AU2005266225A1 - Products containing at least one anticancer active principle with low diffusion and an immunostimulatory active principle - Google Patents

Description

WO 2006/010838 PCT/FR2005/001612 PRODUCTS CONTAINING AT LEAST ONE ANTICANCER ACTIVE PRINCIPLE WITH LOW DIFFUSION AND AN IMMUNOSTIMULATORY ACTIVE PRINCIPLE 5 The present invention relates to products used in combination, for effective treatment of tumors, and more particularly of metastases. The three main anticancer treatments are surgery, 10 radiotherapy and chemotherapy. However, the effectiveness of these therapies is often insufficient in the case of metastatic tumors, because of the size and the number of the tumors. It is therefore necessary to have new therapeutic approaches. 15 Thus, the effectiveness of conventional surgical therapies or cytolytic therapies (chemotherapy and radiotherapy) remains very limited in many cancers. For glioblastomas, for example, the treatment of which is 20 based mainly on surgical exercesis and local cerebral irradiation, median survival is of the order of 12 months. Supplementary chemotherapy prolongs survival in patients less than 60 years old, but very modestly, by less than 3 months. With this triple treatment, median 25 survival remains less than 15 months (Behin et al., Lancet 2003). Immunotherapy in cancer treatment aims to stimulate the immune system in order to destroy the tumor cells. 30 Various approaches can be used: vaccination against one or more tumor antigens, amplification of a pre-existing antitumor response (such as, for example, amplification of tumor-infiltrating cytotoxic lymphocytes), use of monoclonal antibodies directed against a tumor antigen 35 or of dendritic cells loaded with tumor extracts or antigens (review by Turtle CJ et al., Dendritic cells in tumor immunology and immunotherapy. Curr Drug Targets. 2004 5:17-39; Chaput N et al., Exosome-based WO 2006/010838 - 2 - PCT/FR2005/001612 immunotherapy. Cancer Immunol Immunother. 2004; 53:234-9; Parish CR. et al. Cancer immunotherapy: the past, the present and the future. Immunol Cell Biol. 2003 81:106-13). 5 Unfortunately, although the animal models are often convincing, immunotherapy clinical trials in humans have not yet found their place in the therapeutic arsenal and there are still many uncertainties 10 regarding the optimization of these parameters. In particular, the type of tumor antigen and also the route of administration for the latter are still parameters undergoing evaluation. 15 Another strategy consists in activating the immune system broadly, in order to stimulate innate immunity and to prime or amplify a specific immune response. Among these immunostimulatory agents, mention may be made, inter alia, of bacterial extracts (Jaeckle K. A. 20 et al. (1990), J. Clin. Oncol. 8(8) pp 1408-18), IL-2 (Herrlinger U. et al. (1996), J. Neurooncol. 27(3) pp 193-203), IL-12 (Kishima H. et al. (1998,) Br. J. Cancer 78(4) pp 446-53; Jean W. C. et al. (1998), Neurosurgery 42(4) pp 850-6), bacterial DNA (MY-1) 25 (Tokunaga T. et al. (1984) JNCI 72 pp 966-62), or poly(I,C) (Ewel, C. H. et al (1992). Canc. Res. 52:3005). The immunostimulatory properties of DNA were discovered 30 in 1984, when it was observed that the DNA of Mycobacterium tuberculosis activated NK cells in vitro and in vivo [Tokunaga T, et al. J. Natl Cancer Inst 1984; 72: 955-962]. Several studies have subsequently shown that these immunostimulatory properties are 35 generally related to nonmethylated 5'-CG motifs, which motifs are underrepresented in vertebrate DNA [Krieg AM et al. Nature 1995; 374: 546-549; Yamamato S et al J Immunol 1992; 148: 4072-4076; applications WO 2006/010838 - 3 - PCT/FR2005/001612 US 2004-0006034; 2003-0212029; patents US 6,653,292; US 6,239,116; US 6,207,646; US 6,194,388; US 6,427,199, US 6,406,705 US 6,218,371. US 6,214,806 US 6,218,371, US 6,727,230, PCT international applications 5 WO 01/51500, WO 03/035695, European patent application no. 1 162 982). Synthetic oligodeoxynucleotides (ODNs) containing such motifs (CpG-ODNs) maintain marked immunostimulatory properties, particularly nuclease resistant phosphorothioate ODNs. 10 Stimulation of the immune system with CpG-ODNs or bacterial DNA requires intracellular penetration thereof, probably by endocytosis. After degradation in the acidic environment of endosomes, the CpG motifs are 15 recognized specifically by a member of the Toll-like receptor family, TLR9, expressed in the endosomes. Intracellular activation after binding to TLR9 depends on the MyD88, IRAK and TRAF6 proteins, which in turn activate the MAP kinase and NFKB pathways [Hacker H, 20 Curr Top Microbiol Immunol 2000; 247: 77-92; Hemmi H, et al. Nature 2000; 408, 740-745; Takeshita F et al, J Immunol 2001; 167, 3555-8] . In humans, TLR9 expression predominates in B lymphocytes and plasmacytoid dendritic cells (pDCs), whereas the expression is 25 broader in mice and includes the myeloid line and the microglia [Hornung V, Rothenfusser S et al. J Immunol 2002; 168, 4531-4537]. B lymphocytes are activated by CpG-ODNs, resulting in 30 the secretion of cytokines such as, for example IL-6 or IL-10, cell proliferation, the inhibition of apoptosis induced by various agents, and immunoglobulin secretion [review by Klinman DM, Nat Rev Immunol 2004; 4: 249-59; Krieg AM, Curr Oncol Rep 2004; 6: 88-95; Carpentier AF 35 et al, Front Biosci 2003; 8: E115-27]. The activation of human pDCs results in their maturation, the secretion of numerous cytokines such as WO 2006/010838 - 4 - PCT/FR2005/001612 TNFa, interferon alpha or gamma, IL-6 or IL-12, and the expression of costimulatory molecules (CD40, CD80, CD86) and of CCR7, a receptor which controls migration into the lymph node T zones. Although pDCs are directly 5 activated by CpG-ODNs, the activation of human myeloid dendritic cells appears to be indirect via cytokines [review by Klinman DM, Nat Rev Immunol 2004; 4: 249-59; Krieg AM, Curr Oncol Rep 2004; 6: 88-95]. The activation of monocytes by secreted cytokines can 10 induce, inter alia, the secretion of CXCL10 which promotes the antitumor immune response and which also has anti-angiogenic properties (Strieter RM et al (1995), J Biol Chem, 270; 27348-57). 15 The activation of T lymphocytes and of NK cells by CpG-ODNs depends on the cytokines secreted by DCs (dendritic cells) and B lymphocytes. The secretion of IL-12 and of IFN gamma directs the response toward the Thl profile, and can even convert a Th2 response to 20 Thl. In addition, DCs can then activate CD8 lymphocytes, independently of CD4 helper lymphocytes [review by Klinman DM, Nat Rev Immunol 2004; 4: 249-59; Krieg AM, Curr Oncol Rep 2004; 6: 88-95]. 25 The biological activity of an oligonucleotide depends on many variables, which are still not completely understood, for instance the sequences of the nucleotides surrounding the CpG motif or the number of CpG motifs. Some immunostimulatory oligonucleotides 30 without CpG motifs have even been described (for example, in application US 2004-0006032). The chemical modification of the backbone naturally formed in DNA by phosphodiester bonds (sensitive to nucleases) plays an essential role both in improving the stability of ODN, 35 but also in modifying its immunostimulatory properties. Several types of stabilized oligonucleotides have thus been created (Iyer RP (1999) Curr Opinion Mol Therap 1; 344-358) . The CpG-ODNs most commonly used are WO 2006/010838 - 5 - PCT/FR2005/001612 phosphorothioate oligodeoxynucleotides, or mixed phosphorothioate/phosphodiester oligodeoxynucleotides (for example, phosphorothioate ends/phosphodiester centre; or phosphorothioate ODNs with the exception of 5 the cytosine-guanosine bond, which are phosphor diesters). The oligodeoxynucleotides synthesized can also be purified according to their stereoisomerism so as to modify or improve their biological activity. 10 The immunostimulatory activity of synthetic oligonucleotides which are highly modified, owing to the presence of unnatural purine or pyrimidine bases (Kandimalla ER et al. Nucleic Acids Research, 2003, 31; 2393-2400; applications US 2003-0181406; 2002-0137714; 15 patent US 6,562,798; application US 2003-0186912), or of DNA/RNA or of ODN-linker-ODN chimeric molecules (application US 2004-0052763; Bhagat L et al. BBRC, 2003, 300; 853-861; applications US 2003-0225016; 2003-0199466; 2003-0175731) or the addition of non 20 oligonucleotide ligands, has also been reported (review by Uhlmann E. and Vollmer, J., Curr. Opin. Drug Discov. Devel., 2003, 6, 204-217). The precise biological activities of each of these 25 oligonucleotides are not yet completely understood. Three families of CpG-ODNs have already been characterized [review by Klinman DM, Nat Rev Immunol 2004; 4: 249-59; Krieg AM, Curr Oncol Rep 2004; 6: 88-95] . The most conventional, called "type B" (or 30 "K"), is characterized by a strong activation of B lymphocytes and of dendritic cells, but a weak secretion of interferon alpha by pDCs. The "type A" (or "D") ODNs are characterized by a weak activation of B lymphocytes but a strong activation of NKs and 35 secretion of interferon alpha by pDCs. Type C ODNs, the most recently described, combine the properties of the above 2 types [review by Klinman DM, Nat Rev Immunol WO 2006/010838 - 6 - PCT/FR2005/001612 2004; 4: 249-59; Krieg AM, Curr Oncol Rep 2004; 6: 88-95]. The strong immunostimulatory capacity of CpG-ODNs (and, 5 by extension, of the TLR ligands, and more particularly the TLR9 ligands) opens up several therapeutic strategies in cancers. Their use in fact makes it possible to activate the two components of the immune response: 10 - The priming of the immune response: by direct stimulation of dendritic cells and the selection by the immune system of an appropriate antigen with a Thl-type immune response. 15 - The effectiveness of the immune response: the activation of macrophages and of NK cells makes it possible to increase the antitumor cytotoxicity, either direct, or by means of antibodies (antibody-dependent 20 cell cytotoxicity or ADCC) . In addition, by virtue of the local secretion of IFN gamma, the expression, generally weak, of CMH I by the tumor cells can be induced, making them more sensitive to T lymphocyte cytotoxicity. 25 These multiple biological activities have thus made it possible to develop several strategies in cancers: 1. The CpG-ODNs used in local (intratumor or 30 peritumor) or general (= systemic) monotherapy can induce tumor rejection in almost all the tumor models [Carpentier AF, Auf G, Delattre JY. CpG oligonucleotides for cancer immunotherapy: review of the literature and potential applications in malignant 35 glioma. Front Biosci 2003; 8: E115-27]. For example, in a model of intracerebral glioma (CNS1), more than 85% of rats experienced recovery with a single local injection of CpG-ODN [Carpentier AF, Xie J, Mokhtari K, WO 2006/010838 - 7 - PCT/FR2005/001612 Delattre JY, Clin Cancer Res 2000; 6, 2469-2473] . Most studies have underlined the joint role of the nonspecific innate immune system and of the specific immune system in tumor rejection. In vivo depletion of 5 NK cells decreases or abolishes the antitumor effects of CpG-ODNs [Carpentier AF, Chen L, Maltonti F, Delattre JY. Cancer Res 1999; 59, 5429-5432]. CD8+ lymphocytes are necessary in most of the animal models, whereas CD4-cell depletion appears to have less impact 10 [Lanuti M, Rudginsky S, Force SD, et al. Cancer Res 2000; 60, 2955-2963; Kawarada Y, Ganss R, Garbi N, et al. J Immunol 2001; 167, 5247-5253]. Be that as it may, the existence of a long-term immune memory, reported by all the models, suggests a role for memory CD4 15 lymphocytes. Interestingly, it has been reported that ENT tumor-infiltrating pDCs in humans are less sensitive to type A CpG-ODNs than the blood pDCs from the same patient. This resistance would partly be explained by the local secretion of IL-10 by the tumor 20 cells [Hartmann E, Wollenberg B, Rothenfusser S, et al. Cancer Res. 2003; 63: 6478-87] . In an animal model, the combination of CpG and of anti-IL-10 antibodies has, moreover, proved to be synergistic [Vicari AP, Chiodoni C, Vaure C, et al. J Exp Med 2002; 196, 541 25 9]. 2. CpG-ODNs can also be used for the production of vaccines, since they are very effective adjuvants with most antigens, unless the latter are purely 30 polysaccharide in nature [Krieg AM, Annu Rev Immunol 2002; 20:709-60; Krieg AM, Curr Oncol Rep 2004; 6: 88 95]. CpG-ODNs have been found to be the most effective adjuvants for the induction of cytotoxic Thl immune responses with antigen-specific CD4 and CD8 T 35 lymphocytes [Zwaveling S, Ferreira Mota SC, Nouta J, et al. J Immunol 2002; 169: 350-8; Miconnet I, Koenig S, Speiser D, et al. J Immunol. 2002; 68, 1212-1218]. Direct conjugation between the antigen and CpG-ODNs WO 2006/010838 - 8 - PCT/FR2005/001612 makes it possible to reduce the amount of antigen necessary [Tighe H, Takabayashi K, Schwartz D, et al. J Allergy Clin Immunol. 2000; 106: 124-341 . The effectiveness of CpG-ODNs is further improved by 5 combination with other adjuvants, for instance aluminum hydroxide, QS21, MPL or GMCSF [Miconnet I, Koenig S, Speiser D, et al. CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. J Immunol. 2002; 68, 1212-1218; Kim SK, 10 Ragupathi G, Cappello S, et al. Effect of immunological adjuvant combinations on the antibody and Te-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates. Vaccine. 2000; 19: 530-7; Liu HM, Newbrough SE, Bhatia SK, et al. Immunostimulatory CpG 15 oligodeoxynucleotides enhance the immune response to vaccine strategies involving granulocytemacrophage colony-stimulating factor. Blood 1998; 92, 3730-3736]. Unfortunately, the antigenic targets are rarely determined in cancers, limiting the practical advantage 20 of such therapeutic strategies. Furthermore, the spontaneous selection of a clone not expressing the antigen would allow the tumor to evade such a treatment. 25 3. The combination of CpG-ODNs and of monoclonal antibodies is also advantageous. It increases the antibody-mediated cytotoxicity (ADCC) through the stimulation of macrophages and of NK cells; several clinical trials using this combination have recently 30 begun, with Herceptin@ in breast cancers or Rituxan@ in non-Hodgkin's lymphomas. This combination can also use the CpG-ODNs as inducers, in the tumor cell, of the expression of a molecule against which a monoclonal antibody exists. CpG-ODNs induce the expression of CD25 35 (IL2R) at the surface of a B lymphoma line, thus rendering the cells more sensitive to the action of an anti-CD25 immunotoxin [Decker T, Hipp S, Kreitman RJ, et al. Blood 2002; 99, 1320-1326].

WO 2006/010838 - 9 - PCT/FR2005/001612 4. The use of CpG-ODNs for stimulating dendritic cells in vitro or in vivo has been described. This stimulation can be carried out in combination with 5 other immune adjuvants or cytokines, with various tumor antigens or extracts (Krieg AM. Annu Rev Immunol 2002; 20:709-60; Krieg AM, Curr Oncol Rep 2004; 6: 88-95). 5. Finally, the use of TLR9 ligands or of CpG-ODNs 10 for cancer immunotherapy can be combined with other therapies, in particular surgery, radiotherapy, chemotherapies, other immunotherapies and differentiating therapies. 15 Combined use of systemic chemotherapy and of CpG-ODNs (administered locally or systemically) has in particular been described. This combination makes it possible, in theory, to decrease the tumor mass to be eliminated by the immune system, and to induce the 20 release of a large number of tumor antigens which will serve to prime the immune system. Positive results from such approaches have been reported by several authors in some tumor models and with some chemotherapies such as cyclophosphamide and topotecan (Carpentier 2003, 25 mentioned above; Weigel BJ, et al., Clin. Cancer Res., 2003, 9, 8, 3105-14; Balsari A. et al., Eur. J. Cancer, 2004, 40, 8, 1275-81). This combination is not, however, effective in all 30 tumor models. In particular, some tumors are insensitive or relatively insensitive to chemotherapies, thus limiting the clinical usefulness of a combination of CpG-ODNs and conventional chemotherapy. 35 One of the obstacles to be overcome in order to obtain CpG-ODNs that are very effective in cancers is the induction of a specific immunity. This is because the WO 2006/010838 - 10 - PCT/FR2005/001612 activation of nonspecific immunity by CpG-ODNs is local and only persists for the duration of the treatment. The capacity of CpG-ODNs to induce a good specific immune response capable of acting remotely and over 5 time is therefore determining for metastatic cancers. This is illustrated by the preliminary results of a clinical trial in melanomas, where local responses were observed but without any remote response (no systemic effect) (Trefzer U. et al., 2nd International Symposium 10 "Activating immunity with CpG oligos", Amelia Island, Florida, October 7-10, 2001). Surprisingly, the inventors have found that tumor destruction by means of an electrochemotherapy 15 technique combined with an appropriate stimulation of the immune system results in an antitumor therapy with a remote effect (systemic effect) that is particularly effective, in particular with regard to metastases. 20 The inventors have in particular found that the combination of TLR (toll-like receptor) ligands (natural or synthetic), in particular immunostimulatory oligodeoxynucleotides (ODNs), and of an optimized chemotherapy protocol makes it possible to obtain an 25 effective action on metastases. A subject of the present invention is, consequently, products containing at least one anticancer active principle with low diffusion and an immunostimulatory 30 active principle selected from the group consisting of TLR ligands, as a combined preparation for simultaneous, separate or sequential use, for the treatment of benign or malignant tumors, in particular solid tumors, and more particularly metastases, in an 35 individual suffering from a tumor and subjected to an electropermeabilization at the level of said tumor.

WO 2006/010838 - 11 - PCT/FR2005/001612 As a variant, the electropermeabilization is carried out at the level of an appropriately selected normal tissue. 5 In such a case, the combined preparation of said products also comprises antigens, for example in the form of purified antigens specific for a tumor, of tumor extracts, or of irradiated or modified tumor cells, said antigens preferably being administered 10 locally. According to this variant of the invention, the electropermeabilization carried out on a normal tissue has the advantage of locally promoting the recruitment 15 of immune cells. Electrochemotherapy (ECT) (Mir et al. 1991a, Belahradek et al, 1991) combines the in situ administration of nonpermeating (or nondiffusible) anticancer molecules 20 or anticancer molecules with low permeation (or low diffusion) and the application of permeabilizing electric pulses. Among the anticancer molecules that have been used in the context of an ECT, mention may be made of bleomycin, a nonpermeating molecule, which does 25 not diffuse through the cell's membrane, already used in conventional chemotherapy (Orlowski et al, 1988; Poddevin et al, 1991; Gehl et al, 1999) and cisplatin and the other platinum salts that have cytotoxic properties (Sersa et al, 1995; Gehl et al, 1999; Sersa 30 et al, 2003) . Other molecules can also be used in the context of an ECT, such as pro-apoptotic, anti angiogenic, differentiating or immunostimulatory substances; thus, ECT has been used to improve the transfection of antisense oligonucleotides or of 35 plasmids (Ivanov MA et al., J. Gene Mede, 2003, 5, 893 899, patents US 5,547,467, 5,749,847 and 6,763,264) or for the administration of immunostimulatory molecules WO 2006/010838 - 12 - PCT/FR2005/001612 such as interleukin 12 (Kishida T et al., Mol. Ther., 2003, 8, 738-745). Electropermeabilization of cells exposed to electric 5 pulses is a phenomenon that has been known for about thirty years. It results from the transmembrane potential difference generated by an electric field external to the surface of the cells exposed to this field. When this transmembrane potential difference 10 exceeds a certain threshold value (Teissi6 et al, 1993), for a sufficiently long period of time (Kotnik et al, 2003), transient and reversible permeabilization of the cells can be obtained, without resulting in tissue lesions. The intensity of the electric field 15 applied depends on the voltage and on the distance between the electrodes. It can range from 20 to 2000 V/cm. The electric pulses can range from 3 to 8; they generally have a duration of 100 ps and are delivered at a frequency of 1 to 5000 Hz. 20 The electropermeabilization of the cells of a tumor can be obtained, for example, by exposure of this tumor to 3-8 electric pulses of 100 microseconds and 1300 V/cm delivered at the repeating frequency of 1 Hz by means 25 of two electrodes placed on the skin on either side of the tumor nodule to be treated. Quite obviously, this is a local treatment, restricted to the volume between the electrodes and subjected to an electric field intensity greater than the threshold value that makes 30 it possible to permeabilize the cells within this volume: bleomycin, administered beforehand, will penetrate only the electropermeabilized cells and only the latter will be effectively killed. 35 According to an advantageous embodiment of said products, the TLR ligands are immunostimulatory oligodeoxynucleotides (ODNs) regardless of whether their structure is natural, synthetic or chimeric.

WO 2006/010838 - 13 - PCT/FR2005/001612 According to an advantageous arrangement of this embodiment, the TLR ligands are more specifically TLR9 ligands, and more particularly immunostimulatory CpG 5 ODNs. In accordance with the invention, said immunostimulatory CpG-ODNs are selected from the group consisting of a stabilized oligodeoxynucleotide which 10 comprises at least one quadrameric motif of formula Xi-CG-X 2 , in which Xi and X 2 are identical or different and represent T or A. According to an advantageous arrangement of this 15 embodiment, said CpG-ODN is preferably selected from the group consisting of an oligodeoxynucleotide which comprises at least one quadrameric sequence selected from the group consisting of: TCGA, ACGT, ACGA and TCGT. 20 According to an advantageous mode of this arrangement, said CpG-ODN is preferably selected from the group consisting of an oligodeoxynucleotide which comprises at least one hexameric sequence selected from the group 25 consisting of: AACGTT, GACGTC, GACGTT, GTCGTT, TTCGAA, TACGTA, ATCGAT, TTCGTT and ATCGTT. According to another advantageous arrangement of this embodiment, said CpG-ODN is preferably selected from 30 the group consisting of an oligonucleotide which comprises at least the following octameric sequence:

AACGTT-X

3

X

4 , in which X 3

X

4 is AT, AA, CT or TT, and preferably the octameric sequence AACGTTAT. 35 According to an advantageous mode of this arrangement, said CpG-ODN is the CpG-ODN of sequence SEQ ID No. 1.

WO 2006/010838 - 14 - PCT/FR2005/001612 According to another advantageous arrangement of this embodiment, the immunostimulatory active principle is a CpG-ODN selected from the group consisting of a stabilized oligonucleotide which comprises at least one 5 octameric motif of the type: 5'-purine-purine-GC pyridimine-pyrimidine-XiX 2 -3', in which the pair X 1

X

2 is AT, AA, CT or TT. According to an advantageous mode of this arrangement, 10 said CpG-ODN is preferably selected from the group consisting of an oligonucleotide which comprises at least one octameric sequence selected from the group consisting of: GACGTT-XiX 2 , AGCGTT-XiX 2 , GGCGTT-XiX 2 , AACGTC-XiX 2 , GACGTC-XiX 2 , AGCGTC-XiX 2 and GGCGTC-XiX 2 , in 15 which X 1

-X

2 is AT, AA, CT or TT. In accordance with this mode, said CpG-ODN is chosen from the group consisting of the sequences SEQ ID No. 2 to SEQ ID No. 41. 20 Such stabilized CpG-ODNs and also the method for preparing them are in particular described in French application no. 99 03432 and PCT international application no. WO 00/56342. 25 In accordance with the invention, the immunostimulatory oligonucleotides can be used in single-stranded or double-stranded form, and can comprise several adjacent or nonadjacent immunostimulatory sequences. They can 30 also comprise other biologically active sequences, such as antisense sequences or aptamers. The stabilized oligonucleotides can be coupled, via covalent, ionic or weak bonds, to one or more other molecules capable of increasing their tumor affinity, of modifying their 35 biological activity or of increasing their immunostimulatory activity.

WO 2006/010838 - 15 - PCT/FR2005/001612 For the purpose of the present invention, the anticancer active principle is a substance normally used in chemotherapy; anticancer chemotherapy is based on the use of cytotoxic substances belonging to two 5 categories of substances: antimetabolites which interfere in particular with the synthesis of nucleic acids (fluorouracil, methotrexate, doxorubicin) or of proteins (topoisomerase inhibitors) or else other essential metabolic processes such as mitosis 10 (vincristine), and genotoxic substances which modify the structure of DNA, such as cleaving agents (bleomycin) or intercalating agents, in particular alkylating agents (cyclophosphamide, lomustine, temozolomide, platinum derivatives (cisplatin or 15 cisdiaminedichloroplatin, cDDP or DDP), nitrosourea derivatives (fotemustine or (RS)-diethyl [[(chloro-2 ethyl)nitroso-3-ureido]-l-ethyl]phosphate)). The effectiveness of this therapy can vary considerably from one type of tumor to the other and from one 20 individual to the other, due to differences in the sensitivity of the tumor cells to these agents. Other substances are also used in anticancer chemotherapy; they are substances capable of acting on 25 factors other than those specified above and that are involved in cancers: cytolytic (or pro-apoptotic) substances, anti-angiogenic substances and differentiating substances. 30 According to an advantageous embodiment of said products, the anticancer active principle is selected from the group consisting of cytotoxic substances (antimetabolites and genotoxic substances). 35 According to an advantageous arrangement of this embodiment, the anticancer active principle is preferably a genotoxic substance, and even more preferably bleomycin.

WO 2006/010838 - 16 - PCT/FR2005/001612 According to yet another advantageous embodiment of said products, the cells of the tumor are electro permeabilized by exposure of said tumor cells to 5 electric pulses of sufficient strength or intensity and duration to allow the electroporation of said cancer cells; they are preferably electropermeabilized after the administration of said products; said cells can also be electropermeabilized prior to the 10 administration of said products. According to another advantageous embodiment of said products, the intensity of the electric field applied ranges between 20 and 2000 V/cm. 15 According to an advantageous arrangement of this embodiment, the intensity of the electric field applied preferably ranges between 500 and 1500 V/cm, and preferably between 800 and 1300 V/cm. 20 According to yet another advantageous embodiment of said products, the pulse frequency ranges from 0.01 to 10 000 Hz. 25 According to an advantageous arrangement of this embodiment, the pulse frequency preferably ranges from 1 to 5000 Hz. According to yet another advantageous embodiment of 30 said products, the duration of the pulses ranges from 10 pIs to 10 s. According to an advantageous arrangement of this embodiment, the duration of the pulses preferably 35 ranges from 50 ps to 500 ms, and preferably from 100 ps to 1 Ms.

WO 2006/010838 - 17 - PCT/FR2005/001612 According to another advantageous embodiment of said products, the anticancer active principle is administered intratumorally, locally or systemically. 5 According to another advantageous embodiment of said products, the immunostimulatory active principle is administered intratumorally, locally or systemically. In accordance with the invention, the products are 10 administered once or several times or as a continuous release, in particular by means of osmotic micropumps, or associated with any physical or chemical means, in particular with encapsulating agents such as colloidal dispersion systems and polymers. 15 Also in accordance with the invention, said products are also combined with another treatment, such as radiotherapy, another cytotoxic substance, a cytolytic substance, an anti-angiogenic substance, a 20 differentiating substance, an immunostimulatory substance other than an ODN or products derived from cell therapy, such as dendritic cells. Advantageously, the products according to the invention 25 can be used in the treatment of cancers, regardless of their nature and their degree of anaplasia, and they make it possible to obtain, surprisingly, a systemic curative effect, in particular on metastases. 30 Thus, the injection of a single weekly dose of CpG-ODN (CpG) into the tumor volume treated by electrochemotherapy (the first injection being carried out the day after the electrochemotherapy) has made it possible not only to improve the local results of the 35 electrochemotherapy, but also, surprisingly, to obtain remote effects. The demonstration of these systemic effects, obtained by combining two local treatments (electrochemotherapy and a local injection of CpGs), WO 2006/010838 - 18 - PCT/FR2005/001612 has been carried out in mice and rats inoculated with two tumors transplanted into the two flanks of the animals, three days apart. The largest of the tumors (the first to have been implanted) is then treated by 5 electrochemotherapy and receives the injection of CpGs, and the evolution of the two tumors is then followed over the subsequent days. As shown in the reported tables and graphs, with the combination of electrochemotherapy and CpGs, considerable antitumor 10 effects (complete regression) were obtained in the treated tumor and in the contralateral tumor. Besides the above arrangements, the invention also comprises other arrangements which will emerge from the 15 description that follows, which refers to examples of implementation of the method which is the subject of the present invention and also to the attached drawings, in which: 20 - figure 1 illustrates the effect of the treatment by electrochemotherapy (ECT) (DO) combined with a CpG-ODN (SEQ ID No. 1) administered intratumorally (Dl, 8, 15, 22), on two LPB tumors on opposite sides (mean ± SD). A: evolution of the left tumor (treated). B: 25 evolution of the right tumor (not treated); - figure 2 is a detailed representation, mouse by mouse, of the effect of the treatment by electrochemotherapy (DO) and intratumor CpG-ODN (SEQ ID 30 No. 1) (D1, 8, 15, 22), on two LPB tumors on opposite sides. A: evolution of the left tumor (treated). B: evolution of the right tumor (not treated). (S: mouse); - figure 3 illustrates the effect of the combination 35 ECT + CpG-ODN (SEQ ID No. 1) in the B160VA tumor model (7 mice per group); WO 2006/010838 - 19 - PCT/FR2005/001612 - figure 4 illustrates the effect of the electroporation of a CpG-ODN (SEQ ID No. 1) at Dl, 8, 15, 22 intratumorally, on two LPB tumors on opposite sides (mean ± SD) . A: evolution of the left tumor 5 (treated). B: evolution of the right tumor (not treated); - figure 5 is a representation, mouse by mouse, of the effect of the electroporation of a CpG-ODN (SEQ ID 10 No. 1) at Dl, 8, 15, 22 intratumorally, on two LPB tumors on opposite sides. A: evolution of the left tumor (treated) . B: evolution of the right tumor (not treated). (S: mouse); 15 - figure 6 represents the monitoring of the TCD8 response in the homolateral inguinal lymph node of the treated and nontreated, immunized mice. A: visualization of the lymphocytes. B: isolation of the TCD8s. C: isolation of the SIINFEKL-specific TCDs. 20 It should be clearly understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they in no way constitute a limitation. 25 EXAMPLE 1: MATERIALS AND METHODS Animals: female C57BL/6 H-2b mice 8-20 weeks old were used to establish the tumor models. These mice, from 30 the Centre d'Elevage Janvier (Le Genest, St Isle, France), were all bred in the small animals service of the Institut Gustave Roussy in accordance with the guidelines of the Ethical Committee for Animal Experimentation. The mice were all sacrificed humanely 35 by CO 2 inhalation. Tumor cell lines: the LPB and B160VA lines were cultured in vitro at 37 0 C under 5% CO 2 . The LPB cells WO 2006/010838 - 20 - PCT/FR2005/001612 are derived from a sarcoma induced with methyl cholanthrene in C57BL/6 mice. They were maintained in minimum essential medium (Gibco, Cergy-Pontoise, France) supplemented with 8% of heat-inactivated (half 5 an hour at 56*C) fetal calf serum (Gibco) and 100 U/ml of penicillin and 100 ig/ml of streptomycin (Gibco). The stable B160VA line generously provided by Dr. K.L. Rock (UMass Medical School, Worcester, MA, USA), derives from a murine melanoma transfected with the 10 gene encoding the chicken ovalbumin protein (OVA). These cells were maintained in vitro in RPMIl640 medium (Gibco) supplemented with 8% of fetal calf serum (SVF), 5% of L-glutamine (Gibco), 5% of sodium pyruvate (Gibco), 5% of nonessential amino acids (Gibco) and 5% 15 of penicillin and streptomycin. Tumor models: after trypsination of the cells and inactivation of the trypsin (Gibco) with complete medium, the cells were harvested in minimum essential 20 medium (MEM) for the LPBs and in RPMIl640 for the B160VAs. The LPB and B160VA cells (106) were injected subcutaneously into the left flank of the mice. Three days later, 106 cells were again injected into the right flank of the same animals. The mice were then divided 25 up for each experiment before being treated 8 days or 9 days after the first injection, respectively, for the LPB and B160VA tumors. The tumor size was measured 3 times a week using a caliper rule. The tumor volume was determined according to the formula of an ellipse V = 30 a 2 b/r/6 in which a represents the width and b the perpendicular length. Anesthesia: the mice were anesthetized with 150 ptl of a mixture of xylazine (Bayer Pharma, Puteaux, France) at 35 12.5 mg/kg and ketamine (Parke Davis, Courbevoie, France) at 125 mg/kg, injected intraperitoneally, before being treated by electroporation or electrochemotherapy.

WO 2006/010838 - 21 - PCT/FR2005/001612 Electrochemotherapy (ECT): 10 ptg of bleomycin (Roger Bellon, Neuilly, France) in 100 p.l of 0.9% NaCl were injected iv into the retroorbital sinus of the mice. 5 Four minutes after the injection, the electric pulses were delivered to the tumor mass by means of a power source connected to a computer console (prototype). The electroporation was carried out by means of two electrodes 5 mm apart, placed on either side of the 10 tumor, and delivering 8 electric pulses of 100 ps at 1300 V/cm and a frequency of 5000 Hz. Systematic application of a gel to the tumor before the electroporation and the control of each electroporation by graph made it possible to be sure that the current 15 was correctly distributed in the tumor. Immunotherapy: the single-stranded sequence of the oligodeoxynucleotide containing several nonmethylated CpG motifs (5'-TAAACGTTATAACGTTATGACGTCAT-3') (SEQ ID 20 No. 1). 50 micrograms of CpG oligodeoxynucleotides diluted in 100 p.l of 0.9% NaCl were injected intra tumorally the day after the ECT, once a week for four weeks. 25 In vitro cytotoxicity assay: a Chinese hamster lung fibroblast line, DC3F, was maintained in vitro as described above for the LPB line. The cells were harvested in order to resuspend them at a concentration of 2.2 x 10 7 /ml in MEM-S (MEM without calcium, Gibco). 30 Aliquots of 106 cells/45 p.l were mixed with 5 p.l of 0.9% NaCl or 5 p.l of CpG ranging in a concentration from 5 to 60 p.M. The 50 p.l mixture was then placed between 2 electrodes 2 mm apart and the electric shock was immediately delivered (8 pulses of 100 ps for an 35 electric field of 1000 V/cm and a frequency of 1 Hz). After having been left to stand for 5 minutes, the cells were diluted in complete MEM and cultured in triplicate in petri dishes 6 cm in diameter WO 2006/010838 - 22 - PCT/FR2005/001612 (500 cells/ml). The cell proliferation was estimated after 5 days of culture. For this, the cells were first fixed with formaldehyde for 20 minutes and then stained with a crystal violet for 15 minutes. 5 Analysis of the immune response: a tumor model was created by injecting 5 x 105 B160VA cells per 50 pl of RPMI1640 into the right thigh of the mice. After 14 days, the tumor measured approximately 5 mm and was 10 treated with ECT (DO) followed by immunotherapy with CpGs at Dl. Moreover, 3 mice were immunized by injection of peptide directly into the footpad at Dl: 45 pg of SIINFEKL peptide (Eurogentec) derived from ovalbumin, mixed into 50 ig of CpG-ODN. Seven days 15 after this treatment, all the mice were sacrificed in order to remove the inguinal lymph nodes. The lymph nodes were mechanically crushed in 100 pim filters and the lymph node cells were harvested in RPMIl640. Ten thousand cells/well were incubated in a 96-well plate 20 with PRMIl640 supplemented with 1% of murine serum, in the presence or absence of OVA peptide, in order to carry out an in vitro stimulation. After 72 hours of culture at 370C under 5% C02, the supernatant was removed in order to evaluate the secretion of 25 interferon y by means of an ELISA assay (OptEIA

T

" kit from BD Pharmigen) . Some of the cells (1-2 x 106) were labeled with the tetramer H-2b/SIINFEKL coupled to PE (Beckman Coulter, Fullerton, CA) diluted to 1/100th, for half an hour at ambient temperature and in the 30 dark, and then with an anti-CD3-FITC and an anti-CD8 APC (BD Pharmingen, San Diego, CA) diluted to 1/100th, for the same incubation time. The cells were then washed once in 1X PBS and fixed in 200 pl of 1% paraformaldehyde before being analyzed in a FACScalibur 35 cytofluorimeter with the CellquestPro software. Statistics: an analysis of variance with a multiple comparison according to the Krusal-Wallis test was WO 2006/010838 - 23 - PCT/FR2005/001612 carried out for the in vivo and in vitro experiments. The means with the unilateral standard deviations differ significantly for a value z greater than 1.96. Each test had an overall p of less than 0.05 making it 5 possible to compare the groups with one another. EXAMPLE 2: RESULTS Antitumor activity of the combination of ECT and CpG ODN (CpG): 10 e LPB fibrosarcoma model - Analysis per group: 15 In order to test the hypothesis of a systematic therapeutic effect of the ECT-CpG combination, we used a double tumor model. 106 LPB cells were implanted subcutaneously, first into the left flank, followed, 3 days later, by a further 106 cells into the right flank 20 of the same mice. Only the left tumor was treated at DO with EP or ECT and at Dl, D8, D15 and D22 with 0.9% NaCl or CpG intratumorally, as described in the materials and methods section. The effect of the treatment was observed on the 2 tumors. 25 Firstly, local effects of the treatment (left tumors) are processed, followed, secondly, by remote effects (right tumors): 30 The animals were monitored for 30 days after the treatment. The group having received the combined ECT + CpG treatment showed 100% complete regression of the tumors treated at D28 (Figure 1A). On the other hand, when a single treatment was applied, i.e. ECT alone or 35 CpGs alone, the growth of the treated tumors was only slowed down compared to the control group EP + NaCl (Fig. 1A). The evolution of the tumors is statistically different between the ECT + CpG group and the other WO 2006/010838 - 24 - PCT/FR2005/001612 groups (z = 4.18; z = 2.62; z = 2.46 with the respective groups EP + NaCl, EP + CpG and ECT + NaCl at D28). 5 The remote antitumor effect was evaluated by measuring the contralateral tumors (Fig. 1B). An overall tumor stabilization was obtained for 20 days with the ECT + CpG combination in an entirely significant manner with respect to the control group (z = 2.47) . On the other 10 hand, the single treatments did not allow stabilization, but were associated with a tumor proliferation that was slowed down compared to the control group (Fig. 1B). 15 - Analysis per mouse: The presentation of the tumor growth, animal by animal, makes it possible to follow the evolution of the left and right tumors in the same mouse (Fig. 2). The 20 evolution of the contralateral tumor appears to be correlated with the evolution of the treated tumor, without it being possible to generalize this result to all the mice. For example, in the EP + CpG group, the Sl mouse that had been stabilized on the left was also 25 stabilized on the right, whereas the S3 mouse which evolved rapidly on the left progressed slowly on the right. * B160VA melanoma model 30 The fibrosarcoma model was reproduced by also implanting 106 B160VA cells on the left and right flanks of the mice and by observing a period of 3 days between the two injections. The tumors were treated as 35 described above, 9 days after the implantation of the cells. A single experiment which is currently ongoing is described from the day of the beginning of the treatment DO (Fig. 3) . The survival rates at the 18th WO 2006/010838 - 25 - PCT/FR2005/001612 day of treatment are respectively 28.6%, 71.4%, 14.3% and 85.7% for the EP + NaCl, EP + CpG, ECT + NaCl and ECT + CpG groups. 5 Analysis of the effect of the electroporation of the CpGs, in the context of an ECT: The innate immune system detects nonmethylated CpG motifs via the TLR9s in mice (26). The TLR9s are 10 expressed intracellularly precisely at the level of the endoplasmic reticulum (30). Increasing the intracellular concentration of the CpG-ODNs makes it possible to increase their stimulatory activity (31). The hypothesis that the CpG-ODNs will be more effective 15 when they are electroporated in vivo in our double LPB tumor model was tested. Two additional groups in which the CpGs were electroporated into the tumor immediately after 20 injection thereof were realized. The CpGs were injected intratumorally and then electroporated at Dl, D8, D15 and D22 as above. The group of electroporated CpGs combined with ECT 25 [ECT + (CpG + EP)] showed recovery from the treated tumors in 100% of cases (Fig. 4A, 5A). There was therefore no difference in antitumor effect with the ECT + CpG group which had itself also produced the same results. On the contrary, when there was no ECT, the 30 direct effect of the electroporated CpGs showed greater effectiveness (without a significant difference) compared to the nonelectroporated CpGs: 4 tumors out of 5 were stabilized, against only 1 out of 5 (Fig. 4A, SA). 35 In the ECT + (CpG + EP) group, all the nontreated contralateral tumors were stabilized for 20 days after the ECT, as for the ECT + CpG group (Fig. 4B, 5B). The WO 2006/010838 - 26 - PCT/FR2005/001612 growth of the right tumors of the EP + (CpG + EP) and EP + CpG groups was substantially identical. At the statistical level, the ECT + (CpG + EP) group 5 was significantly different on the left with respect to the EP + NaCl and EP + CpG groups. On the right, this group showed a significant difference with respect to the EP + NaCl and ECT + NaCl groups. 10 Study of the induction of cytotoxic T lymphocytes with the combination of ECT and CpGs: To test the ability of the therapeutic combination to induce cytotoxic T lymphocytes specific for the 15 SIINFEKL peptide, a tumor model was created by injecting 500 000 B160VA cells into the thigh of the animals. This model made it possible to easily treat the animals with ECT at DO and intratumor CpG at Dl. Five groups of 3 mice were created: one group in which 20 the mice with no tumor were immunized with the SIINFEKL peptide mixed into CpG, on the day of the immunotherapy, three groups treated with ECT + CpG or ECT + NaCl or CpG alone and one nontreated group. Seven days after the CpG injection, the animals were 25 sacrificed in order to remove the homolateral and contralateral inguinal lymph node of each mouse. The possible presence of specific cytotoxic T lymphocytes in the homolateral lymph node was sought ex vivo by means of labeling with soluble tetramers that recognize 30 H-2b/SIINFEKL. The contralateral inguinal lymph nodes were used as an internal negative control. A single experiment of this type has been carried out until now, but another is ongoing. 35 A cytometry analysis representative of the set of all the groups is shown in Figure 6. In the population of viable cells (Gl window), we selected the CD3/CD8 T lymphocytes (G2 window) in which the percentage of WO 2006/010838 - 27 - PCT/FR2005/001612 lymphocytes recognized by the tetramer was determined. This type of analysis was carried out for all the groups of the experiment; however, no positive labeling was detected, including in the control group immunized 5 with the peptide (Fig. 6C). No secretion of interferon y was found in the supernatant of the cultures stimulated in vitro with the peptide. It emerges from all these results that the combination 10 of ECT combined with CpGs resulted in both local and remote effects. Firstly, complete local regressions were observed in almost 100% of cases (8 cases out of 9 in all the 15 experiments) in the murine fibrosarcoma model. This local effectiveness is greater than the effect of each of the two treatments carried out separately. The substantial tumor reduction induced by the ECT therefore appears to be supplemented by an indirect 20 antitumor effect due to the activation of the immune system by the CpGs (synergistic action), whereas electroporation alone and bleomycin alone at sub therapeutic doses do not have any actual antitumor activity (3). 25 Secondly, regarding the remote effect of the ECT + CpG combination in the fibrosarcoma model, 5 stabilizations of 20 days and 3 complete regressions out of 9 animals in all the experiments were obtained. This combination 30 therefore very probably involves immune mechanisms responsible for a systemic effect in this model. The cell death induced the ECT alone, even though it appears to be associated with some immunological effects, is not sufficiently immunogenic to result in a 35 remote antitumor effect. Several hypotheses have been put forward to explain why the ECT-induced cell death became immunogenic when it was combined with the administration of CpGs, as attested to by the results.

WO 2006/010838 - 28 - PCT/FR2005/001612 The detailed observation of the results animal by animal (Fig. 2) suggests a close relationship between the direct antitumor effect and the remote effect, with 5 the need, it would appear, in order to obtain a contralateral effect, to have obtained a local activity and a substantial tumor reduction locally. Thus, the systemic effect of the treatment appears to be linked to its ability to induce a good local immune response. 10 As suggested above with regard to the local antitumor effect, the T lymphocyte immune response set up on the left (treated tumor) could be responsible for the effect observed remotely. 15 The combination according to the invention, which targets the immune response more globally and upstream by involving both innate immunity via the stimulation of NKs and of macrophages and acquired inner transition by activating CDs, makes it possible to achieve an 20 effective antitumor response. The B160VA melanoma model made it possible to test the effectiveness of the combination according to the invention on a tumor type other than fibrosarcoma, 25 allowing analysis of the immune response by virtue of the expression of the SIINFEKL peptide by the tumor. However, this tumor line was found to be very aggressive and rapidly led to deaths which did not make it possible to evaluate the evolution of the tumors, in 30 the double tumor model, and therefore to draw any conclusions with regard to the local and remote effects of the treatment. Nevertheless, this first experiment showing a survival rate higher than the other groups for the combination ECT + CpG at the 18th day of 35 treatment, encourages the development of a model more suitable for analyzing a local and systemic therapeutic effect.

WO 2006/010838 - 29 - PCT/FR2005/001612 Moreover, when the CpGs were electroporated (EP) in order to see whether access of CpGs to different intracellular compartments had an additional therapeutic effect, results comparable to the other 5 groups were apparent, whether at the local or contralateral level. In all the experiments on the LPB fibrosarcoma, all the mice (n = 9) of the ECT + (CpG + EP) group showed local regression and responded remotely (7 stabilizations and 2 complete regressions). 10 Since the ECT + CpG combination also showed very good results locally (8 recoveries of 9) and remotely (5 stabilizations and 3 recoveries, n = 9) electroporation of the CpGs does not provide any further effectiveness. The only noticeable benefit was observed in the group 15 where the CpGs alone were electroporated (EP + (CpG + EP)), in which group 4 stabilizations of the treated tumors of 5 were observed, against 1 out of 5 in the group treated with CpG alone (EP + CpG). In vitro, electroporation of the CpGs leads to 20 to 30% 20 mortality on the tumor cells without a dose effect. This is perhaps due to a phenomenon of saturation of cell cytotoxicity or of limited penetration into the cells. In vivo, the consequences of the direct cytotoxicity of the electroporated CpGs are difficult 25 to assess. Furthermore, in these experiments, the tumor was electroporated 4 times in a row without it being known exactly what effects this would have on the growth. 30 BIBLIOGRAPHY I. Orlowski, S., and L. M. Mir. 1993. Cell electropermeabilization: a new tool for biochemical and pharmacological studies. Biochim Biophys Acta 1154:51.

WO 2006/010838 - 30 - PCT/FR2005/001612 2. Mir, L. M., H. Banoun, and C. Paoletti. 1988. Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol. Exp Cell Res 175:15-25. 3. Mir, L. M., S. Orlowski, J. Belehradek, Jr., and C. Paoletti. 1991. Electro chemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. EurJCancer 27:68. 4. Mir, L. M., 0. Tounekti, and S. Orlowski. 1996. Bleomycin: revival of an old drug. Gen Pharmacol 27:745. 5. Poddevin, B., S. Orlowski, J. Belehradek, Jr., and L. M. Mir. 1991. Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture. Biochem Pharmacol 42 Suppl:S67. 6. Tounekti, 0., G. Pron, J. Belehradek, Jr., and L. M. Mir. 1993. Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized. Cancer Res 53:5462. 7. Mekid, H., 0. Tounekti, A. Spatz, M. Cemazar, F. Z. El Kebir, and L. M. Mir. 2003. In vivo evolution of tumour cells after the generation of double-strand DNA breaks. Br J Cancer 88:1763. 8. Okino, M., and H. Mohri. 1987. Effects of a high-voltage electrical impulse and an anticancer drug on in vivo growing tumors. Jpn J Cancer Res 78:1319. 9. Ramirez, L. H., S. Orlowski, D. An, G. Bindoula, R. Dzodic, P. Ardouin, C. Bognel, J. Belehradek, Jr., J. N. Munck, and L. M. Mir. 1998. Electrochemo therapy on liver tumours in rabbits, Br J Cancer 77:2104. 10. Mir, L. M., and S. Orlowski. 1999. Mechanisms of electrochemotherapy. Adv Drug Deliv Rev 35:107. 11. Sersa, G., D. Miklavcic, M. Cemazar, J. Belehradek, T. Jarm and L.M. Mir. 1997. Electrochemotherapy with CDDP on LPB sarcoma: comparison of the anti-tumor effectiveness in immunocompetent and immunideficient mice. Bioelectrochemisiry and Bioenergetics 43:279. 12. Mir, L. M., S. Orlowski, B. Poddevin, and J. Belehradek, Jr. 1992. Electro chemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses. Eur Cytokine Netw 3:331.

WO 2006/010838 - 31 - PCT/FR2005/001612 13. Belehradek, M., C. Domenge, B. Luboinski, S. Orlowski, J. Belehradek, Jr., and L. M. Mir. 1993. Electrochemotherapy, a new antitumor treatment. First clinical phase 1-11 trial. Cancer 72:3694. 14. Heller, R., R. Gilbert, and M. J. Jaroszeski. 1999. Clinical applications of electrochemotherapy. Adv Drug Deliv Rev 35:119. 15. Gothelf, A., L. M. Mir, and J. Gehl. 2003. Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev 29:371. 16. Sersa, G., M. Cemazar, V. Menart, V. Gaberc-Porekar, and D. Miklavcic. 1997. Anti-tumor effectiveness of electrochemotherapy with bleomycin is increased by TNF-alpha on SA-1 tumors in mice. Cancer Left 116:85. 17. Mir, L. M., C. Roth, S. Orlowski, F. Quintin-Colonna, D. Fradelizi, J. Belehradek, Jr., and P. Kourilsky. 1995. Systemic antitumor effects of electro chemotherapy combined with histoincompatible cells secreting interleukin-2. J Immunother Emphasis Tumor Immunol I 7:30. 18. Mir, L. M., C. Roth, S. Orlowski, J. Belehradek, Jr., D. Fradelizi, C. Paoletti, and P. Kourilsky. 1992. [Potentiation of the antitumoral effect of electro chemotherapy by immunotherapy with allogeneic cells producing interleukin 21. C R Acad Sci 111 314:539. 19. Roth, C., L. M. Mir, v. Cressent, F. Quintin-Colonna, V. Ley, D. Fradelizi, and P. Kourilsky. 1992. Inhibition of tumor growth by histoincompatible cells expressing interleukin-2. Int Immunol 4:1429. 20. Orlowski, S., D. An, J. Belehradek, Jr., and L. M. Mir. 1998. Antimetastatic effects of electrochemotherapy and of histoincompatible interleukin-2 secreting cells in the murine Lewis lung tumor. Anticancer Drugs 9:551. 21. Andersen, M. H., J. Gehl, S. Reker, L. 0. Pedersen, J. C. Becker, P. Geertsen, and P. thor Straten. 2003. Dynamic changes of specific T cell responses to melanoma correlate with IL-2 administration. Semin Cancer Biol 13:449. 22. Gilboa, E. 1999. The makings of a tumor rejection antigen. Immunity 11:263. 23. Dhodapkar, M. V., J. W. Young, P. B. Chapman, W. I. Cox, J. F. Fonteneau, S. Amigorena, A. N. Houghton, R. M. Steinman, and N. Bhardwaj. 2000.

WO 2006/010838 - 32 - PCT/FR2005/001612 Paucity of functional T-cell memory to melanoma antigens in healthy donors and melanoma patients. Clin Cancer Res 6:4831. 24. Tokunaga, T., H. Yamamoto, S. Shimada, H. Abe, T. Fukuda, Y. Fujisawa, Y. Furutani, 0. Yano, T. Kataoka, T. Sudo, and et al. 1984. Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. J Aatl Cancer Inst 72:955. 25. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546. 26. Hemmi, H., 0. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll like receptor recognizes bacterial DNA. Nature 408:740. 27. Miconnet, I., S. Koenig, D. Speiser, A. Krieg, P. Guillaume, J. C. Cerottini, and P. Romero. 2002. CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. JImmunol 168:1212. 28. Carpentier, A. F., L. Chen, F. Maltonti, and J. Y. Delattre. 1999. Oligodeoxy nucleotides containing CpG motifs can induce rejection of a neuroblastoma in mice. Cancer Res 59:5429. 29. Heckelsmiller, K., K. Rall, S. Beck, A. Schlamp, J. Sciderer, B. Jahrsdorfer, A. Krug, S. Rothenfusser, S. Endres, and G. Hartmann. 2002. Peritumoral CpG DNA elicits a coordinated response of CD8 T cells and innate effectors to cure established tumors in a murine colon carcinoma model. J Immunot 169:3892. 30. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock. 2004. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 5:190. 31. Yamamoto, T., S. Yamamoto, T. Kataoka, and T. Tokunaga. 1994. Lipofection of synthetic oligodeoxyribonucleotide having a palindromic sequence of AACGTT to murine splenocytes enhances interferon production and natural killer activity. Microbial Immunol 38:831.

WO 2006/010838 - 33 - PCT/FR2005/001612 32. Auf, G., A. F. Carpentier, L. Chen, C. Le Clanche, and J. Y. Delattre. 2001. Implication of macrophages in tumor rejection induced by CpG-oligodeoxy nucleotides without antigen. Clin Cancer Res 7:3540. 33. Ballas, Z. K., W. L. Rasmussen, and A. M. Krieg. 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. Jimmunol 157:1840. 34. J. Belehradek Jr, S. Orlowski, B. Poddevin, C. Paoletti and L.M. Mir. Electrochemotherapy of spontaneous mammary tumours in mice. European Journal of Cancer, 2, 73-76 (1991). 35. C.Domenge, S.Orlowski, B.Luboinski, T. De Baere, G. Schwaab, J.Belehradek Jr and L.M.Mir. Antitumor electrochemotherapy : new advances in the clinical protocols. Cancer 77, 956-963, 1996. 36. J.Gehl, T.Skovsgaard and L.M. Mir. Enhancement of cytotoxicity by electropermeabilization: an improved method for screening drugs. Anti-Cancer Drugs 9, 319-325, 1998. 37. T. Kotnik, G. Pucihar, M. Reber~ek, D. MiklavUie and L.M. Mir. The role of pulse shape in cell membrane electropermeabilization in vitro. Biochimica Biophysica Acta - Biomembranes, 1614, 193-200, 2003. 38. LM.Mir, M.Belehradek, C.Domenge, S.Orlowski, B.Poddevin, J.Belehradek Jr, G.Schwaab, B.Luboinski and C.Paoletti. L'dlectrochimiothdrapie, un nouveau traitement antitumoral : premier essai clinique Electrochemotherapy, a novel antitumor treatment: first clinical trial. Compte rendus de l'Acad6mie des Sciences, sdr [11, 313, 613-618 (199 lb). 39. L.M. Mir, F.L. Glass, G.8ersa, J.Teissid, C.Domenge, D. Miklavcic, M.J. Jaroszeski, S. Orlowski, D.S. Reintgen, Z. Rudolf, M. Belehradek, R. Gilbert, M.P. Rols, J. Belehradek Jr, J.M. Bachaud, R. DeConti, B. Stabuc, P. Coninx, M. Cemazar, R.Heller. Effective treatment of cutaneous and subcutaneous malignant tumors by electrochemotherapy. British Journal of Cancer 77, 2336 2342, 1998. 40. Orlowski S, Belehradek J, Jr., Paoletti C, Mir LM. Transient electropermeabilisation of cells in culture. Increase of the cytotoxicity of anticancer drugs. Biochem Pharmacol 37: 4727-4733, 1988.

WO 2006/010838 - 34 - PCT/FR2005/001612 41. Sersa G, Cemazar M, Rudolf Z. Electrochemotherapy: advantages and drawbacks in treatment of cancer patients. Cancer Therapy 1: 133-142, 2003. 42. Teissid J., Rols M. P. An experimental evaluation of the critical potential difference inducing cell membrane dlectropermeabilization. Biophys J, 65:409 13, 1993. As emerges from the above, the invention is in -no way limited to those of its methods of implementation, execution and application that have just been 5 specifically described; on the contrary, it encompasses all the variants thereof that may occur to a person skilled in the art, without departing from the context or the scope of the present invention.

Claims (27)

1. A product containing at least one anticancer active principle with low diffusion and an 5 immunostimulatory active principle selected from the group consisting of TLR ligands, as a combined preparation for simultaneous, separate or sequential use, for the treatment of benign or malignant tumors, and more particularly 10 metastases, in an individual suffering from a tumor and subjected to an electropermeabilization at the level of said tumor.

2. A product containing at least one anticancer 15 active principle with low diffusion and an immunostimulatory active principle selected from the group consisting of TLR ligands, as a combined preparation for simultaneous, separate or sequential use, for the treatment of benign or 20 malignant tumors, and more particularly metastases, in an individual suffering from a tumor and subjected to an electropermeabilization at the level of an appropriately selected normal tissue. 25

3. The product as claimed in claim 1 or claim 2, characterized in that the TLR ligands are immunostimulatory oligodeoxynucleotides (ODNs). 30

4. The product as claimed in any one of claims 1 to 3, characterized in that the TLR ligands are TLR9 ligands.

5. The product as claimed in claim 4, characterized 35 in that the TLR9 ligands are immunostimulatory CpG-ODNs. WO 2006/010838 - 36 - PCT/FR2005/001612

6. The product as claimed in claim 5, characterized in that the immunostimulatory CpG-ODN is selected from the group consisting of a stabilized oligodeoxynucleotide which comprises at least one 5 quadrameric motif of formula Xi-CG-X 2 , in which X, and X 2 are identical or different and represent T or A.

7. The product as claimed in claim 6, characterized 10 in that said CpG-ODN is preferably selected from the group consisting of an oligodeoxynucleotide which comprises at least one quadrameric sequence selected from the group consisting of: TCGA, ACGT, ACGA and TCGT. 15

8. The product as claimed in claim 6 or claim 7, characterized in that said CpG-ODN is preferably selected from the group consisting of an oligodeoxynucleotide which comprises at least one 20 hexameric sequence selected from the group consisting of: AACGTT, GACGTC, GACGTT, GTCGTT, TTCGAA, TACGTA and ATCGAT.

9. The product as claimed in claim 8, characterized 25 in that said CpG-ODN is preferably selected from the group consisting of an oligonucleotide which comprises at least the following octameric sequence: AACGTT-X 3 X 4 , in which X 3 X 4 is AT, AA, CT or TT, and preferably the octameric sequence 30 AACGTTAT.

10. The product as claimed in any one of claims 6 to 9, characterized in that said CpG-ODN is the CpG ODN of sequence SEQ ID No. 1. 35

11. The product as claimed in claim 5, characterized in that the immunostimulatory active principle is a CpG-ODN selected from the group consisting of a WO 2006/010838 - 37 - PCT/FR2005/001612 stabilized oligonucleotide which comprises at least one octameric motif of the type: 5'-purine purine-CG-pyrimidine-pyrimidine-XiX 2 -3', in which the pair X 1 X 2 is AT, AA, CT or TT. 5

12. The product as claimed in claim 11, characterized in that said CpG-ODN is preferably selected from the group consisting of an oligonucleotide which comprises at least one octameric sequence selected 10 from the group consisting of: GACGTT-XiX 2 , AGCGTT-XiX 2 , GGCGTT-XiX 2 , AACGTC-XiX 2 , GACGTC-XiX 2 , AGCGTC-XiX 2 and GGCGTC-XiX 2 , in which Xi-X 2 is AT, AA, CT or TT. 15

13. The product as claimed in claim 11 or claim 12, characterized in that said CpG-ODN is chosen from the group consisting of the sequences SEQ ID No. 2 to SEQ ID No. 41. 20

14. The product as claimed in any one of claims 1 to 12, characterized in that the anticancer active principle is selected from the group consisting of cytotoxic substances selected from the group consisting of antimetabolites and genotoxic 25 substances.

15. The product as claimed in any one of claims 1 to 14, characterized in that the anticancer active principle is preferably a genotoxic substance, and 30 even more preferably bleomycin.

16. The product as claimed in any one of claims 1 to 15, characterized in that the anticancer active principle is administered intratumorally, locally 35 or systemically.

17. The product as claimed in any one of claims 1 to 16, characterized in that the immunostimulatory WO 2006/010838 - 38 - PCT/FR2005/001612 active principle is administered intratumorally, locally or systemically.

18. The product as claimed in any one of claims 1 and 5 3 to 17, characterized in that the cells of the tumor are electropermeabilized by exposure of said tumor cells to electric pulses of sufficient strength or intensity and duration to allow the electroporation of said cancer cells. 10

19. The product as claimed in claim 18, characterized in that the cells of the tumor are electropermeabilized after the administration of said products by exposure of said tumor cells to 15 electric pulses of sufficient strength or intensity and duration to allow the electroporation of said cancer cells.

20. The product as claimed in claim 18, characterized 20 in that the cells of the tumor are electropermeabilized beforehand by exposure of said tumor cells to electric pulses of sufficient strength or intensity and duration to allow the electroporation of said cancer cells. 25

21. The product as claimed in claim 18, characterized in that the intensity of the electric field applied ranges between 20 and 2000 V/cm. 30

22. The product as claimed in claim 18, characterized in that the intensity of the electric field applied ranges between 500 and 1500 V/cm.

23. The product as claimed in claim 18, characterized 35 in that the pulse frequency ranges from 0.01 to 10 000 Hz. WO 2006/010838 - 39 - PCT/FR2005/001612

24. The product as claimed in claim 23, characterized in that the pulse frequency ranges from 1 to 5000 Hz. 5

25. The product as claimed in claim 18, characterized in that the duration of the pulses ranges from 10 pLs to 10 s.

26. The product as claimed in claim 25, characterized 10 in that the duration of the pulses ranges from 50 ps to 500 ms.

27. The product as claimed in claim 2, characterized in that said antigen is selected from the group 15 consisting of purified tumor-specific antigens, tumor extracts, and irradiated or modified tumor cells.