ES2328330A1 - Pharmaceutical composition for cancer treatment - Google Patents

Abstract

The invention relates to compositions containing a first compound having TGF-ss1 inhibitory activity and a second compound in the form of an immune-stimulating cytokine, preferably IL-12 or a functionally equivalent variant of same. Said compositions demonstrate synergistic effects on antitumour properties when compared to components acting separately. The invention also relates to compositions containing the aforementioned compounds and to the medical use of same for the treatment of cancer, particularly hepatocellular carcinoma and gastrointestinal tumours.

Description

Pharmaceutical composition for the treatment of Cancer.

Technical Field of the Invention

The invention relates to compositions therapeutic treatments for cancer and, more specifically, to compositions comprising a TGF? 1 inhibitor and an immunostimulatory cytokine or a polynucleotide comprising a sequence that encodes an immunostimulatory cytokine.

Background

Of the many suppressor factors derived from cancer, transforming growth factor? (TGF?) it is unique given its inhibitory activities exerted on cells immune, and its promtastic influence on cells cancerous TGF? Profoundly alters the functions of T lymphocytes, and is able to inhibit the generation of responses from Cytotoxic T lymphocytes specific for viruses and tumors. Because its potent immunosuppressive properties, tumor cells that actively secrete TGF? may be able to avoid host immunovigilance mechanisms. It has been investigated the negative impact that TGF? exerts on tumor-specific cytotoxic T lymphocyte responses in studies that use the adoptive transfer of T cells that have become insensitive to TGF? signaling, by means of introduction of a transgene of a dominant TGF? receptor negative (dnTGFβRII mice). Such investigations clearly demonstrate that tumor-specific T cells resistant to the suppressive effects of TGF? are more effective than normal T cells in tumor eradication established.

Interleukin-12 (IL-12) is a cytokine capable of increasing antitumor immunity through multiple mechanisms that include: (1) increased T lymphocyte responses cytotoxic, (2) activation of natural cytolytic cells, (3) promotion of the proliferation of natural cytolytic cells and T lymphocytes, (4) induction of the polarization of a subset of TH1 helper cells, and (5) induction of an effect antiangiogenic Many of these activities are mediated by the production and secretion of interferon-? (INF- γ) by natural cytolytic cells and activated T lymphocytes. Despite these properties potent immunomodulators, the use of IL-12 in human cancer patients has been hindered by the unexpected Toxicity and disappointing efficacy. It is believed that the rates of low response to individual agent therapy IL-12 are the result of reduced production of INF-? By effector cells after repeated treatments with IL-12. Besides, the active secretion of immunosuppressive cytokines by cells cancer has the potential to further reduce the production of INF-? By effector cells. Given the importance of INF-? in the mediation of protective immunity, combine IL-12 with strategies that enhance the sustained secretion of INF-? By the effector cells of tumor infiltration may allow the generation of responses superior antitumor immune.

Several documents disclose therapies of combination to treat cancer:

Fan, TM et al ., 2007 (J Immunother 30: 478-489) shows that strategies that combine IL-12 with TGFβ neutralization may be useful for complementary treatment for some human cancer patients.

Chang, CJ, et al ., 2007 (Hepatology, vol. 45: 746-754) discloses that the combined gene therapy of GM-CSF and IL-12 synergistically suppresses the growth of orthotopic liver tumor.

Hwang, L. 2006 (J Biomed Science, vol. 13: 453-468) discloses gene therapy strategies for hepatocellular carcinoma (HCC), which includes the combination of IL-12 and costimulatory molecules such as B7.1.

U.S. patent application US2003 / 0125251 discloses the use of a viral vector encoding an antagonist of TGF? To treat cancer in combination with IL-12, where it is proposed to use IL-12 to block the humoral immune response or cell to viral vector.

U.S. patent application US2003 / 0125251 discloses compositions comprising TGF? antagonists (in particular, the soluble domain of the receptor of TGF-? Type II) and a second component that inhibits cell proliferation and that is selected from the group of temozolomide, nitrosoureas, vinca alkaloids, antagonists of the purine and pyrimidine bases, cytostatic antibiotics assets, derivatives of caftothecin, antiestrogens, antiandrogens and Gonadotropin releasing hormone analogues. These compositions are suitable for the treatment of metastasis.

The international patent application WO2005 / 097832 discloses anti-TGF? Antibodies humanized to treat prostate cancer in combination with drugs that include cytokines such as IL-12.

However, neither US2003 / 0125251 nor W02005 / 097832 disclose the fact that the combined administration of an agent TGFβ1 inhibitor and an immunostimulatory cytokine can result in a therapeutic effect greater than administration of the TGF? 1 inhibitor or the immunostimulatory cytokine when administered alone.

According to this, there is a need in the technique for antitumor compositions based on agents TGF-? 1 inhibitors.

Brief Description of the Invention

In a first aspect, the invention relates to a composition or kit comprising

(i)

a TGFβ1 inhibitor or a polynucleotide comprising a nucleotide sequence encoding an inhibitor agent of TGF? 1, and

(ii)

to the minus an immunostimulatory cytokine or a polynucleotide that it comprises a nucleotide sequence that encodes a cytokine immunostimulatory

In further aspects, the invention relates to to the above compositions for use as a medicine, for treat and / or prevent cancer or tumor diseases and to a pharmaceutical preparation comprising a composition of the invention.

In another aspect, the invention relates to the use of a TGF? 1 inhibitor or a polynucleotide that comprises a nucleotide sequence that encodes an agent TGF? 1 inhibitor to promote the antitumor effects of an immunostimulatory cytokine.

In yet another aspect, the invention relates to a polynucleotide comprising the nucleotide sequence that encodes a TGF? 1 inhibitor and the sequence of nucleotides that encode an immunostimulatory cytokine.

Brief description of the figures

Figure 1 shows the antitumor effect in mice that have MC38 tumors in the liver treated with SFV-enhIL-12 alone or in combination with p17 . Groups of mice that had MC38 tumors in the liver were injected with 10 8 pv of SFV-enhIL-12, 10 8 pv of SFV-LacZ or the same volume of saline solution (left part of the figure) . Parallel groups of animals were inoculated with the same vectors combined with 10 repeated intraperitoneal administrations of 75 µg of p17 given every 48 hours (right part of the figure). Tumor size was measured on the day of intratumoral treatment and 20 days later by laparotomy. The percentage of complete tumor regressions as well as the number of animals treated in each group is indicated below the
graph.

Figure 2 shows the survival of mice that have MC38 tumors in the liver treated with SFV-enhIL-12 alone or in combination with p17 . All mice described in Figure 1 were maintained after the laparotomy performed for tumor size evaluation and followed for at least 200 days after treatment.

Figure 3 shows the evaluation of the induction of protective memory antitumor responses . Animals that rejected MC38 tumors in the liver after treatment with SFV-enhIL-12 alone or in combination with p17 were again exposed 3 months later by injecting 5x105 MC38 cells into the liver (left part of the figure) or subcutaneously (right part of the figure). Tumor size was assessed by laparotomy 20 days after reexposure. The percentage of animals resistant to re-exposure as well as the total number of animals treated in each group is indicated under the graph. Naive, animals that had not had previous contact with MC38 tumor cells.

Figure 4 shows the antitumor effect in mice that have MC38 tumors in the liver treated with HC-Ad-mIL-12 alone or in combination with p17 . Liver metastases were induced in mice by intrahepatic inoculation of MC38 cells. Three days later, 4x10 8 ui of HC-Ad-mIL-12 vector was administered in the liver around the tumors. Six days later, inductions with RU486 were performed as described in the methods. After the sixth induction, a group of animals were treated with ip injections of p17 every 48 hours. Other groups received p17 alone, or no treatment, as indicated. The graph represents the size of liver tumors measured 55 days after the inoculation of the cells, or at the time of death of the animal in case of shorter survival.

Figure 5 shows the survival of mice that have MC38 tumors in the liver treated with HC-Ad-mIL-12 alone or in combination with p17 . All mice described in Figure 4 were maintained after laparotomy performed for tumor size evaluation and followed for at least 100 days after treatment.

Figure 6 shows the antitumor effect in mice that have subcutaneous MC38 tumors treated with SFV-enhIL-12 alone or in combination with p144 . Groups of mice that had subcutaneous MC38 tumors were injected with 10 7 pv of SFV-enhIL-12 or 10 7 pv of SFV-LacZ (right part of the figure). Parallel groups of animals were inoculated with the same vectors, or the same volume of saline, combined with 10 repeated intratumoral administrations of 50 µg of p144 given every 48 hours (left part of the figure). Tumor size was measured on the day of intratumoral treatment and 20 days later by laparotomy. The percentage of complete tumor regressions as well as the number of animals treated in each group is indicated below the graph.

Figure 7 shows the antitumor effect in mice that have MC38 tumors in the liver treated with SFV-enhIL-12 alone or in combination with a TGF? Blocking antibody . Groups of mice that had MC38 tumors in the liver were injected with 10 8 pv of SFV-enhIL-12, or the same volume of saline (right part of the figure). Parallel groups of animals were inoculated with the same saline vector or solution, combined with 4 repeated 200 µg intraperitoneal administrations of a TGF? Blocking antibody given every 72 hours (left part of the figure). Tumor size was measured on the day of intratumoral treatment and 20 days later by laparotomy. The percentage of complete tumor regressions as well as the number of animals treated in each group is indicated below the graph.

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Detailed description of the invention

The present invention relates to the treatment of cancer through the use of different therapeutic agents. The inventors of the present invention have found surprisingly that the combined administration of an agent TGFβ1 inhibitor and immunostimulatory cytokine not only induces more potent antitumor responses than each agent therapeutic separately, but it is also capable of generating a stronger and more widespread memory immune response, which It achieves the eradication of tumors formed in mice. He example accompanying this description shows that the administration of a TGF? 1 inhibitor has the ability to increase the immune response and / or the antitumor effect induced by IL-12.

The present invention thus relates to  treatment of cancer or tumor diseases by means of combined administration of a TGF? 1 inhibitor and a  immunostimulatory cytokine, thus achieving a therapeutic effect greater than if said inhibitor of TGFβ1 and cytokine is They administer alone.

Therefore, in one aspect the invention is refers to a composition or kit, which comprises

(i)

a TGFβ1 inhibitor or a polynucleotide comprising a nucleotide sequence encoding an inhibitor agent of TGF? 1, and

(ii)

to the minus an immunostimulatory cytokine or a polynucleotide that it comprises a nucleotide sequence that encodes a cytokine immunostimulatory

In the present invention "an inhibitory agent TGF? 1 "is understood as any compound capable of prevent signal transmission caused by the interaction between TGFβ1 and its receptor. TGF? 1 inhibitors which can be used according to the present invention include compounds that prevent competitive or allosteric binding of TGF? 1 to its receptor, compounds that bind to TGF? 1 and compounds that inhibit intracellular signaling of TGFβ1. Preferably, the present invention considers the use of agents TGF? 1 inhibitors that prevent TGF? 1 binding with its receptor such as inhibitory peptides, soluble proteins that naturally bind to and inhibit TGFβ1 (LAP, decorin, fibromodulin, lumican, endoglin, a2-macroglobulin), receptors that compete with the endogenous TGF? 1 receptor by ligand binding, as BAMBI, or receptors that increase the binding of TGFβ1 to its type II receptor, such as betaglycan. Between agents TGF? 1 inhibitors that prevent TGF? 1 binding with its receptor you can find monoclonal antibodies and polyclonal specific for the TGFG31 receptor and forms soluble TGFβ1 receptor.

Thus, in an embodiment Particular of the composition or kit of the invention, the agent TGFβ1 inhibitor is selected from peptide inhibitors of TGFβ1, TGFβ1 inhibitor antibody, soluble receptor of TGFβ1 and cell-producing inhibitors of TGF? 1.

The TGFβ1 inhibitor peptides according to the invention are those peptides capable of binding TGFβ1 and inhibiting its activity as explained above, that is to say preventing signal transmission caused by the interaction between TGFβ1 and its receptor. Examples of such peptides are disclosed in international patent applications WO0031135, WO200519244 and WO0393293 which are incorporated herein by reference. In a particular embodiment, TGFβ1 inhibitor peptides are selected from peptides comprising SEQ ID NO: 1 to SEQ ID NO: 6. Suitable assays to determine the inhibitory capacity of TGFβ1 inhibitor peptides they include the in vitro inhibition of the biological activity of TGFβ1 using peptides in Mv-1-Lu cell proliferation assays as well as the in vivo inhibition of the biological activity of TGFβ1 by peptides using a model of induced acute liver damage by CC14 (disclosed in WO200519244).

In the present invention, "an antibody TGF? 1 "inhibitor is understood as any antibody which can specifically bind TGF? 1 protein or TGFβ1 receptor and prevent signal transduction caused by the interaction between TGF? 1 and its receptor. He term "antibody" should be interpreted in a broad sense and includes multispecific, polyclonal, monoclonal antibodies and Fab fragments thereof, F (ab ') 2, provided that can specifically bind TGF? 1 protein or receptor of TGF? 1 and therefore prevent the transmission of signs. Examples of antibodies that can be used in the context of the present are, for example and are not limited to, polyclonal antibodies, monoclonal antibodies, antibodies recombinants, chimeric antibodies, humanized antibodies, fully human antibodies, etc. Examples of antibodies specific for TGFβ1 or its receptor in EP1175445.

Polyclonal antibodies are originally heterogeneous mixtures of antibody molecules produced in the serum of animals that have been immunized with an antigen. Too include monospecific polyclonal antibodies obtained at from heterogeneous mixtures, for example, by means of column chromatography with peptides of an individual epitope of the antigen of interest

A monoclonal antibody is a population homogeneous antibody specific for an individual epitope of the antigen. These monoclonal antibodies can be prepared by means of conventional techniques that have already been described, by example, in Kohler and Milstein [Nature, 1975; 256: 495-497] or Harlow and Lane ["Using Antibodies. A Laboratory Manual "by E. Harlow and D. Lane, Editor: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1998 (ISBN 978-0879695439)].

A chimeric antibody is an antibody monoclonal constructed by means of cloning or recombination of antibodies of different animal species. In a configuration typical but not limiting of the invention, the chimeric antibody includes a part of a monoclonal antibody, usually the variable region (Fv) that includes sites for recognition and antigen binding, and the other part corresponding to an antibody human, usually the part that includes the constant region and the region adjacent to the constant.

A fully human antibody is an antibody or antibodies that have been produced in transgenic animals with a human immune system or by in vitro immunization of human immune cells (including both genetic and traditional immunization with or without adjuvants and pure or impure antigen; or through of any method to expose the antigen to the immune system) or through native / synthetic libraries produced from human immune cells. These antibodies can be obtained and screened from transgenic animals (mice, for example) in which human immunoglobulin genes have been cloned and immunized with the target antigen (with the TGF? 1 protein or the TGF? 1 receptor in the present invention). These antibodies can be obtained by screening single-variable variable regions (scFv) or human antigen-binding regions (Fab) presented in phages and subsequently, cloning and grafting in a human antibody or by any other method known by the skilled in the art, to produce and present the libraries generated by cloning the variable regions of both chains and subsequently cloning / mutation thereof to generate antibody libraries.

A humanized antibody is an antibody monoclonal constructed by means of cloning and grafting of Hypervariable Complementarity Determining Regions (CDR) of a murine monoclonal antibody in a human antibody in replacement of their own hypervariable CDR regions.

Furthermore, in the context of the present invention, the term "antibody" also includes variants with a altered glycosylation pattern, as described in WO2006088447 as well as glycosylated antibody fragments or not glycosylates that have been obtained from the protein or through recombination technology, which may consist of (i) areas antibody variables linked together by a peptide of junction (scFv), (ii) the variable area adjacent to the constant area CHI of the heavy chain (Fd) attached to the light chain by means of cysteines or by means of binding peptides and disulfide bridges (scFab), (iii) new variants, such as heavy chains alone, or (iv) any modification made to the fragments of antibody with the purpose of making them more similar, less immunogenic (humanized) or more stable in biological fluids and so that they have the ability to bind TGF? or its receptor and  to inhibit or prevent the transmission of signals produced by the interaction of TGFβ1 and its receptor.

The TGF? 1 inhibitor antibody described in the present invention it can be obtained by techniques Conventional recombination or genetic engineering techniques Conventional for the production of antibodies, extraction and purification of biological fluids or tissues, or any other conventional technique to obtain proteins and antibodies, which are widely known to those skilled in the art. When the TGFβ1 inhibitors are antibodies, they can be use the following techniques, among others, for its production, without this being any limitation: immunization techniques in animals, including transgenic animals for the genes of the human immunoglobulins, production of monoclonal antibodies through hybridomas, production through libraries of antibodies, which can be native, synthetic, or derived from organisms immunized against the antigen of interest and that they could screen through different presentation methods (phage presentation, ribosome presentation, etc.) and subsequently, through genetic engineering techniques, could redesign and express in vectors designed to produce recombinant antibodies with size, composition and structure different. A review of the methods can be found main to produce and purify antibodies in, by example:

"Handbook of Therapeutic Antibodies ", by S. Dübel. Publisher: Wiley-VCH, 2007, Vol: I to III (ISBN 978-3527314539);

"Antibodies: Volume 1: Production and Purification "by G. Subramanian Ed., Editor: Springer, 1st Ed, 2004 (ISBN 978-0306482458);

"Antibodies: Volume 2: Novel Technologies and Therapeutic Use "by G. Subramanian Ed., Publisher: Springer, first edition, 2004 (ISBN 978-0306483158);

? Molecular Cloning: a Laboratory Manual "by J. Sambrook and D.W. Russel Eds., Editor: Cold Spring Harbor Laboratory Press, third edition, 2001 (ISBN 978-0879695774).

In the present invention, a soluble receptor of TGF? 1 is understood as the extracellular domain of the receptor of TGF? 1, which can be obtained physiologically by proteolytic processing of endogline or betaglycan (receptors Type III), or by recombinant technology expressing only the  extracellular domain of type I TGF? 1 receptors and type II

In the context of the present invention, the inhibitors of TGFβ1 producing cells are understood as those compounds capable of inhibiting the production of TGF? 1 cytokine by TGF? 1 producing cells, such as macrophages or lymphocytes, thrombospondin antagonists,  antioxidants that prevent the activation and overexpression of TGFβ1, TGFβ1 specific silencing agents (RNA antisense, RNAi, siRNA and the like) or plant extracts such as silibinin and tranilast (Capper, E.A., 2000, J. Pharmacol and Exp Therapeutics, vol. 295 (3): 1061-1069).

In a particular embodiment, the inhibitors of TGFβ1 producing cells are those FoxP3 inhibitor peptides.

As mentioned above, the composition or kit of the invention comprises (i) an inhibitory agent of TGF? 1, and (ii) an immunostimulatory cytokine or a polynucleotide comprising a nucleotide sequence that encodes an immunostimulatory cytokine.

In the present invention, "a cytokine immunostimulatory "is understood as any compound that stimulates an increase in the activity of any component of immune system including those components that are part or are involved in the cellular immune response, immune response humoral and complement system. Preferably, the cytokine immunostimulatory is selected from the group of IL-12,  IL-2, IL-15, IL-18, IL-24, GM-CSF, TNF-?, CD40 ligand, IFN-?, IFN-?, IFN-? And functionally equivalent variants from the same.

IL-12 is a heterodimeric cytokine consisting of two covalently linked p35 and p40 subunits. Therefore, as the person skilled in the art will understand, the polynucleotide comprising a sequence encoding IL-12, or a functionally equivalent variant thereof may include a polynucleotide comprising the nucleotide sequence encoding said p35 and p40 subunits of IL-12, ie both coding sequences included in the same polynucleotide and operably linked to separate promoter regions that may be identical or different between both coding sequences. In the present invention "a functionally equivalent variant of IL-12" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids of the IL-12 sequence, and polypeptide that at less in part it retains the ability to increase antitumor immunity, determined for example by means of the test mentioned in the Example or the test cited in Zabala, M. 2007 et al ., [J Hepatology, vol. 47 (6): 807-815].

IL-2 is a cytokine that is produced by antigen presenting cells after antigen binding to the T cell receptor and that stimulates the growth, differentiation and survival of the cytotoxic T cells selected by the antigen. In the present invention, "a functionally equivalent variant of IL-2" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids from the IL-2 sequence, and polypeptide that at least in part it retains the ability to stimulate the growth, differentiation and survival of cytotoxic T cells, determined for example by means of the assay described in Gillis et al . (J Immunol, 1978, 120: 2027-2032) based on the extent of [3 H] -thymidine incorporation into CTLL-2 cells expressing the IL-2 receptor.

IL-15 is a cytokine secreted by mononuclear phagocytes after virus infection that induces cell proliferation of natural cytolytic cells. In the present invention, "a functionally equivalent variant of IL-15" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids of the IL-15 sequence, and polypeptide that at least in part it retains the ability to induce cell proliferation of natural cytolytic cells, determined for example by means of the assay described in Gillis et al . (J Immunol, 1978, 120: 2027-2032) based on the measure of the incorporation of [3 H] -thymidine into CTLL-2 cells expressing the IL-2 receptor in response to the IL- variant fifteen.

IL-18 is a cytokine produced by monocyte line cells, increases the activity of NK cells, increases proliferation of T cells and encourages NK and T cells to secrete INF-γ as well as GM-CSF. In addition, this cytokine also increases the production of Th1 type responses associated with CTL antitumor responses. In the present invention, "a functionally equivalent variant of IL-18" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids of the IL-18 sequence, and polypeptide that at least in part it retains the ability to induce the production of INF-γ in human myelomonocytic cell lines, determined for example by means of the assay described in Konishi et al . (J Imunological Methods, 1997, 209: 187-191).

IL-24 (also known as a melanoma differentiation associated gene 7, mda-7) is produced by activated monocytes, T cells and other cells of the immune system, as well as other cells such as cancer cells under specific conditions such as differentiation after treatment with INF-? and mezerein (reviewed in Pharmacol Ther, 2006, 111: 596-628). Apart from its ability to stimulate immune responses, overexpression of this cytokine has the ability to induce apoptosis preferentially in cancer cells. In the present invention, "a functionally equivalent variant of IL-24" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids of the IL-24 sequence, and polypeptide that at least in part it retains the ability to activate functions related to immunity and induce apoptosis in cancer cells determined for example by means of the assays described in Caudell et al . (J Immunol, 2002, 168: 6041-6046) based on measuring the stimulation of IFN-γ expression after treatment of cells with variants of IL-24, and Lebedeva et al . (Oncogene 2002, 21: 708-718), based on the quantification of apoptotic cells by annexin V binding.

GM-CSF is a cytokine that stimulates the differentiation of stem cells to granulocytes (neutrophils, eosinophils, and basophils) and monocytes. In the present invention, "a functionally equivalent variant of GM-CSF" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids from the GM-CSF sequence, and polypeptide that at least in part it retains the ability to stimulate the differentiation of stem cells into neutrophils and monocytes, determined for example by means of the assay described in McCormack et al . (Blood, 2000, 95: 120-127) based on microscopically measuring the differentiation of FDB1 cells to neutrophils, monocytes and megakaryocyte-like cells in the presence of GM-CSF.

TNF-? Is a cytokine mainly produced by macrophages that is involved in systemic inflammation and stimulates the acute phase reaction, acts on neutrophils that helps stick to endothelial cells for migration and on macrophages to which It helps by stimulating phagocytosis, and the production of oxidants of IL-1 and the inflammatory lipid prostaglandin E2 PGE2. In the present invention, "a functionally equivalent variant of TNF-?" Is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids from the TNF-? Sequence, and polypeptide that at least in part retains the ability to stimulate respiratory burst in neutrophils, determined for example by means of the assay described in Menegazi et al . (Blood, 1994, 84: 287-293) based on detecting the production of O2 produced by neutrophils grown on fibronectin coated surfaces in response to TNF-? Using cytochrome c reduction that is inhibited by superoxide dismutase (SOD)

The CD40 ligand (also called CD154) is a cytokine that binds to its related receptor protein found in antigen presenting cells. In macrophages, the activation of CD40 by the CD40 ligand leads to an increase in the expression of more CD40 and TNF receptors on its surface. In B cells, the binding of the CD40 ligand to the receptor leads to the activation of B cells and their transformation into plasma cells. In the present invention, "a functionally equivalent variant of the CD40 ligand" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids from the CD40 ligand sequence, and polypeptide that at least in part it retains the ability of the CD40 ligand to stimulate an increase in inositol 1,4,5-triphosphate levels in B cells or increase the activation of tyrosine kinases in B cells, both determined for example by means of the assay described in Uckum et al . (J. Biol. Chem., 1991, 266: 17478-17485).

IFN-?, IFN-? Are type I interferons that act by binding to common receptors (IFNAR) expressed in a wide range of cells through activation of the JAK-STAT signaling pathway. Type I interferons act by activating antiviral immunity but also inducing immunomodulatory effects on T cells and dendritic cells (CDs). In the present invention, "a functionally equivalent variant of INF-? Or INF-?" Is understood as any polypeptide whose sequence can be obtained by insertion, substitution or deletion of one or more amino acids from the INF sequence. -? or INF-?, and polypeptide that at least partly retains the ability of INF-? or INF-? in any of the assays known to those skilled in the art to detect IFN-like activity, for example , the assays described in table 1 in Runkel et al . (J. Biol. Chem. 1998, 273: 8003-8008).

IFN-? Is a type II interferon that acts by binding to the IFN-? Receptor through the activation of the JAK-STAT signaling pathway. IFN-γ is secreted primarily by NK cells, TH1 CD4 + T cells and CD8 + T cells inducing various immunomodulatory effects on both innate and acquired immunity. In the present invention, "a functionally equivalent variant of INF-γ" is understood as any polypeptide whose sequence can be obtained by insertion, substitution or elimination of one or more amino acids from the INF-γ sequence, and polypeptide that at least in part retains the ability of INF-γ to protect WISH cells from death by infection with vesicular stomatitis virus or encephalomyocarditis virus (ECMV) as described in Langer et al . (Proc. Natl. Acad. Sci. USA, 1994, 91: 5818-5822).

The variants of the immunostimulatory cytokines preferably have a sequence identity with said immunostimulatory cytokine of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% . The degree of identity between variants and immunostimulatory cytokines is determined using computer algorithms and methods that are widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLASTM Annual, Altschul, S., et al ., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al ., J Mol Biol, 215: 403-410 (1990)].

Those skilled in the art will understand that mutations in the nucleotide sequence that encodes the immunostimulatory cytokines that lead to substitutions amino acid preservatives in non-critical positions for protein functionality are evolutionarily neutral mutations that do not affect their overall structure or functionality.

The components of the invention can be provide as polypeptides or, alternatively, one or both of said components can be provided as a polynucleotide comprising a nucleotide sequence encoding said components.

Therefore, in one embodiment In particular, the composition or kit of the invention may comprise a polynucleotide comprising a nucleotide sequence that encodes an immunostimulatory cytokine and / or a polynucleotide that comprises a nucleotide sequence encoding an inhibitor of TGF-? 1.

In a more preferred embodiment, the polynucleotide encoding the immunostimulatory cytokine and / or the nucleotide sequence encoding the inhibitor of TGF-? 1 is (s) comprised in a vector.

In some cases, the cytokine Immunostimulatory comprises more than one polypeptide chain. By example, IL-12 is a heterodimer that is formed by two covalently linked glycosylated chains of 40 kD (p40) and 35 kD (p35). In this case, the polynucleotides that encode the different polypeptide chains can be provided in the same vector.

As the expert in the field will understand, in instead of the combined administration of a polynucleotide that comprises the nucleotide sequence encoding an agent TGFβ1 inhibitor and a polynucleotide comprising a nucleotide sequence that encodes the cytokine immunostimulatory for the treatment of cancer, another form of Carrying out the present invention consists in administering a vector comprising both polynucleotides, that is, a vector that comprises the nucleotide sequence encoding an agent TGFβ1 inhibitor and the nucleotide sequence it encodes an immunostimulatory cytokine. When the vector is expressed in the receiving organism, will thus produce the corresponding proteins that carry out the aforementioned therapeutic effect for the treatment of cancer. If the immunostimulatory cytokine It consists of more than one type of polypeptide chain, the polynucleotides encoding said different types of chains polypeptides may be located in the same vector as the polynucleotide encoding the TGF? 1 inhibitor. By therefore, in a particular embodiment, the polynucleotide encoding the TGF? 1 inhibitor is a polynucleotide and said polynucleotide and the polynucleotide that encodes the immunostimulatory cytokine are included in the same vector.

Suitable vectors to accommodate the polynucleotides that form the compositions and kits of the invention include a plasmid or a vector that, when introduced into the host cell, integrates or not into the genome of said cell. Said vector can be obtained by conventional methods known to the person skilled in the art and can be found in, for example, Sambrook et al ., 2001 (cited above).

However, in the scope of this invention, the vector of the invention is preferably a vector viral or non-viral suitable for use in gene therapy; by way of non-limiting illustration, said vectors may be vectors virals based on retroviruses, adenoviruses, alphaviruses, etc., or on the case of non-viral vectors, vectors can be complex DNA-liposome, DNA-polymer, DNA-polymer-liposome etc. [watch "Nonviral Vectors for Gene Therapy", edited by Huang, Hung and Wagner, Academic Press (1999)]. Such viral vectors and not viral comprises a nucleotide sequence that encodes a TGFβ1 inhibitor can be administered directly to the body of the human or animal being by methods conventional.

In a particular embodiment, the vector is a viral vector, which in another embodiment particular is an alphavirus, such as the forest virus of Semliki, Sindbis and Venezuelan equine encephalitis (VEE); a high capacity adenovirus; a replication adenovirus conditional or an adeno-associated virus. In one embodiment preferably particular, the alphavirus is a forest virus of Semliki

Such vectors may alternatively be used to transform, transfect or infect cells, preferably mammalian cells, including human cells, for example, ex vivo , and subsequently implant them in the human or animal body to obtain the desired therapeutic effect. For administration to the subject, said cells will be formulated in a suitable medium that does not adversely affect its viability. Likewise, as those skilled in the art will understand, said vector may have, among others, multiple cloning sites, sequences that regulate expression, suitable origins of replication for the host cell into which the vector is to be introduced, selection markers, etc.

The vector of the present invention may have expression regulatory sequences that precede the polynucleotide that are operably linked to said polynucleotide As used in this description, the expression "operably linked" means that the polynucleotide that encodes the TGF? 1 inhibitor and the polynucleotide that encodes the immunostimulatory cytokine are within the framework of correct reading for expression under the control of said regulatory sequences

The regulatory sequences useful for the present invention may be nuclear promoter sequences or, alternatively, enhancer sequences and / or other regulatory sequences that increase the expression of the heterologous nucleic acid sequence. The promoter can be constitutive or inducible. If constant expression of the heterologous nucleic acid sequence is desired, then a constitutive promoter is used. Examples of well known constitutive promoters include the immediate early cytomegalovirus (CMV) promoter, the Rous sarcoma virus promoter, and the like. Some other examples of constitutive promoters are well known in the art and can be used in carrying out the invention. If controlled expression of the heterologous nucleic acid sequence is desired, then an inducible promoter must be used. In an uninduced state, the inducible promoter must be "silent." It is understood that "silent" means that in the absence of an inductor, little or no expression of the heterologous nucleic acid sequence is detected; in the presence of an inducer, however, the expression of the heterologous nucleic acid sequence occurs. The level of expression can be controlled frequently by varying the concentration of the inductor. By controlling the expression, for example, by variation in the concentration of the inducer such that an inducible promoter is stimulated more strongly or weakly, the concentration of the transcribed product of the heterologous nucleic acid sequence can be affected. In the case of the heterologous nucleic acid sequence encoding a gene, the amount of synthesized protein can be controlled. It is thus possible to vary the concentration of the therapeutic product. Examples of well-known inducible promoters are: a promoter that responds to androgens or estrogens, a promoter that responds to doxycycline, a metallothionein promoter, or a promoter that responds to ecdysone. Other examples are well known in the art and can be used to carry out the invention. In addition to the constitutive and inducible promoters (which normally function in a wide variety of cell or tissue types), tissue specific promoters can be used to achieve the expression of the specific nucleic acid heterologous sequence in cells or tissues. Well-known examples of tissue-specific promoters include liver-specific promoters such as albumin, alpha-1-antitrypsin, or hemopexin promoters (Kramer et al ., (2003), Molecular Therapy 7: 375-385), various specific promoters of muscle including: the skeletal α-actin promoter, the cardiac actin promoter, the skeletal troponin C promoter, the slow-contracting cardiac troponin C promoter and the creatine kinase promoter / enhancer, or tumor-specific promoters such as the α-fetoprotein promoter for human hepatocellular carcinoma or the prostate specific antigen promoter for prostate cancer. There are several muscle-specific promoters that are well known in the art and that can be used to perform the invention (for a review of muscle-specific promoters see Miller et al ., (1993) Bioessays 15: 191-196).

In another particular embodiment, the composition or kit of the invention further comprises a compound Chemotherapeutic

In the present invention, "compound chemotherapeutic "is understood as those compounds or agents used in the treatment of cancer, such as agents alkylating agents, alkaloids, antimetabolites, antitumor antibodies, nitrosoureas, analogue antagonists / agonists, immunomodulators, Enzymes and others. Tables 1 and 2 of the patent application WO2004 / 093831 describe chemotherapeutic agents useful for Carry out the present invention.

According to the present invention, the administration combined with a TGF? 1 inhibitor and a cytokine immunostimulatory in the treatment of cancer or diseases tumor has a therapeutic effect greater than if said agent TGFβ1 inhibitor and the immunostimulatory cytokine were They administer alone. Therefore, in another aspect, the invention is refers to a composition or kit according to the present invention for its Use as medicine.

In another aspect, the invention relates to the use of a composition or kit according to the present invention for the Treatment and / or prevention of cancer and tumor diseases.

In another aspect, the invention relates to the use of a composition or kit according to the present invention to prepare a medical product for the treatment and / or prevention of cancer and tumor diseases

In the context of the invention, it is understood "antitumor treatment", "cancer treatment" or "cancer prevention" as the combined administration of a TGFβ1 inhibitor and an immunostimulatory cytokine to prevent or delay the onset of symptoms, complications or biochemical indications of cancer or tumor, mitigate its symptoms or stop or inhibit its development and progression, such as onset of metastasis, for example. The treatment can be a prophylactic treatment to delay the onset of the disease or to prevent the manifestation of your clinical symptoms or subclinical, or a therapeutic treatment to eliminate or mitigate the symptoms after the manifestation of the disease or in relation to its surgical or radiotherapeutic treatment.

The cancer to be treated with the composition or kit of the composition can be any type of cancer or tumor. These  tumors or cancers include, and are not limited to, cancers hematologic (leukemia or lymphomas, for example), tumors neurological (astrocytomas or glioblastomas, for example), melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors (stomach, pancreas or colon cancer, for example), liver cancer (for example, carcinoma hepatocellular), renal cell cancer, genitourinary tumors (ovarian cancer, vaginal cancer, cervical cancer, cancer bladder, testicular cancer, prostate cancer, for example) bone tumors and vascular tumors.

Thus, in an embodiment In particular, the cancerous disease to be treated or prevented using the composition or kit of the invention is a carcinoma Hepatocellular or a gastrointestinal tumor.

The composition of the invention or the principles assets of the kit of the invention can be administered by different methods, for example, intravenously, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, oral, intranasal or intrabronchial, and can be administered  locally or systemically or directly at the target sites. It can find a review of the different administration methods of active ingredients, of the excipients to be used and of the process to produce them in the Galician Pharmacy Treaty, C. Faulí i Trillo, Luzán 5, S.A. of Editions, 1993 and in Remington Pharmaceutical Science (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

In a particular embodiment, the TGFβ1 inhibitor or polynucleotide comprising a nucleotide sequence encoding an inhibitor agent of TGF? 1 is administered systemically which, in another form of particular embodiment, said systemic or localized route comprises intraperitoneal, intravenous, oral, intratumoral or intrahepatic or at any location around the tumor.

In another particular embodiment, the immunostimulatory cytokine or polynucleotide comprising a nucleotide sequence that encodes the cytokine Immunostimulatory is administered in a localized way.

In the present invention, a "form localized "is understood as the local administration of the polynucleotide comprising a nucleotide sequence that encodes the immunostimulatory cytokine to a specific place in the human or animal body

The dose schedule will be determined by the doctor and clinical factors As is well known in medicine, the doses depend on many factors including physical characteristics of the patient (age, size, sex), the method of administration used, the severity of the disease, the particular compound used and the pharmacokinetic properties of the individual.

When the components of the composition or kit of the invention, that is, the TGF? 1 inhibitor Immunostimulatory cytokine are included in a viral vector, the amount of vector in the composition or kit of the invention may vary between 10 5 and 10 13 viral particles per dose depending on the viral vector used. In addition, said vector is can be administered between 1 and 10 times, also depending on the viral vector used. On the other hand, if the vector is a vector non-viral, for example, a plasmid, the amount of vector in the composition or kit of the invention may vary between 100 ng and 5 mg by dose.

The composition or kit of the invention may have an amount of antitumor agents that may vary within a  wide range, but always in therapeutically amounts effective.

In the present invention, a "quantity therapeutically effective "is understood as the amount of agent TGFβ1 inhibitor and immunostimulatory cytokine which is enough to cause a delay in tumor growth or inhibition thereof, or to induce an increase in response Immune antitumor.

The composition or kit of the invention may have thus a quantity of antitumor agents that varies between 0.1 and 2000 mg, preferably in the range of 0.5 to 500 mg and, even more  preferably between 1 and 200 mg. The appropriate doses of Compositions can vary between 0.01 and 100 mg / kg of weight body, preferably between 0.1 and 50 mg / kg body weight, more preferably, between 0.5 and 20 mg / kg body weight. The composition can be administered a variable number of times at day, particularly from 1 to 4 doses a day.

The combinations of compounds according to the present invention, that is, TGFβ1 inhibitor and immunostimulatory cytokine, have proven useful in the anti-tumor treatment Therefore, in another aspect, the invention refers to a pharmaceutical preparation comprising the composition or kit of the invention and a pharmaceutically support  acceptable.

For use in medicine, combinations of Compounds of the invention may be in the form of prodrug, salt, solvate or clathrate, either in isolation or in combination with additional active ingredients Compound combinations according to the present invention can be formulated together with a excipient that is pharmaceutically acceptable. Preferred excipients for use in the present invention They include sugars, starches, cellulose, gums and proteins. The Pharmaceutical preparation of the invention can be formulated in a solid pharmaceutical dosage form (e.g., tablets, capsules, sugar-coated tablets, granules, suppositories, sterile amorphous or crystalline solids that can be rebuild to provide liquid forms, etc.), liquid (by example, solutions, suspensions, emulsions, elixirs, lotions, ointments etc.) or semi-solid (gels, ointments, creams and the like). Examples of pharmaceutically acceptable carriers are known. in the prior art and include buffered saline solutions with phosphate, water, emulsions such as oil in water emulsions, different types of wetting agents, sterile solutions, etc. Compositions comprising said supports can be formulated by conventional processes known in the previous state of The technique.

In the present invention, a "kit" is understood as a product that contains the different principles or active compounds of the invention (ie, inhibitor of TGFβ1 and the immunostimulatory cytokine) forming the packaged composition so that it can be transported, storage and its simultaneous or sequential administration. The kits of the invention can thus contain one or more suspensions, tablets, capsules, inhalers, syringes, patches and the like, which contain the active ingredients of the invention and that can be prepared in an individual dose or as multiple doses The kit may additionally contain a support suitable to resuspend the compositions of the invention such as an aqueous medium, such as saline, Ringer's solution, Ringer's lactate solution, dextrose and sodium chloride, media Water soluble, such as alcohol, polyethylene glycol, propylene glycol and water insoluble supports such as oil corn, cottonseed oil, peanut oil, oil sesame, ethyl oleate, isopropyl myristate and benzoate benzyl Other components that may be present in the kit is a package that allows to maintain the formulations of the invention within certain limits. The right materials for Prepare such containers include glass, plastic (polyethylene, polypropylene, polycarbonate and the like, bottles, vials, paper, Sachets and similar.

The kit of the invention may contain additionally instructions for simultaneous administration, sequential or separate from the different pharmaceutical formulations present in the kit. Therefore, in one embodiment In particular, the kit of the invention further comprises instructions for simultaneous, sequential or separate administration of Different components These instructions can be found at form of printed material or in the form of electronic support that you can store the instructions so that they can be read by a subject, such as electronic storage media (disks magnetic, tapes and the like), optical media (CD-ROM, DVD) and the like. The media can additionally or alternatively contain websites on the Internet providing such instructions.

As already mentioned at the beginning of the present description, the invention shows that the administration of a TGF? 1 inhibitor has the ability to increase the immune response and / or the antitumor effect induced by a immunostimulatory cytokine.

In this way, in another aspect, the invention is refers to the use of a TGF? 1 inhibitor or a polynucleotide encoding the TGF? 1 inhibitor agent for promote the anti-tumor effects of a cytokine immunostimulatory

In a preferred embodiment, the immunostimulatory cytokine is selected from the group of IL-12, IL-2, IL-15, IL-18, IL-24, GM-CSF, TNF-?, CD40 ligand, INF-?, IFN-?, INF- γ and functionally equivalent variants from the same.

In a particular embodiment, the TGFβ1 inhibitor is selected from the peptide group  TGF? 1 inhibitors, TGF? 1 inhibitor antibody, soluble TGF? 1 receptor and cell inhibitors TGF? 1 producers.

In another particular embodiment, the TGFβ1 inhibitor peptides are selected from the peptides comprising SEQ ID NO: 1 to SEQ ID NO: 6.

In another particular embodiment, the inhibitors of TGFβ1 producing cells are peptides FoxP3 inhibitors.

Where a polynucleotide comprising the nucleotide sequence that codes for inhibitors of  TGF? 1, said polynucleotide may be comprised in a vector.

In another particular embodiment, the vector is a viral vector that in another embodiment particular is an alphavirus, such as the forest virus of Semliki, Sindbis virus and Venezuelan equine encephalitis (VEE); a high capacity adenovirus; a replication adenovirus conditional or an adeno-associated virus. In one embodiment preferably particular, the alphavirus is a forest virus of Semliki

Finally, in another aspect the invention is refers to a polynucleotide comprising a sequence of nucleotides encoding a TGF? 1 inhibitor and a nucleotide sequence encoding a cytokine immunostimulatory In a preferred embodiment, the nucleotide sequence that encodes the cytokine immunostimulator encodes a cytokine selected from the group of IL-12, IL-2, IL-15, IL-18, IL-24, GM-CSF, TNF-?, CD40 ligand, INF-?, IFN-?, INF- γ and functionally variants equivalents thereof. Preferably, the cytokine Immunostimulatory is IL-12.

The following example illustrates the invention and not It should be considered as limiting its scope.

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Example

1. Materials and methods Animals and cell lines

Female C57BL / 6 mice 4 to 6 weeks old were purchased from Harlan (Barcelona, Spain) and were maintained in standard pathogen-free conditions according to the institution's guidelines. The MC38 mouse adenocarcinoma cell line (Tirapu, I. et al ., 2004, Int J Cancer 110: 51-60), hamster BHK-21 cells (Rodríguez-Madoz, JR, et al ., 2005, Mol Ther 12: 153-63), 293Cre4 humans (Parks, RJ et al ., 1996, Proc Natl Acad Sci USA 93: 13565-70) and 293Cre66 (Morral, N. et al ., 1998 Hum Gen Ther 9: 2709-16 ) were grown as previously described. All culture media were supplemented with 2 mM glutamine and antibiotics.

Plasmids and virus production

Plasmids pSFV-enhIL-12 and pSFV3-LacZ, which contain the genes encoding the subunits of mouse interleukin 12 and β-galactosidase, respectively, have been previously described (Rodríguez-Madoz, JR, et al . , 2005, Mol Ther 12: 153-63; Liljestrom, P. and Garoff, H. 1991, Biotechnology (NY) 9: 1356-61). In vitro RNA synthesis and transfection in BHK-21 cells by electroporation was performed as previously described (Liljestrom, P. and Garoff, H. 1991, Biotechnology (NY) 9: 1356-61). The packaging of the recombinant RNA into viral particles SFV (Semliki forest virus) was performed as described (Smerdou, C. and Liljestrom, P. 1999, Curr Opin Mol Ther 1: 244-51). Briefly, BHK-21 cells were co-electroporated with the recombinant SFV vector RNA (SFV-enhIL-12 or SFV-LacZ) and the RNAs of the helper-S2 and helper-C-S219A SFVs, which provide the capsid proteins and spicules in trans , respectively. After 24 hours the cell supernatants were collected and the SFV viral particles were purified by ultracentrifugation as previously described (Fleeton, MN, et al ., 1999, J Gen Virol 80: 1189-98). Indirect immunofluorescence of infected BHK-21 cells was performed to determine the titer of recombinant virus stores (Salminen, A., et al ., 1992, J Cell Biol 116: 349-57). A rabbit polyclonal antiserum specific for the nsp2 subunit of the SFV replicase was used as the primary antibody. SFV-LacZ reserves were titrated by infecting BHK-21 cells with serial dilutions of the virus and counting the β-galactosidase positive cells after staining with X-gal. Titers of approximately 5x10 9 pv / ml were usually obtained after purification.

The GL-Ad / RUmIL-12 vector (also called HC-Ad-mIL-12), was produced as previously described (Parks, RJ et al ., 1996, Proc Natl Acad Sci USA 93: 13565-70; Wang , L., et al ., 2004 Gastroenterology 126: 278-89). Briefly, 293Cre66 cells were transfected with a linearized plasmid containing the HC-Ad genome and subsequently infected with the AdLC8cLuc helper adenovirus containing the packaging signal flanked by loxP sites. Additional amplification steps were performed by coinfection of new 293Cre66 or 293Cre4 cells with a lysate of previous steps plus new helper adenoviruses. The purification of HC-Ad was done by double gradient in CsCl.

Induction and treatment of tumor nodules in experiments with  SFV

To generate intrahepatic tumor nodules, MC38 cells were collected, washed 3 times with HBSS and resuspended in the same buffer at a final concentration of 1.25x10 7 cells / mL. C57BL / 6 mice were inoculated with 40 µl of cells (5x105 cells) in the liver lobe left and after 5-7 days of implantation of the cells a single tumor nodule of about 4-5 mm in diameter. Tumors were treated by intratumoral injection with the indicated doses of SFV viral particles in a total volume of 60 µl of saline buffer The treatment evaluation was carried out 4 weeks after inoculation of the virus by laparotomy. Similarly, to generate subcutaneous tumor nodules, it collected MC38 cells, washed and resuspended to a final concentration 5x10 6 cells / mL. Mice were inoculated C57BL / 6 with 100 µl of cells (5x105 cells) on the flank right and after 7-9 days nodules were observed individual tumors of about 4-5 mm of diameter. The tumors were treated as described above. by intratumoral injection with the indicated doses of SFV viral particles. Tumor size was estimated as the mean of two diameters of each tumor nodule and the survival of The animals were checked daily.

Induction and treatment of tumor nodules in experiments with  HC-Ad

Hepatic cancer metastases were established of colon by intrahepatic inoculation of 5x105 cells MC38 (resuspended in a volume of 50 µl) in female mice C57BL / 6. Three days later, the vector was injected intrahepatically HC-Ad expressing murine IL-12 (GL-Ad / RUmIL-12) in the area that surrounded the tumors at a dose of 4x108 infectious units (ui). Six days after the viral injection, the IL-12 expression by injection intraperitoneal of Mifepristone (RU486). Inductions are performed daily for 11 days with individualized amounts and crescents of RU486. Dose adjustments were made of RU486 to counteract individual variations in the effectiveness of transduction in order to reach therapeutic concentrations of IL-12 (between 100 and 1000 ng / ml) and avoid toxicity. The induction protocol was performed as follows: a initial dose of 125 µg / kg to all animals in the group of treatment, and this dose was repeated the next day. Ten hours after the first administration of RU486, a quantification of IL-12 in animal serum. The dose of RU486 was adjusted to 250, 500 or 1000 µg / kg, based on the concentration of IL-12. This dose RU486 individualized remained for 3 days in a row, and then it was increased 2 times to compensate for the loss of inducibility in the system. This progressive increase was repeated in two additional rounds of 3 days. In a typical induction pattern, RU486 doses are as follows (in / mug / kg per day): 125; 125; 250; 250; 250; 500; 500; 500; 1000; 1000; 1000

Peptides and antibodies

Two different peptides that inhibit TGF-? Were used in this study. P17 peptide (KRIWFIPRSSWYERA) [SEQ ID NO: 2] (Dotor, J. et al ., 2007 Cytokine 39: 106-15) was selected from a phage display peptide library for its ability to bind TGF- [ 3. Peptide p144 (TSLDASIIWAMMQN) [SEQ ID NO: 1] (Ezquerro, IJ et al ., 2003 Cytokine 22: 12-20) was predicted from the sequence of the TGF-β1 type III receptor (amino acids 1-853) , which could join TGF-3l. The predictions were made using a program developed in the laboratory of Dr. Borras-Cuesta. Both peptides were synthesized by NeoMPS SA (Strasbourg, France) using the solid phase method (Merrifield, RB 1963 J Am Chem Soc 85: 2149-2154) using the Fmoc alternative (Atherton, E., et al ., 1989 J Chem Soc Perkin Trans 1: 538-546). The peptides were pure at least 95% according to HPLC analysis. Animals treated with p17 received a total of 10 doses of 75 µg of peptide (SFV protocol) or 4 doses of 50 µg of peptide (HC-Ad protocol) diluted in 200 µl of saline buffer. These doses were given intraperitoneally every 48 hours, the first being inoculated 2-3 hours after intratumoral injection of the vectors or saline solution (SFV) or 6 days after the start of the induction of IL-12 (HC-Ad). Animals treated with p144 received 10 doses of 50 µg of peptide given intratumorally at the same intervals as p17. A polyclonal chicken antibody against TGF-3 (R&D systems, ref. AB-101-NA) was also used to neutralize the bioactivity of TGF-?. The neutralizing anti-TGF- [3 antibody was injected intraperitoneally every 3 days at a dose of 200 µg in a volume of 200 µl of saline buffer until a total of 4 doses per animal was completed. Control groups that did not receive inhibitory treatment of TGF-? Were inoculated intraperitoneally with the same volume of saline buffer.

II. Results Intraperitoneal administration of the peptide inhibitor of TGF-? P17 increases the antitumor effect of a SFV vector expressing IL-12

Previous studies had shown that an SFV vector expressing IL-12 at high levels (SFV-enhIL-12) was capable of inducing a potent antitumor effect in the MC38 1 murine model of colon adenocarcinoma (Rodríguez-Madoz, JR, et al ., 2005, Mol Ther 12: 153-63). In these studies, intratumoral injection of a single dose of 10 8 pv of SFV-enhIL-12 induced the complete regression of 92% of tumor nodules established subcutaneously in syngeneic C57BL / 6 mice. However, when similar MC38 nodules were established in the liver and treated by intratumoral injection of 10 8 pv of SFV-enhIL-12 the antitumor efficacy was reduced to 50% of complete tumor regressions (previous results and Figure 1 ). This minor antitumor response could be due to the presence of a richer immunosuppressive medium in the liver. TGF-? Has been proposed as one of the most potent immunosuppressive cytokines, being able, among other things, to inhibit the maturation and presentation of antigens by dendritic cells as well as the activation of T cells. TGF-? it can be produced in the tumor medium both by regulatory T cells and also by tumor cells (Wahl, SM, et al ., 2006 Immunol Rev 213: 213-27; Gorelik, L. and Flavell, RA 2002 Nat Rev Immunol 2 : 46-53). To assess whether the anti-tumor response induced by SFV-enhIL-12 against liver tumors could be increased by inhibition of TGF-t3, mice that had MC38 tumors were treated with a single intratumoral injection of 10 8 pv of SFV- enhIL-12 and 10 intraperitoneal doses of 75 µg of the TGF-? p17 inhibitor peptide (Dotor, J., et al ., 2007 Cytokine 39: 106-15) given every 48 hours. Control groups received only the intratumoral injection of SFV-enhIL-12 or only the 10 intraperitoneal doses of 75 µg of the p17 peptide. Two additional groups of control animals received a single inoculation of 10 8 pv of SFV-LacZ alone or in combination with 10 75 µg intraperitoneal doses of the p17 peptide, and a third control group received no treatment. The efficacy of the different treatments was evaluated by measuring the size of the tumor 20 days after the inoculation of the vector (Figure 1). While SFV-enhIL-12 by itself was capable of inducing complete tumor regressions in 50% of tumors (n = 32), the combination of SFV-enhIL-12 and p17 greatly increased this effect, inducing complete regressions of tumor in 92% of tumors (n = 26). The additional effect provided by p17 took place only in the presence of IL-12 expression since tumor growth was very similar in all control groups and no complete tumor regressions were observed in animals receiving p17 alone or in combination are SFV. -LacZ. In addition, all the animals in which the tumors were rejected survived for the entire time of the experiment (more than 200 days) (Figure 2).

Intraperitoneal administration of the peptide inhibitor of TGF-? P17 mediates the induction of an immunity strongest systemic memory antitumor in treated animals with SFV-enhIL-12

To determine whether treatment with the vector SFV-enhIL-12, alone or in combination with the TGF-? p17 inhibitor, it had generated a long-lasting antitumor memory, animal groups who had removed the primary MC38 tumor were exposed again to the same tumor cells in the liver or subcutaneously 3 months after tumor rejection. After the restatement all Naive control animals developed MC38 nodules in the liver or subcutaneously, depending on the route of inoculation (Figure 3). On the contrary, 87.5% (n = 8) of the mice that had rejected the MC38 tumors and were exposed again in the liver were resistant against the development of tumors, regardless of the type of treatment they had received (Figure 3, left panel). However, when the restatement of MC38 cells were carried out subcutaneously mice that were had healed after treatment with the combination of SFV-enhIL-12 and p17 showed a degree of protection much higher (71%, n = 14) than those animals that had healed after treatment with SFV-enhIL-12 alone (21.4%, n = 14) (Figure 3, right panel). These results indicate that the combination of SFV-enhIL-12 and the TGF-? p17 inhibitor not only induces more potent antitumor responses; it is also capable of generate a stronger and more immune memory response generalized

Intraperitoneal administration of the peptide inhibitor of TGF-? P17 increases the antitumor effect of a high capacity adenovirus vector (HC-Ad) that express IL-12

To determine whether the peptide inhibitor of TGF-? P17 could also increase antitumor responses induced by an adenovirus vector that Expressing IL-12, the following experiment was performed: C57BL / 6 mice were injected into the liver with 5x105 cells MC38 and three days later the animals were inoculated intrahepatically with 4x10 8 infectious units (ui) of a HC-Ad vector expressing IL-12 murine (GL-Ad / RumIL-12). Six after injection, the expression of IL-12 by intraperitoneal injection of mifepristone (RU486). Inductions were performed daily for 11 days with individualized and increasing amounts of RU486 as described in the methods. The dose adjustment of RU486 was performed to counteract individual variations in transduction efficiency in order to reach concentrations Therapeutics of IL-12 and avoid toxicity (see methods). Six days after the start of induction of IL-12, the animals received 4 doses 50 µg intraperitoneal peptide inhibitor TGF-? P17 given every 48 hours. Animals controls received only the vector GL-Ad / RumIL-12 or saline solution followed by the same induction protocol of RU486. The size of liver tumors were determined 55 days after the inoculation of cancer cells, or at the time of death of animal. As shown in Figure 4, the vector GL-Ad / RumIL-12 by itself was capable of inducing 40% of complete tumor regressions (n = 10) but its effect increased to 60% when the animals received the vector in combination with p17. In addition, the combined treatment achieved greater protection from death related to the disease that GL-Ad / RumIL-12 alone, at least during the first 2 months of the experiments (Figure 5). From interesting way, the reduction in survival in points of later times was not always due to the progression of liver tumors Lung metastasis and other complications explain why the final survival rate is lower than the proportion of animals free of liver tumors on day 55. In together, these results suggest that the inhibition of TGF-? With p17 could be a new method general to enhance the vector-mediated antitumor effect of IL-12 expression.

The TGF-? P144 inhibitor peptide or a TGF-? blocking antibody also increase the antitumor effect of SFV-enhIL-12

The effect that p17 peptide has shown increasing antitumor responses caused by SFV-enhIL-12 or GL-Ad / RumIL-12 strongly indicates that inhibition of TGF-? in combination with the expression of IL-12 could represent a new Powerful strategy to increase antitumor responses. For confirm that this effect is not specific for peptide p17 is evaluated the effect of two other inhibitors of TGF-? On the antitumor effect provided by SFV-enhIL-12 in the MC38 model: a TGF-? inhibitor peptide different (p144) and a blocking antibody of TGF-?. In the first case mice were treated who had subcutaneous MC38 tumors with an intratumoral injection single 10 7 pv of SFV-enhIL-12 and 10 doses 50 µg intratumoral peptide inhibitor TGF-? P144 given every 48 hours. In this case the peptide was inoculated intratumorally due to nature highly hydrophobic of p144. Since it was not possible administer 10 intratumoral doses of the peptide in tumors of Liver subcutaneous model of MC38 was chosen for this study. The dose of SFV-enhIL-12 was reduced to 10 7 pv because in previous studies this dose had resulted in approximately 60% of total regressions in MC38 tumors subcutaneous, leaving a wide margin for therapeutic improvement. The control groups received well only one injection intratumoral 10 7 pv of SFV-enhIL-12 well only 10 doses 50 µg intratumoral peptide p144. Although the effect p144 enhancer was lower than that observed with p17 the combination of SFV-enhIL-12 and p144 was able to increase the antitumor effect from 60% of total tumor regressions (SFV-enhIL-12 only) up to 80% of Complete regressions with combined therapy (Figure 6). He peptide by itself showed no antitumor effect.

In the second model mice were treated that had MC38 tumors in the liver with an intratumoral injection individual 10 8 pv of SFV-enhIL-12 and 4 doses intraperitoneal 200 µg of a blocking antibody of TGF-? Given every 72 hours. The groups controls received well only an intratumoral injection of 10 8 pv of SFV-enhIL-12 well only 4 intraperitoneal doses of 2000 µg of the antibody TGF-? blocker. Combining SFV-enhIL-12 and the antibody TGF-? blocker induced complete regression of tumor in all treated animals (n = 8) while the vector itself induced 67% regression of tumors (n = 7) (Figure 7). In this case the antibody itself induced a complete regression (16%, n = 6), with no apparent effect on the growth of the rest of the tumors.

These results indicate that the use of different strategies to inhibit TGF-? both systemically and locally it can be very efficient for increase the antitumor effect induced by IL-12

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<400> 3

         \ hskip1cm
      

10

         \ vskip0.400000 \ baselineskip
      

<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 12

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of amino acids that comprises amino acids 1 to 12 of the p17 peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

         \ hskip1cm
      

eleven

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 13

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of amino acids that comprises amino acids 1 to 13 of the p17 peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

         \ hskip1cm
      

12

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 14

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence of amino acids that comprises amino acids 1 to 14 of the p17 peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

         \ hskip1cm
      

13

Claims (32)

1. A composition or kit comprising

(i)

a TGFβ1 inhibitor or a polynucleotide comprising a nucleotide sequence encoding an inhibitor agent of TGF? 1, and

(ii)

to the minus an immunostimulatory cytokine or a polynucleotide that it comprises a nucleotide sequence that encodes a cytokine immunostimulatory

2. Composition or kit according to claim 1, wherein the TGF? 1 inhibitor is selected from the group of TGFβ1 inhibitor peptides, antibody inhibitor of TGFβ1, soluble TGFβ1 receptor and inhibitors of TGFβ1 producing cells. 3. Composition or kit according to claim 2, wherein the TGFβ1 inhibitor peptides are selected from Peptides comprising SEQ ID NO: 1 to SEQ ID NO: 6. 4. Composition or kit according to claim 2, wherein the inhibitors of TGFβ1 producing cells they are FoxP3 inhibitor peptides. 5. A composition or kit according to claims 1 to 4 wherein the immunostimulatory cytokine is select from the group of IL-12, IL-2, IL-15, IL-18, IL-24, GM-CSF, TNF-?, CD40 ligand, INF-?, IFN-?, INF- γ and functionally variants equivalents thereof. 6. A composition or kit according to claim 5 where the immunostimulatory cytokine is IL-12 or a functionally equivalent variant thereof. 7. Composition or kit according to any of the claims 1 to 6 wherein the composition or kit comprises a polynucleotide comprising a nucleotide sequence that encodes an immunostimulatory cytokine and / or the composition or kit comprises a polynucleotide comprising a sequence of nucleotides encoding a TGF? 1 inhibitor and wherein the polynucleotide encoding an immunostimulatory cytokine and / or the nucleotide sequence encoding the TGF? 1 inhibitor It is included in a vector. 8. Composition or kit according to claim 7 wherein the polynucleotide comprising a nucleotide sequence encoding an immunostimulatory cytokine and the composition or kit comprising a polynucleotide comprising a nucleotide sequence encoding a TGFa1 inhibitor are comprised therein.
vector. 9. Composition or kit according to the claims 7 or 8, where the vector is a viral vector. 10. Composition or kit according to claim 9, where the viral vector is an alphavirus, a high adenovirus ability, a conditional replication adenovirus or a virus adenoassociated. 11. Composition or kit according to claim 10, where the alphavirus is a virus from the Semliki forest. 12. Composition or kit according to any of the claims 1 to 11, further comprising a compound Chemotherapeutic 13. A composition or kit according to any of claims 1 to 12 for use as a medicament. 14. A composition or kit according to any of claims 1 to 12 for treating and / or preventing cancer and Tumor diseases 15. Composition or kit according to claim 14, where the cancerous disease is a hepatocellular carcinoma or a gastrointestinal tumor. 16. Composition or kit according to the claims 14 or 15, wherein the TGF? 1 inhibitor or the polynucleotide comprising a nucleotide sequence that encodes a TGF? 1 inhibitor agent is administered through of a systemic or localized route. 17. Composition or kit according to claim 16, where the systemic or localized pathway comprises pathway intraperitoneal, intravenous, oral, intratumoral or intrahepatic or at any location around the tumor. 18. Composition or kit according to any of the claims 14 to 17, wherein the immunostimulatory cytokine or the polynucleotide comprising the nucleotide sequence that encodes the immunostimulatory cytokine is administered located. 19. A pharmaceutical preparation comprising a composition according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier. 20. In vitro use of a TGF? 1 inhibitor or a polynucleotide encoding a TGF? 1 inhibitor to stimulate the anti-tumor effects of an immunostimulatory cytokine. 21. In vitro use according to claim 20 wherein the immunostimulatory cytokine is selected from the group of IL-12, IL-2, IL-15, IL-18, IL-24, GM-CSF, TNF-?, CD40 ligand , INF-?, IFN-?, INF-? And functionally equivalent variants thereof. 22. In vitro use according to claim 21 wherein the immunostimulatory cytokine is IL-12. 23. In vitro use according to any of claims 20 to 22 wherein the TGFβ1 inhibitor is selected from the group of TGFβ1 inhibitor peptides, TGFβ1 inhibitor antibody, soluble TGFβ1 receptor and inhibitors of TGFβ1 producing cells. 24. In vitro use according to claim 23, wherein the TGFβ1 inhibitor peptides are selected from the peptides comprising SEQ ID NO: 1 to SEQ ID NO: 6. 25. In vitro use according to claim 23, wherein the inhibitors of TGFβ1 producing cells are FoxP3 inhibitor peptides. 26. In vitro use according to any of claims 20 to 25, wherein a polynucleotide encoding the TGFβ1 inhibitor is used and said polynucleotide is comprised in a vector. 27. In vitro use according to claim 26, wherein the vector is a viral vector. 28. In vitro use according to claim 27, wherein the viral vector is an alphavirus, a high capacity adenovirus, a conditional replication adenovirus or an adeno-associated virus. 29. In vitro use according to claim 28, wherein the alphavirus is a virus from the Semliki forest. 30. A polynucleotide comprising the sequence nucleotide encoding a TGF? 1 inhibitor and the nucleotide sequence encoding a cytokine immunostimulatory 31. A polynucleotide according to claim 30, wherein the TGF? 1 inhibitor is selected from the TGFβ1 inhibitor peptide group, antibody inhibitor of TGFβ1, soluble TGFβ1 receptor and inhibitors of TGFβ1 producing cells. 32. A polynucleotide according to the claims 30 or 31 wherein the immunostimulatory cytokine is selected from group of IL-12, IL-2, IL-15, IL-18, IL-24, GM-CSF, TNF-?, CD40 ligand, INF-?, IFN-?, INF- γ and functionally variants equivalents thereof.