider using a drug derived from acyclovir and, in particular, ganciclovir. The cell population may, in addition, express a positive selection marker, for example a gene for resistance to an antibiotic facilitating its selection. There may be mentioned the neo (neomycin) gene conferring resistance to the antibiotic G418, the dhfr (dihydrofolate reductase) gene, the pac (puroacetyl transferase) gene (Morgenstern and Land, 1990, Nucleic Acids Res. 18, 3587- 3596), the hygromycin B gene and the gpt (xanthine phosphoribosyl) gene.

The present invention also relates to an encapsidation cell derived from the 293 line and comprising: the gag/pol genes of the FMuLV virus (Friend Murine Leukemia Virus) of the FB29 strain and the env gene of the amphotropic virus 4070A, and a vector allowing the expression of a gene encoding IL-2.

An appropriate vector may consist of the vector pTG5324 described below in which the expression of IL-2 is directed by the CMV virus (Cytomegalovirus) early promoter. However, other expression vectors may of course 10 be used.

The subject of the present invention is also a retroviral vector characterized in that it comprises from 5' to 3': a 5' LTR, an encapsidation region, a gene encoding gamma-interferon, a constitutive internal promoter, a gene encoding thymidine kinase of the HSV-1 virus, an internal ribosome entry site (IRES), a gene encoding a positive selection marker, and a 3' LTR.

A vector according to the invention may derive from any retrovirus. There may be mentioned, by way of examples, the avian retroviruses such as the avian erythroblastosis virus (AEV), the avian leukemia virus (ALV), the avian sarcoma virus (ASV), the spleen necrosis virus (SNV) and the Rous sarcoma virus (RSV), bovine retroviruses, feline retroviruses, murine retroviruses such as the murine leukemia virus (MuLV), Friend's virus (F-MLV) and the murine sarcoma virus (MSV) and primate retroviruses.

However, the use of the Moloney murine leukemia virus (MoMuLV) is most particularly preferred. The numerous retroviral vectors derived from the latter, which are described in the literature, in particular the N2 vector or one of its derivatives, may be used within the framework of the present invention.

The present invention also relates to the use of an antitumor composition, of an encapsidation cell or of a retroviral vector according to the invention, for the manufacture of a medicament intended for the treatment or for the prevention of cancer or of a cancerous condition in humans or in animals. The cancers which could thus be treated are advantageously solid tumors such as kidney, breast, lung and colon cancers and melanomas.

11 The medicament derived from the present invention maybe administered by any general route in common use, in particular by the parenteral route such as the systemic, intramuscular subcutaneous or intraperitoneal route. In general, the intratumor route is recommended as being particualrly advantageous. In general, the administration may take place in a single dose or in a dose repeated once or several times after a certain time interval. According to a preferred embodiment of the invention, the medicament will comprise, in addition, a pharmaceutically acceptable carrier.

It may also comprise a pharmaceutically acceptable vehicle, diluent or adjuvant. The appropriate dosage varies according to various parameters, for example the route of adminstration, the individual to be treated, the nature and the severity of the tumor condition, and the type of therapeutic genes used.

Finally, the subject of the present invention is also a ooooo method for treating cancer in mammals, according to which a 20 pharmaceutically effective quantity of an antitumor composition, an encapsidation cell or a retroviral vector according to the invention is injected into an individual oooo requiring such a treatment.

25 Further, in all of the above embodiments, the invention may be limited to a cell population of human origin.

In one aspect the invention provides an antitumor composition comprising a cell population, characterised in that the said cell population allows the expression of at least three antitumor therapeutic genes and comprises a mixture of cells allowing the expression of the said antitumor therapeutic genes.

MAW:NP:#29458.RS 1 18 December 2000 12 directed by the murine PGK promoter.

Figure 3 illustrates the retroviral vector pTG9326 which is derived from the vector pTG9344 by insertion of the gene encoding canine IFNy.

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 cloning steps using bacterial plasmids are preferably carried out in the E. coli strain XL1-Blue or DH5a (Gibco BRL). The M13 vectors are amplified in the E. coli strain JM110 or NM522. As regards the repair of the restriction sites, the procedure is carried out by filling the protruding 5' ends with the aid of the large fragment of the DNA polymerase I of E. coli (Klenow) or by digestion with S1 nuclease followed by treatment with Klenow. The PCR (Polymerase Chain Reaction) amplification techniques are known to the person skilled in the art (see for example PCR Protocols A guide to methods and applications, 1990, published by Innis, Gelfand, Sninsky and White, Academic Press Inc.).

In the following examples, use is made of the murine cell line NIH 3T3 (ATCC CRL 1685), the canine line MDCK (ATCC CCL 34), the B16 line derived from a murine melanoma (ATCC CRL 6322), the P815 line derived from a murine mastocytoma (ECACC No. 870 42 301), the E17 line (described in detail in international application PCT/IB96/00439) and the PA317 line (Miller and Buttimore, 1986, Mol. Cell. Biol. 6, 2895-2902). As a guide, the E17 line is a retroviral encapsidation line which is derived from the human 293 line (Graham et al., 1977, J. Gen.

13 Virol. 36, 59-72) by transfection of vectors expressing, respectively, the gag/pol genes of the FMuLV virus (Friend Murine Leukemia Virus) of the FB29 strain and the env gene of the amphotropic virus 4070A. The cells are transfected according to standard techniques well known to persons skilled in the art. There may be mentioned the calcium phosphate technique, but any other protocol may also be used, such as the DEAE dextran technique, electroporation, methods based on osmotic shocks, microinjection or methods based on the use of liposomes.

As for the culture conditions, use is made in general of the DMEM medium (Dulbecco's Modified Eagle's Medium, Gibco BRL) containing 10% (vol/vol) foetal calf serum (FCS), 3 g/ml of glucose, 2 mM glutamine, 1% of nonessential amino acids and 40 Ag/ml of gentamycin with the exception of the MDCK and P815 cells for which use is made of the RPMI-1640 medium containing 10% of FCS, 2 mM glutamine, 1% of nonessential amino acids and 40 Ag/ml of gentamycin. The cells transduced with the retroviral vectors expressing the neo gene are cultured in the presence of G418 at a final concentration of 1 mg/ml or mg/ml. The E17 encapsidation cells are cultured in the presence of the selection agent puromycin (1 Ag/ml).

EXAMPLE 1: Construction of a retroviral encapsidation line producing human interleukin-2 (hIL-2) (E17-TG5324) The sequences encoding for hIL-2 are isolated from the vector pTG36 (described in French Patent 85 09480) in the form of a PstI fragment, are subcloned into the vector M13TG130 (Kieny et al., 1983, Gene 26, 91-99) and are subjected to site-directed mutagenesis so as to introduce an Sail site 12 nucleotides downstream of the stop codon (Amersham mutagenesis Kit, RPN 1523). The hIL-2 cDNA is purified from the mutated vector by Sail digestion and inserted into the XhoI site of pBCMGneo (Karasuyama and 14 Melchers, 1988, Eur. J. Immunol. 18, 97-104) situated in 3' and 5' respectively of the splicing and polyadenylation signals of the rabbit 6-globin gene.

pTG5320 is obtained.

In parallel, a BamHI-HindIII fragment carrying the CMV virus (Cytomegalovirus) early promoter purified from the pLNCX (Miller and Rosman, 1989, BioTechniques 7, 980-988) is introduced into the vector p polyIII-I* (Lathe et al., 1987, Gene 57, 193-201) treated with the same enzymes.

The SalI-BamHI fragment purified from pTG5320 carrying the A-globin intron, the hIL-2 cDNA and the /-globin polyadenylation signal is introduced between the SalI and BglII sites situated downstream of this promoter. The resulting vector pTG5321 is linearized with the enzyme BamHI and a BamHI-BglII fragment containing the selectable gene pac placed under the control of the early promoter and the polyadenylation signal of the SV40 virus is inserted. The vector thus obtained is designated pTG5322.

Finally, the vector pTG5324 (Figure 1) is generated by inserting into the preceding vector, linearized with BamHI, a BamHI fragment comprising the murine 12 S mitochondrial sequences (Luftalla et al., 1985, Som.

Cell. Mol. Genet. 11, 223-238).

20 Ag of plasmid pTG5324 are used to transfect the E17 cells at a density of 40 to 50% according to the conventional calcium phosphate technique. The next day, the transfected cells are placed in the presence of puromycin. After two weeks in selective medium, the resistant clones are subcultured, propagated and frozen in liquid nitrogen while waiting to check their capacity to secrete hIL-2. To do this, 6-well culture plates are used into which 4 x 105 cells to be tested are inoculated. The next day, the medium is changed and harvested 24 h later. The quantity of hIL-2 present in 15 the cellular supernatant is estimated by ELISA (R&D Systems Minneapolis, D2050). About one quarter of the clones tested secrete quantities of IL-2 exceeding 2 Ag/ml/10 6 cells/24 h. The most productive clone, designated E17-5324-clone 2 and which secretes 3 Ag/ml/106 cells/24 h of hIL-2 is selected for subsequent studies.

Since the E17-5324 cells are intended for a human therapeutic use, it is advantageous to test their capacity for resistance to inactivation by human complement. To do this, 5 x 104 cells are placed in culture in an appropriate support. On the next day, the medium is removed and the cells are exposed to 0.5 ml of fresh human serum or heat-inactivated human serum (negative control) collected from two different individuals or alternatively FCS (negative control).

After 150 min, the culture is continued in a conventional medium for 24 h. The viable cells are counted after treating with trypsin and staining with trypan blue. The experiment is also carried out in parallel on the 293 cells (line from which the E17 cells are derived), the encapsidation line PA317 and the murine cells 3T3. As expected, the viability of all the lines tested is not affected by the exposure to the FCS or to the inactivated serum. On the other hand, the treatment with fresh serum induces the death of the 3T3 and PA317 cells (viability 0.015%). This phenomenon is not observed with the 293 cells (viability of 80 and 100% depending on the serum) nor with the E17-5324 cells (viability of 75 and EXAMPLE 2: Construction of the retroviral vector pTG9344.

The retroviral vector pTG9344 allows the bicistronic expression of a cytotoxic gene, in this case the HSV-1 tk gene and of the positive-selection neo gene. The parent vector is pLXSP which is derived from pLXSN (Miller and 16 Rosman, 1989, supra). The PGK promoter obtained from the plasmid PKJ-I (Adra et al., 1987, Gene 60, 65-74) in the form of an EcoRI-PstI fragment (positions -517 to relative to the site of initiation of transcription) is introduced downstream of the encapsidation region. The tk gene is isolated from the vector pTK-1 (Spandidos et al., 1982, Exp. Cell. Res. 141, 149-158; Wagner, 1981, Proc.

Natl. Acad. Sci USA 78, 1441-1445) and subcloned into the BamHI site of pBR328 (Covarrubias and Bolivar, 1982, Gene 17, 79-82). The noncoding 3' region is eliminated by SmaI-XbaI digestion and an SmaI-XbaI adaptor resulting from the reassociation of the oligonucleotides oTG4322 and oTG4323 (SEQ ID NO: 1 and 2) is introduced, which makes it possible to reconstitute the last 7 codons situated in 3' of the SmaI site and to create an XbaI site 4 base pairs (bp) after the stop codon. The tk gene thus modified is then isolated in the form of a BglII- XbaI fragment of 1191 bp and inserted downstream of the PGK promoter.

The IRES site of the EMCV virus is then introduced downstream of the tk gene in order to allow the reinitiation of the translation of the second cistron of the bicistronic mRNA. It is isolated from the vector IRES-~geo (described in international application WO 94/24301) by XbaI-NcoI digestion. Finally, the neo gene is cloned downstream of the IRES sequence in the form of a BgII-SmaI fragment produced from (Colbere-Garapin et al., 1981, J. Mol. Biol. 150, 1-14) cleaved with these same enzymes. The final vector pTG9344 is presented in Figure 2.

It is known that the reinitiation of translation from the IRES site is less efficient than the initiation from capped mRNAs. As regards cells transduced by particles derived from the vector pTG9344, resistance to the selection agent G418 depends on the efficiency of the reinitiation from the EMCV IRES. Consequently, when the 17 culture is carried out in a selective medium, only the cells producing large quantities of bicistronic mRNAs are able to survive, thereby ensuring a high level of expression of the tk gene.

EXAMPLE 3: Construction of the retroviral vector pTG9326 containing canine IFNy The vector pTG9344 comprises a unique EcoRI restriction site which allows the insertion of an additional gene under the control of the 5' LTR retroviral promoter.

The canine IFNy is cloned from the cellular RNA isolated from canine T lymphocytes stimulated by concanavalin A.

The cellular RNA is reverse-transcribed using the degenerate primer oTG4031 (SEQ ID NO: The specific fragment is amplified in two stages. First of all, an internal fragment is produced with the aid of the primers oTG4169 and oTG4170 (SEQ ID NO: 4 and Next, two specific oligonucleotides oTG4321 and oTG4319 (SEQ ID NO: 6 and 7) are used in combination with two oligo d(T) adaptors and primer to generate the 5' and 3' fragments according to the RACE method (Frohmann et al., 1988, Proc. Natl. Acad. Sci. USA 85, 8998-9002). The two PCR fragments are cleaved with SphI and AflII before being inserted into the SphI site of M13TG131 (Kieny et al., 1983, supra) and subjected to sequence analysis by conventional techniques. The data confirm a sequence identical to that published by Zucker et al. (1992, J.

Interferon Res. 12, 191-194). The preceding vector M13TG8173 is used as template to isolate, by PCR, the IFNy sequences with the aid of two mutagenic primers oTG6727 and oTG6788 (SEQ ID NO: 8 and 9) which make it possible to introduce an EcoRI site upstream and downstream of the initiation and stop codons respectively. The PCR product after cleaving with EcoRI is cloned into the vector pTG9344 to give pTG9326 (Figure 3).

18 EXAMPLE 4: Construction of the retroviral vector pTG9337 containing murine IFNy The murine IFNy is isolated by PCR on the basis of the sequence data (Gray and Goeddel, 1983, Proc. Natl. Acad.

Sci. USA 80, 5842-5846) with the aid of the primers oTG7295 and oTG7296 (SEQ ID NO: 10 and 11). The retroviral vector pTG9337 results from the insertion of the amplification fragment cleaved with EcoRI inside the EcoRI site of pTG9344.

EXAMPLE 5: Construction of a retroviral vector containing human IFN The human IFNy is isolated by PCR from the vector M13TG2437 which results from the subcloning, into the vector M13TG131, of the IFN coding sequence isolated from pTG23 (Tessier et al., 1984, Nucleic Acids Res. 12, 7663- 7676). The primers oTG6147 and oTG4983 (SEQ ID NO: 12 and 13) are used. The amplified fragment is treated with S1 nuclease and then with Klenow before being cloned into an intermediate vector from which it can be isolated so as to be inserted into the EcoRI site of pTG9344.

EXAMPLE 6: Construction of an encapsidation line secreting hIL-2 and producing the retroviral vector pTG9326 The E17 cells are transfected with 10 ig of each of the plasmids pTG9326 and pTG5324 by the calcium phosphate method and cultured in selective medium (1 Ag/ml of puromycin and 1 mg/ml of G418) 24 h after the transfection. Ten to 14 days later, the resistant cells are subcloned by limiting dilution (placing in culture, in a 96-well plate, of 200 Ml/well of a dilution at a density of 1.5 cells per ml). The cell clones are recovered after two weeks of culture and amplified in a conventional manner. The clones are placed in culture in 19 an amount of 4 x 10 5 cells. After a change of medium, the 24 h culture supernatant is harvested, from which the secreted quantities of hIL-2 and IFNy are estimated.

As regards hIL-2, the ELISA method (R&D Systems, Minneapolis, D2050) previously described is used. The IFN is assayed by the method of inhibition of the cytopathic effect of the VSV virus (Vesicular Stomatitis Virus Indiana strain, ATCC VR 158) on the dog kidney epithelial cell line MDCK (Steward II, in The Interferon System, pp 17-19, Springer-Verlag, NY; Familletti et al., 1981, Methods Enzymology 78, 387). Briefly, 3 x 104 MDCK cells/well are placed in culture in a 96-well microtiter plate and then serial dilutions of the supernatants obtained from the clones resistant to puromycin and to G418 are added. Next, the cells are exposed to 103 cfu (for colony forming unit) of VSV virus and the cytopathy is determined 24 h later. The IFNy makes the cells resistant to VSV infection. The results are given as arbitrary units which correspond to the reciprocal of the dilution for which a 50% protection is obtained. The control cells show a cytopathy greater than Finally, the production of retroviral particles by these clones is evaluated by infecting 1 to 2 x 10 s permissive 3T3 cells. After 24 h of culture, they are exposed for 90 min to 200 Al of 10-fold serial dilutions of cellular supernatant to be tested and 200 tl of medium containing 16 Ag/ml of polybrene (Sigma). The culture is continued in a conventional medium to start with and then in a selective medium (5 mg/ml of G418) 24 h after the infection. The colonies which are resistant after two weeks are stained with crystal violet (0.05% in a water mixture). The number of retroviral particles present in the supernatant of producing clones may be calculated from the number of 3T3 colonies resistant to G418. The clone designated hereinafter E17- TG5324&TG9326 #28, which produces 1 Ag/ml/10 6 cells/24 h 20 of hIL-2, 32 U/ml of IFNy and 4.5 x 106 cfu/ml of viral particles, is selected.

EXAMPLE 7: Construction of an encapsidation line secreting hIL-2 and producing the retroviral vector pTG9337 The cells E17-5324 are transfected with 20 gg of plasmid pTG9337 and the resistant clones are selected in G418 medium (1 mg/ml). After subcloning, the most productive clones in terms of secretion of hIL-2, of murine IFNy and of viral particle titer are evaluated. The methods used are described in the preceding example with the exception of the technique for assaying murine IFNy which is quantified by the ELISA test (PerSeptive Diagnostics, Cambridge, MA, No. 8-6716). The clone selected E17- TG5324&TG9337 #33 produces 1 Ag/ml/10 6 cells/24h of hIL- 2, 60 ng/ml/10 6 cells/24h of IFNy and exhibits a viral titer of 1.3 x 106 cfu/ml.

EXAMPLE 8: Transduction of target cells It is checked that the viral particles produced from the producing lines E17-TG5324&TG9326 and E17-TG5324&TG9337 are capable of transducing target cells and that the expression of the therapeutic genes is not adversely affected in the cellular context. This study is carried out on an established murine line (3T3 cells) and on two primary lines (P3D6M and P3D4M cells). The latter are derived from dog melanomas and subjected to one passage on immunodeficient SCID mice in order to generate homogeneous primary lines. 2 x 10 7 cells at passage 5 stage are injected into the animals by the subcutaneous route. The tumors are removed 3 weeks later and the melanoma cells are maintained in culture under conventional conditions.

The target cells are placed in culture in an amount of 1 to 2 x 10 s cells per well and, on the next day, infected 21 with 0.5 ml of producing cell culture supernatant previously filtered through a 0.45 Am membrane. The infection is allowed to continue for 1 to 2 h and then the cells are again placed in fresh medium. Normally, two transduction cycles are carried out on the same day and the transduced cells are cultured in the presence of G418 mg/ml). The medium is changed every three days until the appearance of resistant colonies whose sensitivity to ganciclovir is evaluated. The tests use 5 x 106 cells which are placed, on the day following their placing in culture, in the presence of ganciclovir at concentrations of between 0 and 1000 AM. The viability of the cells is determined after one week by the trypan blue test. The number of cells counted in the wells in which the culture was carried out in the absence of ganciclovir represents 100%. The results indicate that the three types of target cell transduced by the particles derived from the retroviral vectors pTG9326 and pTG9337 as well as the corresponding producing lines are sensitive to ganciclovir. In other words, their viability is affected in the presence of low doses of ganciclovir (LD50, that is to say the ganciclovir concentration for which viability is obtained, less than 0.1 AM) whereas the nontransduced cells are resistant to much higher concentrations (LD50 over 10 AM). These results show that the level of expression of the tk gene in the transduced tumor cells is sufficiently high to make them sensitive to gancoclovir doses which are nontoxic to normal cells.

EXAMPLE 9: Bystander effect The capacity of the viral particles of vector pTG9344 to induce a cytotoxic bystander effect in the transduced cells is checked. In a first instance, the primary P3D6M cells are transduced with the viral particles pTG9344 and the infected cells are selected in the presence of G418 (1 mg/ml). Next, a coculture is produced containing nontransduced P3D6M cells and a certain percentage of the 22 transduced culture 10, 30, 50, 80 and 100% respectively). The coculture is maintained for 7 days in the presence of Ganciclovir (1 AM) and the viability of the cells is estimated by staining with trypan blue. It is indicated that this concentration is chosen so as to be toxic to the transduced cells expressing thymidine kinase of surviving cells for the 100% test) whereas it does not affect or only slightly affects the viability of the cells not expressing the suicide gene (86% of surviving cells for the 0% test). As regards the coculture tests, ganciclovir exerts a notable toxic effect even at a low percentage of transduced cells. A drastic reduction in viability is already observed when the cellular mixture contains only 10% of transduced cells (18% of surviving cells for the 10% test and 5% for the 30% test). These results indicate that the vector pTG9344 expresses a sufficient quantity of TK to induce cytotoxicity of the infected cell and to propagate this effect to the neighboring nontransduced cells.

EXAMPLE 10: Induction of the expression of the class I and II MHC antigens in the transduced cells This study is intended to check the functionality of the IFN7 genes carried by the vectors pTG9326 and pTG9337.

One of the biological effects of IFNy is to induce the expression of the class I and II MHC antigens.

The P815 (murine mastocytoma) and B16 (murine melanoma) cells are transduced with the vector pTG9344 (tk-neo) as negative control or pTG9337 (murine IFNy-tk-neo) and the presence of class I and II antigens at the surface of the infected cells is determined by conventional immunofluorescence and flow cytometry (FACS) techniques. The class II MHC antigens present at the surface of the P815 and B16 cells are detected by an anti-mouse antibody coupled to FITC (for Fluorescein isothiocyanate) 23 (Pharmingene, San Diego, CA). For the detection of the class I antigens, an anti-H2kb antibody (Pharmingen) is used for the B16 cells and an anti-H2kd antibody (Pharmingen) for the P815 cells.

After infection and maintenance in selective medium (G418 mg/ml) for about two weeks, the cells are detached by the action of 10 mM EDTA in phosphate-buffered saline (PBS) and washed twice in the following buffer (PBS, 1% bovine serum albumin, 0.1% human gammaglobulin, 10 mM EDTA and 0.02% sodium azide). 1 x 10' cells are incubated for 45 minutes at 4 0 C and in the dark in the presence of 200 Al of a 1:50 dilution of each of the antibodies mentioned above or, as negative control, in the absence of antibody or in the presence of a nonspecific antibody (anti-mouse CD8). After washing, the cells are fixed (2% formaldehyde in PBS buffer) and analyzed by FACS (Becton Dickinson cytometer, San Jose, CA).

The results indicate that the expression of the class I MHC antigens at the surface of the cancerous murine cells B16 and P815 (80% for the P815 cells and 95% for the B16 cells) is highly induced by the transfer of the vector pTG9337, showing that the latter expresses a functional IFNy. On the other hand, the P815 and B16 cells transduced by the vector pTG9344 exhibit no fluorescence Furthermore, the fluorescence intensity for the transduced cells expressing IFNy is about 30 to 40 times greater than that obtained with the untreated cells.

Thus, the large number of cells positive for the class I antigens and the intensity of the expression at the surface of these cells due to the expression of IFNy should contribute to a better recognition of the immune effector cells for the purpose of eliminating cancer cells. The expression of the class II MHCs is weakly induced.

A similar experiment is undertaken on the primary canine 24 cells P3D6M infected with the vector pTG9326 (canine IFNy-tk-neo) or, as negative controls, pTG9344 (no expression of IFNy) and pTG9337 (expression of murine IFNy). The class I and II MHC antigens are respectively detected by the monoclonal antibodies H58A recognizing the anti-H2kk molecules and TH14B which is directed against the DR allelic equivalent from several species including dogs (VRMD Inc, Pullman, WA) (Davis et al., 1987, Vet. Immunol, Immunopathol. 15, 337-376). The immunostaining is carried out as described above, with the difference that after the incubation with the antibody solution, the cells are placed in the presence of 200 Al of washing buffer containing a 1:256 dilution of an anti-mouse immunoglobulin G rabbit antibody conjugated to FITC. After 45 min of contact at 4 0 C and in the dark, the cells are washed, fixed and the presence of the class I and II antigens evaluated by FACS.

The results indicate that the expression of the class I and II MHC antigens is strongly induced at the surface of more than 95% of the primary cancer cells P3D6M transduced by the vector pTG9326. The fluorescence intensity for the transduced cells expressing IFNy is about 25 times greater for the I MHCs and 300 times greater for the II MHCs than that obtained with the untreated cells. No induction is produced in the P3D6M cells transduced with the vectors pTG9344 and pTG9337.

EXAMPLE 11: Evaluation in vivo The murine B16 cells are transduced by the vectors pTG9344 and pTG9337 or, as negative control, by a retroviral vector expressing the LacZ marker gene encoding 0galactosidase (pTG5391). The infected cells are selected in the presence of G418 for 14 days, trypsinized and resuspended at a density of 2 x 107 cells/ml in PBS buffer. 100 Al of this suspension are subcutaneously injected into immunocompetent F1 generation B6/D2 mice.

25 Two days later and for the next 6 days, the animals receive an intraperitoneal injection of ganciclovir (100 mg/kg/day) and the number of tumors and their size are examined up to 44 days after the implantation. As controls, there are used mice treated in parallel with nontransduced cells and mice into which there have been implanted cells which are transduced but which are not treated with ganciclovir As summarized in the following table, 5 groups of 10 animals were constituted in total.

Group Cell line Ganciclovir Number of mice with injected treatment no tumor day day day day 21 30 34 44 1 2 3 4 B16 +GC 0 -A B16-TG5391 +GC 0 -A -A (beta Gal) B16-TG9344(TK- +GC 10 3 1 0 Neo) B16-TG9337 +GC 10 9 9 9 (IFNmouse-TK- Neo) B16-TG9337 -GC 0 0 0 0 (IFNmouse-TK- Neo) Smice which died or which were sacrificed because of the large size of the tumors.

Comparison of groups 4 and 5, both resulting from the implantation of cells transduced by the vector pTG9337, but treated or otherwise with ganciclovir, clearly shows the effect of the latter on the development of the tumors. Moreover, the mice into which the cells transduced by a vector expressing therapeutic genes (pTG9344 and pTG9337) were implanted do not develop any tumor before the 25th day (groups 3 and 4) whereas the animals 26 which received no vector (group 1) or which received a nontherapeutic vector (group 2) are decimated well before the 21st day. Furthermore, the advantage of combining the action of several therapeutic genes is clearly evident from this analysis. Indeed, the expression of the tk gene alone slows down tumor development (group 3) whereas the great majority (9/10) of the animals which received the B16-9337 cells (concomitant expression of the tk and IFNy genes) exhibit no tumor more than 44 days post-implantation. These data indicate that the combination of the immunostimulatory effect of IFNy and of the cytotoxic effects of thymidine kinase and of ganciclovir drastically reduces the frequency of tumors.

27 SEQUENCE LISTING GENERAL INFORMATION

APPLICANT:

NAME: Transgene S.A.

STREET: 11 rue de Molsheim CITY: Strasbourg COUNTRY: France POSTAL CODE: 67082 TELEPHONE: (33) 88 27 91 00 TELEFAX: (33) 88 27 91 11 (ii) TITLE OF INVENTION: Novel antitumor compositions (iii) NUMBER OF SEQUENCES: 13 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Tape COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG4322 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGAGATGGGG GAGGCTAACT GAAACT 26 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide 28 oTG4323 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CTAGAGTTTC AGTTAGCCTC CCCCATCTCC INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG4031 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: NGATGCTCTC CGGCCYTCGA AA 22 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG4169 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AAGAATTCTT RGAHATTTKG ARGAAYTGG 29 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

3 (iii) ANTI-SENSE: YES 29 (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG4170 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAGAATTCRT GCAYCACTYK GATGAGYTC 29 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG4321 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACCTGCAGAT CGTTCACAGG AATTTG 26 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG4319 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TGGAATTCTC TACTTGAAAC TGTTTGACAA CT 32 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) S(iii) HYPOTHETICAL: NO 30 (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG6727 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CTTCGGCCGA ATTCTCTGAA AC 22 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG6788 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TCAAATATTG AATTCAGGAT GACC 24 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG7295 (xi) SEQUENCE DESCRIPTION: SEQ.ID NO: CTGCGGCCGA ATTCTGAGAC AA 22 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 31 (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG7296 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTTATTGGGA GAATTCCTTC C 21 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG6147 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TCGGAAACGA TGAAATATAC A 21 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: synthetic oligonucleotide oTG4983 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TATTGCAGCT GGGACAA 17

Claims (15)

1. 8. Antitumor composition according to claim 7, charac- terized in that the immunostimulatory polypeptide is selected from the group consisting of interleukin IL-4, IL-6, IL-10, IL-12, colony stimulating factors (CSF) of the granulocyte type (G-CSF) or of the granulocyte-macrophage type (GM-CSF), alpha- interferon, gamma-interferon, costimulatory factors, in particular, the polypeptides B7.1 and B7.2 and the factors activating the expression of class II histocompatibility antigens.
2. 9. Antitumor composition according to claim 7, charac- terized in that the cytotoxic polypeptide is chosen from the group consisting of a thymidine kinase, in particular the thymidine kinase (TK) of the Herpes Simplex virus type I (HSV-1), a cytosine deaminase, cyctochrome P450 2B1, purine nucleoside phosphorylase from E.coli, nitroreductase and 3- glucoronidase.
3. 10. Antitumor composition according to one of claims 1 20 to 7, characterized in that the said cell population allows the expression of the genes encoding the S..polypeptides HSV-1-TK, IL-2 and gamma-IFN. S* 11. Antitumor composition according to claim 10, charac- terized in that the said cell population comprises 25 a vector allowing the expression of a gene encoding eo IL-2 and a retroviral encapsidation line allowing the production of infectious particles comprising a retroviral vector allowing the expression of a gene encoding thymidine kinase of the HSV-1 virus and of a gene encoding gamma-interferon.
4. 12. Antitumor composition according to either of claims 2 and 10, characterized in that the said cell popu- Slation comprises a mixture of cells derived from the -34 Vero line, one portion allowing the expression of the IL-2 gene and the other portion allowing the expression of the IFN-y gene.
5. 13. Antitumor composition according to claim 12, charac- terized in that it is, in addition, combined with a retroviral encapsidation line allowing the produc- tion of infectious particles or with" infectious particles comprising a retroviral vector allowing the expression of the HSV-1-TK gene.
6. 14. Antitumor composition according to one of claims 1 to 13, characterized in that the said cell population is sensitive to a drug allowing its elimination. 0* 0*
7. 15. Antitumor composition according to claim 14, charac- 15 terized in that the said drug is a derivative of S. acyclovir and, in particular, ganciclovir. 0
8. 16. Antitumor composition according to one of claims 1 to 15, characterized in that the said cell popula- tion allows, in addition, the expression of a posi- 20 tive selection marker. *0
9. 17. Antitumor composition according to any of claims S" characterized in that the said encapsidation line is derived from the 293 line and comprises: the gag/pol genes of the FMuLV virus (Friend Murine Keukemia Virus) of the FB29 strain and the env gene of the amphotropic virus 4070A, and a vector allowing the expression of a gene encoding IL-2.
10. 18. Antitumor composition according to claim 6, charac- terized in that the said retroviral vector is char- acterized in that it comprises from 5' to 3': a 5' LTR, an encapsidation region, a gene encoding gamma-interferon, a constitutive internal promoter, a gene encoding thymidine kinase of the HSV-1 virus, an internal ribosome entry site (IRES), a gene encoding a positive selection marker, and a 3' LTR.
11. 19. Use of an antitumor composition according to one of claims 1 to 18, for the manufacture of a medicament intended for the treatment or for the prevention of cancer or of a cancerous condition in humans or in animals.
12. 20. Use according to claim 19, according to which the said antitumor composition is intended to be administered by the intratumor route.
13. 21. Antitumor composition in accordance with any one of claims 1 20 to 18 substantially as hereinbefore described with reference to the examples.
14. 22. Use according to any one of claims 19 or 20 substantially as S hereinbefore described with reference to the examples.
15. 23. A method of treating or preventing cancer in humans or in other animals including the administration of an antitumor composition in accordance with any one of claims 1 to 18. 30 24. A method in accordance with claim 23 wherein said adminstration is by intratumor route. DATED: 4 April 2000 CARTER SMITH BEADLE Patent Attorneys for the Applicant: R ATRANSGENE S.A. 4 April 2000