MX2008005254A - Use of tgf-$g(b)1 inhibitor peptides in the preparation of an immune response modulating agent - Google Patents

Abstract

The invention relates to a TGF-β1 inhibitor peptide which is selected from among:peptide p144 having a sequence that corresponds to SEQ ID NO:1, peptide p17 having a sequence that corresponds to SEQ ID NO:2, a peptide that has at least 90%homology with said peptides, or fragments of same. The invention relates to the use of said peptide in the preparation of an immune response modulating agent.

Description

USE OF TGF-β1 INHIBITOR PEPTIDES IN THE PREPARATION OF AN IMMUNE RESPONSE MODULATING AGENT TECHNICAL FIELD OF THE INVENTION The present invention is encompassed in the field of the preparation of modulators of the immune response.

STATE OF THE ART TGF-β1 (transforming growth factor β-1) is a potent immunomodulator that is present in all phases of the immune response generating different effects. It is currently recognized as a potent regulator of cells of the mitapoita system, including lymphocytes, macrophages and dendritic cells (Letterio J.J., 1998). The biological activity of TGF-β1 varies greatly depending on the type and state of cellular differentiation, as well as the presence of other cytokines, suggesting that an alteration in the balance of this set of cytokines can also affect TGF-β1 and contribute to the development of pathologies associated with dysfunction of the immune system TGF-β1 regulates the immune response in a complex and context-dependent manner, which has been demonstrated by using experimental models of different diseases, as well as by the evaluation of genetically modified mice in Regarding the expression of TGF-ßl, its receptors, or regulatory proteins TGF-ßl regulates the function and interaction of cells of the immune system in the development of humoral, cytotoxic, immunotolerance responses and the pathological origin of many infectious diseases. autoimmune T lymphocytes are clearly regulated by TGF-β1 at all stages of their development (Fontana A. et al., 1992). The effect of TGF-β1 varies depending on the state of differentiation of the lymphocyte, and the type of activation signal received by it. The first studies on the effect of TGF-β1 on human lymphocytes revealed the ability of these cells to produce and secrete TGF-β1 as an inhibitor of IL-2-dependent proliferation and cytolytic function (Pardoux C. et al., 1997) .

Dendritic cells are a leukocyte population clearly differentiated by their function as antigen presenting cells in the activation of T lymphocyte responses. They are a highly specialized cell population, which includes the Langerhans cells of the epidermis and the follicular dendritic cells of the nodules. lymphatics, and in which TGF-ßl regulates both its differentiation and its activity (Strobl H. Knapp., 1999). It has been identified that TGF-ßl enhances the functional differentiation in vi tro of dendritic cells, from CD34 + precursors, induced by the presence of other cytokines (TNF-a, SCF, and GM-CSF). TGF-ßl also acts by increasing the viability of dendritic cells in culture. On the other hand, the role of TGF-β1 in this cell type also seems to be related to a regulatory mechanism that inhibits ba specific responses to avoid autoimmune processes In the differentiation, proliferation and production of Ig (immunoglobulins) of B cells, TGF-ßl has a regulatory role by inhibiting the levels of certain surface molecules, including the major histocompatibility complex type II (MCH-II). In pre-B lymphocytes as in mature B cells On the other hand, TGF-ßl inhibits Ig secretion in general but clearly induces IgA production and therefore plays an important role in the immune response associated with mucous membranes. Studies on the effect of TGF-β1 as an inhibitor of the production of all types of Ig have been carried out. However, the need for certain levels of TGF-β1, which act at the autogene level, has also been described in lymphocyte cultures. For the effective production of IgG and IgE Thus, the function of TGF-β1 in the induction of antibodies is shown, as in many other processes, dual and opposite depending on the context of the immune response (Le. Bman D A, Edmiston J S, 1999) In the case of macrophages, the effect of TGF-ßl at the tissue level is generally suppressive and contributes to the end of the inflammatory response., the most relevant effect of TGF-β1 on the inactivation of macrophages, is due to its ability to limit the production of reactive oxygen species and metabolic intermediates of nitrogen by cells activated by IFN-α. or LPS. The enzyme responsible for the production of NO (nitric oxide) by activated macrophages is the mducible form of the enzyme nitric oxide synthase (iNOS). The regulation of the activity of this enzyme by different cytokines, including TGF-β1, makes it possible to regulate the immune response in general and, in particular, the response of macrophages to microorganisms and tumor cells. TGF-β1 inhibits the iNOS enzyme both at the trans-occlusional level, decreasing mRNA levels, and suppressing the activity of the protein. TGF-β1 also inhibits the production of intermediate reactive oxygen species and oxidative cytotoxicity, by inactivating macrophages and controlling peripheral blood monocytes (Ashcroft G.S., 1999). Additionally, the activation or production of TGF-β1, as well as the alteration of its signaling pathway, is described in many diseases caused by the infection of different microorganisms, including Lei shmania, Trypanosus a cruzi, the human immunodeficiency virus, the hepatitis C virus ...

The documents of the state of the art closest to the present invention are the Patent ES 2 146 552 and the patent application ES200302020 The first document refers to the use of antagonistic peptides of the binding of TGF-β1 to its receptors in the organism, characterized by presenting partial amino acid sequences similar or identical to those of TGF-β1 itself and / or its receptors; as well as its use to make an application composition in liver diseases, specifically for liver fibrosis. In this document, peptide p44 (SEQ ID NO: 1) is protected as well as its previously indicated use, although its use in the preparation of an immune response modulating agent that constitutes the object of the present invention is not mentioned. Patent application ES200302020 protects peptides inhibitors of the biological activity of TGF-β1 which have been obtained from a phage library, and its use for the treatment of diseases that involve a deregulated expression of TGF-β1 , especially the fibrotic alterations. In this document, peptide p7 (SEQ ID NO: 2) and the described use are protected, but again it does not refer to its use in the preparation of an immunomodulatory agent. This modulating effect of the immune system is very important since it allows to stimulate or inhibit different aspects of the immune response depending on the needs, and may even possess applications as a vaccination adjuvant.

Another relevant document is the patent application WO 2005 / 059133A2, which refers to a pharmaceutical composition comprising at least one stimulator of the function of immune cells and at least one substance that inhibits cell proliferation and / or induces cell death . As a stimulator of the function of the immune system, a TGF-β1 antagonist selected from: oligonucleotides that hybridize with the mRNA or with the DNA encoding TGF-β1, TGF-β1 inhibitory proteins, and peptides having a lower molecular weight are employed at 100 kDa inhibitors of TGF-β1. Additionally, this document refers to the use of said pharmaceutical composition in the treatment of neoplasms. However, in this document no peptide is used as inhibitor of TGF-β1, but an oligonucleotide, although it can be deduced that a peptide of these characteristics would, in principle, have a similar effect. This can not be affirmed without considerable experimentation.

The following is a list of the bibliography cited in the present application: Ashcroft GS. (1999). Bidirectional regulation of macrophage function by TGF-beta. Microbes Infect. Dec; 1 (15): 1275-82.

Fontana A, Constam DB, Frei K, Malipiero U, Pfister H. Modulation of the immune response by transforming growth factor beta. (1992) Int Arch Allergy Immunol.; 99 (1): 1-7.

Lai, M.Z., Ross, D.T., Guillet, J.G., Briner, T.J., Gefter, M.L., Smith, J.A. (1987). T lymphocyte response to bactepophage lambda repressor cl protein. Recognition of the same peptide presented by the molecules of different haplotypes. J Immunol 139, 3973-80.

Letterio, J.J., Roberts, A.B. (1998). Regulation of immune responses by TGF-beta. Annu Rev Immunol 16, 137-61.

Lebman DA, Edmiston JS (1999). The role of TGF-beta growth, differentiation, and maturation of B lymphocytes. Microbes Infect. Dec; 1 (15): 1297-304.

Pardoux, C, Ma, X., Gobert, S., Pellegpni, S., Mayeux, P., Gay, F., Tpnchieri, G., Chouaib, S. (1999).

Downregulation of mterleukm-12 (IL-12) responsiveness m human T cells by transformmg growth factor-beta: relationship with IL-12 signalmg. Blood 93, 1448-55.

Schini, V.B., Durante, W., Elizondo, E., Scott-Burden, T., Junquero, D.C., Schafer, A.I., Vanhoutte, P.M. (1992). The mduction of nitpc oxide synthase activity is mhibited by TGF-beta 1, PDGFAB and PDGFBB m vascular smooth muscle cells. Eur J Pharmacol 216, 379-83.

Strobl H, Knapp W. (1999). TGF-betal regulation of dendritic cells. Microbes Infect. Dec; 1 (15): 1283 -90.

Teicher B A. (2001) Malignant cells, directors of the malignant process. Role of transformmg growth factor-beta Cancer and Metastasis Reviews 20, 133-143 DESCRIPTION OF THE INVENTION To facilitate the understanding of the present text it is indicated that the term "peptide pl44" refers to a peptide inhibitor of TGF-ßl characterized by its sequence of amino acids corresponds to that defined in SEQ ID NO: 1. Also the term "peptide pl7" refers to a peptide inhibitor of TGF-β1 activity, characterized in that its amino acid sequence corresponds to that defined in SEQ ID NO: 2 The "incomplete Freund's adjuvant" refers to a composition well known to a person skilled in the art, characterized in that it is composed of an oil-in-water emulsion, which acts as an adjuvant by delaying the release of antigen. The present invention relates to an immune response modulating agent characterized in that it comprises a peptide inhibitor of TGF-β1 selected from: peptide pl44 whose sequence corresponds to SEQ ID NO: 1, peptide pl7 whose sequence corresponds to SEQ ID NO: 2, a peptide having at least 90% homology to them, or fragments thereof. In a specific embodiment of the invention, said fragment of a TGF-β1 inhibitor peptide is selected from: the fragment pl7 (1-11) defined in the sequence SEQ ID NO: 3, the fragment pl7 (1-11) am corresponds to SEQ ID NO 4, and the Acpl7 (1-11) am fragment defined by the sequence SEQ ID NO: 5 On the other hand, the invention also relates to the use of said modulating agent in the regulation of humoral, cellular immune responses or both. In a preferred embodiment, the invention relates to the use of the modulating agent as a vaccination adjuvant. In a specific embodiment of the invention, the immune response modulating agent is characterized in that it also comprises incomplete Freund's adjuvant.

In a preferred embodiment, the invention relates to the use of said modulating agent in the preparation of a pharmaceutical composition for the treatment of pathologies selected from: pathologies related to microorganisms that induce an immunosuppression mediated by TGF-β1 and cancer. Preferably, said microorganisms are selected from: Lei sh ania, Trypanosoma cruzii, the human immunodeficiency virus, the hepatitis C virus, the influenza virus, and the herpes simplex virus. Also, in a particular embodiment of the invention, the composition described above is for the treatment of a cancer selected from: breast cancer, prostate cancer, colon carcinoma, pancreatic cancer, skin cancer, hepatocarcoma, multiple myeloma and cancer of stomach. The present invention relates to the use of a TGF-β1 inhibitor peptide selected from: the peptide pl44 whose sequence corresponds to SEQ ID NO: 1, the peptide pl7 whose sequence corresponds to SEQ ID NO-2, a peptide having at least 90% homology with the same, or fragments of the above, in the preparation of an immune response modulating agent. On the other hand, the invention relates to a method of using a TGF-β inhibitor peptide selected from: peptide pl44 whose sequence corresponds to SEQ ID NO: 1, peptide pl7 whose sequence corresponds to SEQ ID NO: 2, a peptide having at least 90% homology thereto, or fragments thereof, to prepare a agent modulator of the immune response.

Additionally, the invention relates to the use of a fragment of a TGF-β1 inhibitor peptide obtained from the peptide pl7 selected from: the fragment pl7 (1-11) defined in the sequence SEQ ID NO: 3, the fragment pl7 ( 1-11) a corresponding to SEQ ID NO: 4, and the fragment Acpl7 (1-11) am defined by the sequence SEQ ID NO: 5. To facilitate the understanding of the text, it is indicated that pl7 (l-ll) am corresponds to a fragment corresponding to amino acids 1 to 11 of peptide pl7, wherein the amino acid in position 11 (tryptophan) is amidated; Acpl7 (1-11) am corresponds to the above fragment which also possesses the amino acid in position 1 (lysine) acetylated The present invention also relates to peptides having at least 70% homology to said peptides, and preferably having at least 80% homology with them, as long as they maintain the ability to inhibit the biological activity of TGF-β1. As well as any fragment of the previous ones that maintains the ability to inhibit the biological activity of TGF-β1.

In a preferred embodiment, the present invention relates to the use of a TGF-β inhibitor peptide described above, characterized in that the aforementioned modulating agent regulates humoral, cellular immune responses, or both. In a specific embodiment of the present invention said modulating agent possesses a stimulating effect of the immune response, preferably as a vaccination adjuvant.

On the other hand, a preferred embodiment of the present invention is characterized in that said modulating agent possesses an inhibitory effect of the immune response.

Additionally, the invention relates to the use of a DNA sequence coding for a TGF-β inhibitor peptide selected from: peptide p144 whose sequence corresponds to SEQ ID NO: 1, peptide pl7 whose sequence corresponds to SEQ ID NO: 2, a peptide having at least 90% homology to them, or fragments thereof, to make an immune response modulating agent. Also, the invention also relates to the use of a recombinant expression system encoding peptide p144, peptide p17, a peptide having at least 90% homology to them, or fragments thereof, to manufacture a peptide. immune response modulating agent In a preferred embodiment of the invention, said immune response modulating agent possesses an effect selected from, stimulator and inhibitor of the immune response On the other hand, the invention relates to the use of a TGF-β1 inhibitory peptide whose sequence corresponds to SEQ ID NO. 1, a peptide having at least 90% homology to it, or fragments of one of the above in the preparation of a composition for the treatment of selected pathologies between pathologies related to microorganisms that induce an immunosuppression mediated by TGF- ßl and cancer In a particular embodiment of the present invention, said microorganisms are selected from: Lei shmania, Trypanosoma cruzn, the human immunodeficiency virus, the hepatitis C virus, the influenza virus, and the herpes simplex virus .

A specific embodiment of the present invention is characterized in that said composition would have an effect on the induction of immune responses against established tumors, inhibiting the immunosuppressive effect associated with the production and / or activation of TGF-β1 in various types of tumors (Teicher BA, 2001) breast cancer, prostate cancer, colon carcinoma, pancreatic cancer, cancer p, hepatocarcoma, multiple myeloma and stomach cancer The modulator agent object of the present invention, can be used in all kinds of mammals, including rodents and primates. And in a preferred embodiment in humans.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Inhibition of the expression of different markers of dendritic cell maturation after incubation with pl44 (full bars) or with neutralizing antibodies of TGF-β1 (empty bars). The expression of markers was measured by flow cytometry. Figure 2. Effect of the administration of pl44 and / or RAd-IL12 to BALB / c mice on serum levels of IFN-? at days 0, 3 and 6. 108 pfu of mouse RAd-IL12 (empty bars) or of RAdIL-12 and pl44 (filled bars) was administered mtraperitoneally. Figure 3. Levels of NO (μM) in serum at days 0 and 6 after administration of 108 pfu of mouse RAd IL-12, by tracheitoneal route together with p44 (full bars) or without p14 (empty bars). Figure 4. Humoral response to RAd LacZ induced at day 15 in BALB / c mice after a subcutaneous immunization (in IFA) with RAd LacZmact in the presence or absence of pl44 Figure 5. Humoral response to RAd LacZ induced in BALB mice. c after a second subcutaneous immunization (in IFA) with RAd LacZmact, in the presence or absence of p44.

Figure 6. Humoral response induced in the mice of Figure 5 on day 7 after intravenous infection with 4 x 108 pfu of RAd LacZact. The control group corresponds to mice administered once intravenously with 4 x 108 pfu of RAd LacZact. Figure 7. X-gal stains of histological sections of liver samples of the mice of Figure 6, 7 days after the administration of 4 x 108 pfu of RAd LacZact intravenously. Figure 8. Effect of the inclusion of pl44 in immunization mixtures with FIS on the levels of IL-2 (A) and IFN-? (B) in supernatants of lmfocyte cultures, derived from nodules obtained from mice immunized only with FIS or with FIS + pl44. Cytokine production was measured in vitro after restimulation of the cultures with 6 μM (empty bars) or 30 μM (full bars) of FIS or pl44. Figure 9. Survival of BALB / c mice that were administered 5 x 10 5 CT26 cells intravenously and that received different treatments (i) - (v). With the exception of the control group (i) that received only 5 x 10 5 CT26 cells on day 10, the remaining three groups were also immunized with 50 μg of AHI in IFA subcutaneously on day 0. Groups (m) and (ív) they also received 50 μg of p44 by the intraperitoneal route on alternate days between days 4-20 and 10-20, respectively. Figure 10. The addition of the TGF-β1 blocking peptide pl7 to the culture medium of mouse NK cells inhibits their proliferation in response to high concentrations of IL-2. In all cases, "Natural Killer" lymphocyte cultures established in exactly the same way with and without p7 peptide are compared. A) Cell count at the indicated times from a culture of mouse total splenocytes RAGl ~ _. The presented value corresponds to the average of the counts of 2 wells of 3.5 cm diameter in each case in absolute number. B) Counting at the indicated times of a culture of magnetically purified DX5 + cells from mouse splenocytes RAGl "_, which lacks T and B lymphocytes, represented as mean of the 2-well counts of 0.4 cm in diameter at Each case C) Proliferation of microculture cells similar to that represented in B and at those same times, measured as tritiated thymidine incorporation in a 6-hour test Figure 11. The p7 peptide reduces the membrane expression levels of the markers of CD25 and CD69 activation in mouse NK cells.The histograms show the expression levels by flow cytometry of these markers in cells cultured with and without peptide and activated with IL-2. mean fluorescence (IFM) of each marker Figure 12. The peptide pl7 increases the cytotoxicity of mouse NK cells, activated with IL-2 against various tumor lines. s the percentages of lysis of cell lines with different sensitivity to NK cytotoxicity are shown. The effector cells were maintained with and without peptide for 6 days in culture and during the time of the chromium release assay on the target cells indicated at the corresponding proportions between effector cells and target cells Figure 13. The graphs show the measured cell proliferation, as incorporation of tritiated thymidine, depending on the amount of dendritic cells present per well, in the absence or presence of different previous stimuli and in the presence and absence of peptide pl7 (150 μg / ml) The peptide pl7 increases the proliferation lmfocitaria in assays of Mixed response leucocitapa (MLR) with CD not stimulated or stimulated with LPS or pIC. Figure 14. The CD25 + population generates a suppressive effect on the proliferation of splenocytes activated by Ant? -CD3 antibodies (0.5 μl / poc.) (Diamonds), CD25- (square) cells are unable to generate this effect allowing cell proliferation compared to the basal proliferation of splenocytes in the absence of proliferative stimuli (triangles) Figure 15. The inhibitory peptides of TGF-β (truncated and modified from pl7) in a co-culture of regulatory T cells and mouse activated splenocytes inhibit the suppressive action of the Cellular proliferation exerted by regulatory T lymphocytes The dose-dependent effect of the peptides pl7 (l-ll) am and Acpl7 (l-11) am on the inhibitory action of regulatory Tmmphocytes can be observed The inhibition exerted is dose-dependent, at a concentration of 50 μM the pl7 (1-11) is able to inhibit the suppressive effect by 20%, the pl7 (1-11) am by 128% and the Acpl7 (1-11) by a % at a concentration of 25 μM. Figure 16. The inhibitory peptides of TGF-ßl, p444 and p17 (1-11) a administered mT per day between day 6 and 10 (50 μg / mouse / 48h) delay tumor growth from subcutaneous inoculation of 50,000 CT26 cells at day 0, in animals immunized 10 days before with AHI.

MODE OF EMBODIMENT OF THE INVENTION The following are some examples of the operation of the invention with illustrative character and in no way limiting the scope thereof.

EXAMPLE 1 In this example, the effect of peptide p144 is studied in a system in which exogenous TGF-β1 is used as an inducer of the differentiation of a population of splenocytes into dendritic cells.

Isolation and culture of dendritic cells After sacrificing a male C57 mouse, for 8 weeks, the spleen was removed under sterile conditions and homogenized in a plate with clean medium to obtain a cell suspension. The cells were centrifuged for 5 minutes at 1,000 rpm and the cell pellet obtained was resuspended with 1 ml / spleen of ACK lysis solution (0.15 M NH 4 Cl, 1 mM KHCO 3, 0.1 mM sodium salt-EDTA, pH 7.2 -7.4) for 1 minute at 37 ° C. Next, the cells were centrifuged and washed with 10 ml of cold RIO medium (RPMI-1640, 10% FBS, Glutamine, 2xl0 ~ 5 M 2-Mercaptoethanol) to centrifuge and wash once more. Finally, the cells were resuspended in 50 ml of RIO medium. 6-well plates (Costar # 3471) were prepared by treating them with 1 ml per well of medium for differentiation of splenocytes to dendritic [RIO + 10 ng / ml of mouse GM-CSF (Peprotech, EC LTD, London, UK) + lng / ml of TGF-β1 (RD Systems, Minneapolis, USA)], for 15 minutes at room temperature. Next, 2 ml of the cell suspension was added to each well and incubated at 37 ° C and 5% C02. During the first weeks the medium was changed twice, by eliminating 1 ml of supernatant and adding 1 ml of fresh medium (RÍO + GM-CSF + TGF-ßl) for dendritic. After these two weeks the medium was separated. 1 ml per well of dissociation medium in enzyme-free PBS (GIBCO BRL) was added to the plates and incubated 10 minutes at 37 ° C, then removed and 2 ml of medium added (RIO + GM-CSF + TGF). -ßl) tempered. The cells were then detached by slow pipetting, and the detached cells were centrifuged at 1,000 rpm and resuspended in fresh medium (RIO + GM-CSF + TGF-β1). The cells thus obtained can be reamplified by plating, repeating the initial process.

Treatments of dendritic cells From a 18-day culture of dendritic cells derived from splenocytes, the following treatments were performed for 72 hours: -Control group: cells treated with dendritic medium (GM-CSF + TGF-ßl), with 0.25% DMSO. -Antibody (TGF-ßl): cells treated with dendritic medium (GM-CSF + TGF-ßl), with 0.25% DMSO, to which was added a neutralizing antibody ant? - (TGF-ßl) (Pharmingen) at a concentration of 20 μg / ml. -Pd44 peptide: cells treated with dendritic medium (GM-CSF + TGF-β1), to which peptide pl44 is added in solution with DMSO, leaving a final peptide concentration of 50 μg / ml at 0.25% DMSO . After 48 hours, the media corresponding to each treatment was renewed and at the end of the treatment the cells were collected by pipetting, after treatment with dissociation medium (GIBCO BRL).

Analysis of surface markers by flow cytometry The determination of surface markers of the splenocytes cultured with the different treatments was carried out by flow cytometry (FACScalibur, Becton-Dickmson, San José, CA, USA). The cells were washed with 2 ml of saline per well and then 1 ml of dissociation medium in enzyme-free PBS (GIBCO BRL) was added and incubated for 10 minutes at 37 ° C. The medium was then removed and the medium was added. my from PBS. The cells were detached by slow pipetting and the detached cells were centrifuged at 1,000 rpm and resuspended at a concentration of 2 x 106 cells / ml in PBS. 100 μl / well of the cell suspension obtained was incubated with 1 μl of the vials of monoclonal antibodies (Becton-Dickmson, Pharmmgen), anti-CD80, anti-CDllc and mouse anti-MHC I conjugated with FITC (1 mg / ml), in 96-well plates for 30 minutes at 4 ° C and in the dark. Next, the cells were washed 3 times with PBS by centrifugation of the plate at 1,500 rpm (Centpfuge 5810R, eppendorf) for 5 minutes, removing the supernatant and resuspending the cells in 100 μl of PBS. As a negative control, a non-reactive monoclonal antibody conjugated with FITC (Becton-Dickinson, Pharmmgen) was used. During the maturation of splenocytes in vi tro, the presence of certain factors and cytokines in the medium, can differentiate the cells towards different phenotypes leucocitapos In this case the TGF-β1 is described as a necessary factor for certain cell types to express on the surface, markers associated with dendritic cells. When we studied the effect of incubation of dendritic cells for 72 hours in the presence of p144 on the markers of these cells, it was observed that p14 had a negative effect on the expression of MHC I, CDII, CD80 This effect goes in the same direction and with similar magnitude, that produced when the cells are incubated in the presence of anti- (TGF-ßl) antibodies Indeed, as can be seen in Figure 1, the fluorescence levels associated with each marker are similarly reduced by the treatment with peptide p144 or anti-TGFßl antibody. This result highlights the importance of TGF-β1 in the maturation of dendritic cells, and suggests that the use of inhibitors of this cytokine in immunization protocols could have important effects both in the induction of humoral and cellular responses.

EXAMPLE 2 In vivo activity of a recombinant adenovirus for mouse IL-12 In this example the effect of peptide p144 is studied in an in vivo system in which TGF-β1 acts as a supposed antagonist of the cytokines induced in this model. The production of mouse IL-12 by the expression of a transgene, included in the recombinant adenovirus, induces an inflammatory state through the induction of a cascade of factors, among which the IFN-γ stand out. and nitric oxide. TGF-ßl has been described as an inhibitor of the production and biological action of IL-12, IFN-γ. and NO (Pardoux C. et al., 1999; Schini V. B. et al., 1992). In this model, lxlO8 pfu of mouse RAd IL-12 was administered by intraperitoneal route in 500 μl of saline to groups of 3 BALB / c mice of 4 to 8 weeks of age (Harian), distributing the animals in the following groups- - RAd IL-12: these animals received 1x10 pfu of RAd IL-12 from mouse on day 0. - RAd IL-12 + pl44: these animals received the Same treatment as the previous group but they were administered for 5 days, after administration of the adenovirus, the peptide p144 at a daily dose of 100 μg in 500 μl of SF containing 0.66% DMSO.

Blood samples were taken from both groups on days 6 and 9 after immunization for the subsequent quantification of IFN-α levels. and NOT in serum.

Measurement of IFN-α levels The amount of IFN-? was measured by a commercial ELISA (Mouse IFN-? Duoset ELISA Development System, Genzyme, Cambridge and OPTEIA Mouse IFN-? Set, Pharmmgen, San Diego, USA) according to the manufacturer's instructions. The results were expressed as pg / ml of IFN-? using a standard curve of known amounts of IFN- ?.

Measurement of nitric oxide levels The levels of NO production are taken as an indirect measure of serum nitrite and nitrate levels. The measurement is carried out by means of a chemiluminescence test using the Sievers NOA 280 nitric oxide detector, following the method recommended by the manufacturer (Sievers Instruments Inc. 1996). The technique used allows measuring nitrates or nitrates + nitrites depending on the reduction procedure of NO used. The nitrites present in the samples were reduced to NO by incubation with 350 mM of Nal in glacial acetic acid according to the following reaction: I + N02 + 2 H + > NO + 1/2 I2 + H20 In the measurement of nitrites and nitrates, these were reduced to NO by incubation with 50 mM of VC13 in IN HCl at 90 ° C according to the following reaction- 2 N03 + 3 V + 3 + 2 H20 > 2 NO + 3 V02 + + 4 H + The reduction to NO occurs in the detector cuvette. The NO resulting from either of the above reactions is transported to the detector by a vacuum pump. The chemiluminescence reaction between NO and ozone occurs in the detector: NO + 03 > 02 + N02 * > N02 + h The quantified emission and the obtained data are collected and processed by a computer. The administration to mice of a recombinant adenovirus (RAd) that expresses mouse IL-12, generates a cascade of responses among which a significant increase in serum levels stand out. of IFN-? and NO. Since the induction processes of IFN-? and from NO are affected by TGF-ßl levels (Schini V.B. et al., 1992), we also studied the effect of the administration of peptide p44 on said levels. In FIG. 2 and FIG. 3, the levels of IFN-α are respectively indicated. and NO in the serum of mice given a dose of lxlO8 pfu of recombinant adenovirus for mouse IL-12 (RAd IL-12) with or without p144. In Figure 2 it is shown that the administration of peptide p144 together with RAd IL-12, increases the levels of IFN-α. induced with respect to those achieved after the administration of only RAd IL-12. Figure 3 shows the effect of the administration of RAd IL-12 with or without p44 on the serum levels of NO. The joint administration of RAd IL-12 and pl44 generates a higher NO level at day 6, with respect to the one generated only with the administration of RAd IL-12. The effect of p44 in this induction model of cytokines proinflamtopas, can be explained based on the action that TGF-ßl exerts on the regulation, expression and activity of IL-12 and the processes that this cytokine activates. described that TGF-β1 exerts an antagonistic action on the expression and activity of IL-12 and IFN-? . Therefore, if p144 neutralizes TGF-β1, the antagonistic effect of this cytokine on the expression and activity of IL-12 and IFN-α is eliminated. and consequently increase the serum levels of IFN-? (Fig. 2). In summary, in this model, TGF-β1 acts to slow both the expression of IL-12 and (concomitantly) the expression of IFN-β. Accordingly, the inhibition of TGF-β1 by pl44 has the effect of increasing the expression of IFN-β. With respect to the effect that the p144 has on the NO increase, we believe that it can also be explained based on the inhibition of TGF-ß1 by p44. In fact, since TGF-β1 inhibits the expression and activation of the enzyme iNOS, responsible for the production of NO, it is logical to conclude that if the cytokine is inhibited, NO levels will tend to increase, as seen in the Figure 3 to day 6. Since the IFN-? induces the expression and activity of the iNOS, the results of Figures 2 and 3 are consistent, since on day 6 it is observed that the administration of pl44 results respectively in an increase in the levels of IFN-α. and NO.

EXAMPLE 3 Antibody Induction To analyze the immunomodulatory effect of peptide p144 on the humoral response, female BALB / c mice (Harian, Barcelona) were used between 6 and 8 weeks. For the induction of specific antibodies, a recombinant adenovirus (RAd-LacZ) inactivated with heat was inoculated in a bath at 100 ° C for 10 minutes.

Groups of animals and treatments Three mice were immunized per group, by intraperitoneal injection of a 200 μl mixture containing 1 × 10 8 pfu of inactivated RAd-LacZ adenovirus, physiological saline (SF) or with 50 μg of peptide p44 , all emulsified in Freund's complete adjuvant (CFA) in volumetric ratio 1: 1 as indicated in Table M4. Thirty days after the first immunization the animals were reinmunized with the same mixtures, but emulsified in Freund's incomplete adjuvant (IFA). Blood samples were taken from the retrorbital plexus on days 15 and 45 to quantify the an i -adenovirus antibodies generated in each animal. In order to study the possible induction of antigenic tolerance in mice treated with pl44, on day 50 after the injection, the mice were inoculated intravenously with 4 x 108 pfu of active RAd-LacZ in 100 μl of RPMI-1640 , including a new group of control mice (cont iv) that had only received the intravenous dose of the active LacZ adenovirus. Blood samples were taken at 7 days in all groups. The animals were then sacrificed to include liver samples in 0CTs (Tissue-Tek®, SAKURA, The Netherlands) for the subsequent evaluation of LacZ expression in the liver.

Table 1. Composition of the immunization mixtures of the different groups with heat-inactivated adenoviruses.

Groups RAd-LacZ p144 CFA / IFA SF RAd-LacZ 50μl (1x108 pfu) - 100μl 50μl RAd-LacZ + p144 50μl (1x108 pfu) 50μl (50μg) 100μl Quantification of anti-RAd LacZ antibodies in serum Detection of serum antibodies against RAd-LacZ was performed by ELISA assays using 96-well flat bottom Maxisorp plates (Nunc, Roskilde, Denmark), based on the streptavidin-biotin system using 2, 2 '-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as a developer. Plates were incubated overnight at 4 ° C with 50 μl per well of a 75 μl solution of RAd-LacZ 1010 pfu / ml in 10 ml 0.1 M Na 2 CO 3 (pH = 10.5). Subsequently, 3 washes were carried out with 200 μl per well of PBST washing buffer (phosphate buffered saline pH = 6 with 0.1% Tween 20). Nonspecific junctions were blocked by incubation of the plates for one hour at room temperature with 400 μl per well of PBST with 1% milk powder (PLT). The plates were emptied and three washes were made with PBST. 4 μl of serum in 100 μl of PLT was added making 8 serial double dilutions and the plates were incubated 1 hour in an oven at 37 ° C. It was washed three times with PBST and incubated for one hour at 37 ° C with 50 μl per well, of a Viooo dilution in PBST of goat anti-mouse IgG biotinylated antibody (Amersham). It was washed three times with PBST and 50 μl was added per well of a Vsoo solution of streptavidin-peroxidase (Amersham). After 1 hour of incubation, three washes with PBST were carried out and the plate was developed. The ABTS was used as the substrate for the development reaction, which gives green coloration in the presence of hydrogen peroxide and the enzyme peroxidase. A solution was prepared with 10 ml of 0.6% acetic acid (pH = 4.6), 7.5 μl of 33% H202 (v / v) and 100 μl of 40 mM ABTS. 100 μl per well was added and after one hour the plate was read at 405 nm on a Multiskan Plus MKII reader (Labsystem, Helsinki, Fmland).

Staining in si tu of the expression of the transgene (X-gal stain) The cuts of cpostato (6 μm) from the preparations of hepatic samples included in a compound for optimal cut in cryostat ("optimal cutting temperature compound", OCT), dried at room temperature. Then, they were fixed with 0.5% glutaraldehyde for 10 minutes, adding 200 μl per preparation. Next, 3 washes were made in PBS, to then add 200 μl of the dyeing mixture. K3Fe (CN) 6 30 mM, K4Fe (CN) 6 30 mM, X-Gal 20 ng / ml and MgCl2 in PBS The preparations were incubated for 12 hours at 37 ° C, after which 3 washes were made in PBS and one Once dried, the preparations were assembled The use of certain recombinant viruses as gene therapy tools has the disadvantage that they can rarely be used due to the induction of antibody responses to the virus. Indeed, if the virus is administered more than once, its effect is noticeable. (or completely) depleted because the antibodies induced in the first administrations are able to neutralize the virus administered in subsequent treatments. For this reason, we decided to study the role of pl44 in a process of induction of antibodies against a recombinant adenovirus. The basic idea behind this experiment was to study whether the neutralization of TGF-ß1 by p44, could or could not inhibit the production of antibodies, or even induce immunological tolerance against adenovirus in successive administrations. Thus, in a first experiment we immunized mice with recombinant adenovirus for Lac Z inactivated by heat (RAd LacZmact) This immunization was carried out in the presence or absence of p144. As shown in Figure 4, the first immunization did not have a quantifiable effect by ELISA on the production of anti-RAd Lac Z antibodies. However, after a second immunization a very different effect was observed (Fig 5). second immunization with RAd LacZ inactivated in the absence of p144, induced high titers of antibodies against RAd LacZ. However, when pl44 was included in the immunization mixture, the antibody titre against RAd LacZ was clearly lower than that obtained only with RAd LacZ inactivated. After these results, it was decided to study whether the mice treated with p44 had been able to develop some degree of tolerance to the antigens presented in the form of inactivated adenovirus. For this, the groups of mice mentioned above were inoculated intravenously on day 20 (after the second immunization) with active LacZ adenovirus (RAd LacZact), in the absence of peptide p144. Seven days later the expression of the transgene was measured, and the presence of antibodies in the sera and the expression of LacZ in the livers were analyzed. As can be seen in Figure 6, the antibody titer was approximately equal in the two groups of mice, indicating that no tolerance had been induced and that pl44 is able to inhibit the humoral response, only when it is included in the immunization mixture. with the antigen, in the present case the adenovirus inactivated by heat. The histological analysis on day 7 of the livers of the mice of Figure 6, showed that only the control group infected intravenously with RAd LacZact, was positive to LacZ staining, suggesting that in the other groups, the presence of Anti-adenovirus antibodies in serum was sufficient to neutralize the administration of RAd LacZact virus, preventing infection in the liver and the consequent expression of the LacZ gene (Fig. 7).

EXAMPLE 4 In this example, the presence of peptide p144 in an immunization mixture together with a peptide (FIS), which acts as a "T helper" determinant, is studied. This peptide induces a cytokine profile that favors the production of antibodies against different antigens.

Induction of "T helper" responses The FIS peptide is characterized as a "T helper" determinant derived from sperm whale myoglobin, amino acids (106-118). This peptide has been widely used for the induction of antibodies against haptenic peptides. We wanted to analyze the effect of the p144 peptide in the induction of a cytokine profile characterized among others by the increase of IFN-α and IL-2. In this model, groups of 3 female BALB / c mice from 4 to 8 weeks of age were administered subcutaneously (Harán, Barcelona), distributing the animals in the following treatments: -FIS: mice that received an emulsion 1 subcutaneously. : 1 of incomplete Freund's adjuvant and saline containing 50 μg of FIS. -FIS + pl44: mice subcutaneously receiving a 1: 1 emulsion of incomplete Freund's adjuvant and saline containing 50 μig of FIS and 50 μg of p44.

Ten days after the immunization, the animals were sacrificed and the popliteal, inguinal and periaortic lymph nodes were removed. The nodules were homogenized with a syringe and washed three times at 4 ° C with washing medium (clean RPMI 1640 medium). The cells were then resuspended in complete medium (RPMI 1640 with 10% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 5x10-5 M β-mercaptoethanol, 25 mM Hepes and sodium pyruvate, at a concentration of 5.3 x 10 cells / ml, adding 150 μl in each well of a 96-well flat-bottom plate The different concentrations of peptide (6 and 30 μM) were added in triplicate and in a volume of 100 μl per well The cells were cultured in an oven at 37 CC and with 5% C02 for two days, and at 24 hours 50 μl of the supernatant was collected in a 96-well plate to measure the IL-2 produced by the cells. cells, and at 48 hours 50 μl of the supernatant was collected to measure IFN-γ. These supernatants were frozen at -20 ° C until the concentration of the cytokines was quantified.

Measurement of IFN-α levels The amount of IFN-? was measured by a commercial ELISA (Mouse IFN-? Duoset ELISA Development System, Genzyme, Cambridge and OPTEIA Mouse IFN-? Set, Pharmingen, San Diego, USA) according to the manufacturer's instructions. The results were expressed as pg / ml of IFN-? using a standard curve of known amounts of IFN- ?.

Measurement of IL-2 levels The amount of IL-2 in each supernatant was measured by studying the proliferation of the CTL.L cell line, whose growth is dependent on IL-2 (Lai M. et al., 1987). it was maintained in culture with complete medium supplemented with IL-2 at a concentration of 10 U / ml. To perform the assay, the supernatants were cultured with 3000-5000 CTL.L cells per well, diluted to a final volume of 100 μl. After 24 hours of culture, 0.5 μCi (25 Ci / mmol) of tritiated thymidine (Amersham) was added per well and the cells were collected 20 hours later to filter plates (Un? F? Lter-96 GF / C, Perkm. Elmer) with a collector (Filtermate 196 Harvester, Packard). The radioactivity was quantified in a scintillation counter (Top Count, Microplate Scmtillation Counter, Packard) after addition of 25 μl of scintillation fluid (MICROSCINT, Packard, Bioscience Company) per well. The results of the counts were expressed as mU / ml of IL-2 interpolating the counts of each well in a standard curve.

The FIS peptide is a "T helper" determinant spanning residues 106 to 118 of the sperm whale myoglobin sequence. Immunization of BALB / c mice with FIS induces the activation of IFN-α producing T-cells. and IL-2 in response to the peptide Since TGF-β plays a role in the induction of immune responses, the effect of p44 on the production of cytokines after immunization of mice with FIS was studied in the presence and absence of p44. For this, BALB / c mice were immunized with 50 μg of FIS only, or with 50 μg of FIS in the presence of 50 μg of p44. As can be seen in Figure 8, the presence of peptide p144 in the immunization mixtures decreases the production of IL-2 and IFN-? compared to FIS It is important to indicate that pl44 is also probably presented by the MHC class II molecules of the BALB / c mouse as a DTh, since by restimulating in vi tro versus pl44 IL-2 production is observed and also some IFN-? The results suggest that the inclusion of pl44 in the immunization mixture has a negative effect on the "helper" capacity of the DTh, which is probably due to the neutralization of TGF-ßl that would be necessary at the time of the induction of responses mmunitapas EXAMPLE 5 A Effect of peptide p144 in an immunization mixture with a DTc (AHI) and a DTh (LVQ) of the tumor antigen A DTc is a peptide that is presented by the MHC-II on the surface of the host cell, and a DTc a peptide that is presented by the MHC-I on the surface of the host cell and in tumor cells. It was known that co-immunization with a DTc (peptide AHÍ) and a DTh (peptide LVQ), both peptides from the gp70 protein of the tumor antigen expressed by the CT26 cells, was able to protect against the subcutaneous growth of 500,000 tumor cells CT26 . Given that TGF-β is important in the process of inducing the immune response, we wanted to study the effect of peptide p44 on the induction of the response responsible for protection against growth with CT26 cells. As indicated in Table 1, three groups of BALB / c mice were immunized with the following mixtures in incomplete Freund's adjuvant: (i) with AHÍ + LVQ, (ii) with AHÍ + LVQ + pl44 and (iii) only with adjuvant incomplete by Freund. It was observed that only the immunization with AHÍ + LVQ was able to protect the mice and that therefore the incorporation of p44 in the immunization mixture had a negative effect on the protection against growth with CT26 cells.

Table 2. The administration of pl44 together with AHÍ + LVQ has a negative effect on the protection against the growth of CT26 cells achieved after immunization of BALB / c mice with AHÍ + LVQ.

,,. . Degree of protection immunization mixture (AHÍ + LVQ) in IFA 3/3 (AHÍ + LVQ + pl44) in IFA 0/3 IFA 0/3 Since peptide p144 is capable of blocking the activity of TGF-β1, the results obtained suggest that the cytokine plays a crucial role in the induction of an effective antitumor response and that its blockade at this stage has a negative effect on the induction of anti-tumor protective responses. This result is in line with another previous one (Fig. 8) in which it is shown how the administration of pl44 together with DTh FIS blocks the activation of the Th response to FIS.

B. Effect of peptide pl44 after immunization with a DTc (AHÍ) in a lung metastasis model.

It was speculated that the neutralization of TGF-ßl, once the mmunitapa response was induced, could have a beneficial effect on the evolution of the same. To test this concept, we studied the effect of administering pl44 at different times and different immunization protocols, on the survival of mice in a lung metastasis model induced by administering 5 x 10 5 CT26 cells intravenously. It was known from previous experience , that immunization only with the DTc THERE, produced some delay in the appearance of tumors after intravenous administration of CT26 cells For this reason, in a survival experiment, the inclusion of pl44 at different times after immunization with THERE. Thus, animals of group (i) received only the administration of CT26 cells, groups of animals (11), (111) and (IV) were immunized on day 0 subcutaneously with 50 μg of AHI in IFA (incomplete adjuvant de Freund) and subsequently they were administered 5 x 105 CT26 cells intravenously on day 10. The group (ni) also received 50 μg of p44 in 500 μl of SF (physiological saline) by intraperitoneal route every other day between days 4 and 20. The group (vv), as well as the group (ni), received 50 μg of pd44 in 500 μl of SF via the intraperitoneal route every other day, only between days 10 and 20. As can be seen in the Figure 9, immunization with AHI (n) only mediates a slight delay in mortality compared to the control group (i) not immunized. In the animals immunized with AHI, the treatment with p44 reinforces the survival effect, especially in the group (v) in which pl44 was administered between days 10 and 20 C Effect of p144 peptides after immunization with a DTc (AHI) in a subcutaneous tumor model Next, we studied the effect of p44 in a less aggressive model of tumor progression than intravenous administration of CT26. In this new model mice were immunized with 50 μg of AHI on day 0 and ten days later they were administered 5 × 10 5 CT26 cells subcutaneously. In addition, in order to test the effect of blocking TGF-β1 on protection against CT26 tumor cells, two other groups of mice (groups 2 and 3) were treated every other day intraperitoneally between days 10-30 with 50 μg of p44.

Table 3 The administration of pl44 after an immunization with AHI has a positive effect on the protection against growth of CT26 cells in BALB / c mice with AHI.

Protected animals Groups Day 10 Day 10 Day 10-30 on day 50 50 μg AH ≤ 5 x KP CT26 SF 0/10 50 μg AH ≤ 5 ≤ 105 CT26 50 μg p144 every 48 h 4/10 As can be seen in Table 3, blocking TGF-β 10 days after immunization with AHI generates protection against tumor growth measured on day 50 after subcutaneous administration of the tumor cells. This protection reached 40% of the animals Given the increase in protection efficacy against the growth of tumor cells, due to the neutralization of TGF-β1, it is of great interest and may be a strategy to adopt in order to obtain better responses antitumor EXAMPLE 6 Modulation of NK cells MATERIALS AND METHODS NK cell cultures Were carried out from spleen cells of RAG1 mice, lacking T and B lymphocytes. In some cases, the total splenocytes were cultured in 6-well plates at 4 × 10 6 cells per ml in RPMI medium enriched with 10% SBF, L-glutamma, antibiotics, non-essential amino acids, β-mercaptoethanol and human recombinant mterleuquma-2 (Chiron) at 6000 IU / ml. In half of the wells peptide pl7 was added at a concentration of 150 μg / ml. After 48 h, the medium was removed and the wells were washed with RPMI medium to discard the non-adherent cells. Fresh medium was then added with / without peptide. A day +5 the medium was changed again, this time replacing all the cells and the peptide pl7 in the corresponding cultures The cell counts with blue tppan were carried out at days +5 and +6 of this culture In other cases, NK cells were purified from mouse splenocytes by means of the immunomagnetic selection using the MmiMACS system, antigen pellets. ? -DX5 and MS columns (Miltenyi Biotec) according to the manufacturer's instructions. The cells thus obtained were plated in 48-well plates at 5 × 10 5 / ml in the medium described above with / without peptide p7 at 150 μg / ml. At 48 h, new peptide was added to the wells carrying p17, and the counts were made to days +2 and +4.

Flow cytometry The following anti-mouse rat monoclonal antibodies labeled with PE were used: ant? -CD25, ant? -CD69 and an isotype control antibody, all from Pharmingen (BD). The acquisition and analysis of the samples were carried out using a FACScalibur and the CellQuest program.

Proliferation assay with tritiated thymidine For this assay, DX5 + cells were used at days 2 and 4 of culture. Briefly, triplicates of 10000 cells were plated per well with and without peptide, their tritiated thymidine incorporation being measured in the usual culture medium with IL-2 6000 IU / ml at 6 h after the addition of thymidine.

Chromium release assay The cytotoxicity of the NK was checked by standard 4.5 h 51Cr release assays. Briefly, the targets were incubated with 50 μCi of 51 Cr for 2 h, washed (3 times) and then the effector cells at different proportions, the maximum being 40: 1 (effectors: targets). Finally, the release of 51 Cr due to lysis by NK cells after 4.5 h was measured in a TopCount scintillation counter (Perkm-Elmer). The cytotoxicity was measured as the percentage of Cr released with respect to the total acquired by the cells.

Cell lines The following tumor lines were used as targets for the NK cytotoxicity test: MC38 (colon carcinoma) and CLL (lung carcinoma), originated in C57BL / 6 mice, and CT26 (colon carcinoma) and RENCA (renal carcinoma) ) from BALB / c mice. LLC and RENCA were cultured in RPMI supplemented with fetal bovine serum, antibiotics and L-glutamine and MC38 and RENCA in DMEM supplemented in the same manner.

RESULTS The peptide pl7 exerts a clear antiproliferative effect on the population of NK cells obtained from RAG1 ly and cultured in vi tro mice (F? G.10), in proliferation quantification assays by direct cell counting or DNA synthesis (incorporation of thymidine tptiada). When analyzing the effect of pl7 on the cell surface expression of different markers, it was found that pl7 reduces the levels of CD25 and CD69 (Fig.ll) measured as mean fluorescence intensity. The CD25 and CD69 markers mediate immunosuppression, and both are induced by TGF-β1. So the peptide pl7 works by blocking the effect of TGF-β1, on the induction of these two markers (CD25 and CD69), in this cell population of the mmunitapo system.

On the other hand, in cytotoxicity trials facing this cell population to different mouse tumor lines (F? G.12), the presence of peptide pl7 improves the cytotoxic activity of this population of "Natural Killer" cells to a greater or lesser extent. From all these experimental models it is concluded that the peptide pl7 exerts a clear biological activity on the proliferation, differentiation and effector phase of NK cells EXAMPLE 7 Modulation of dendritic cells MATERIAL AND METHODS Obtaining dendritic cells (CD) from mouse bone marrow: First the legs were separated and placed on a plate with RPMI 10% FBS on ice. To obtain the marrows, it is necessary to cut the heads of the femurs and pass half inside the bone to drag the marrows to a plate with RPMI 10% FBS. The marrows were then undone with the help of a syringe and the contents were collected a Falcon tube that was centrifuged at 2000 rpm for 5 minutes, after centrifugation, the supernatant was removed and the erythrocytes were lysed, which was carried out with the ACK lysis buffer. Once the cells were lysed, depletion of those cell populations that were not of interest was carried out, for which both commercial antibodies and antibodies obtained from combined ascites with rabbit complement were used. The depletion was performed at a cell concentration of 2x10 cells / ml to which the following mixture was added: -Ascites ant? CD4 at lOOμg / ml. - Anti-CD8 antibodies at 100μg / ml - B220 Abbonation in 1/20 dilution (antilimfocytes B) - 10μl / ml of GR1 (antigranulocytes) - Supplement in 1/20 dilution.

This mixture was incubated for 50 minutes at 37 CC stirring approximately every 15-20 minutes. After this time, a washing with RPMI clean medium was carried out and the number of cells obtained was quantified. Finally the cells were seeded in 12-well plates at a final concentration of one million cells per milliliter. (3ml / well) and 20 μg / ml of the cytokines IL-4 and GM-CSF.

On day 3 and 5 after sowing, 2 ml of each of the wells that were replaced with fresh medium were withdrawn together with the concentration of cytokines corresponding to that volume. On day six the CDs were collected, quantified and plated. of 12 wells at a concentration of 1 million / ml (3ml / pound) and 20μg / ml of the cytokines IL-4 and GM-CSF and the following treatment was performed: - without stimulus - without p7 - with p7 (150ug / ml) - LPS (lOug / ml) - without pl7 - with pl7 (150ug / ml) - p (I: C) (lOOug / ml) - sm pl7 - with pl7 (150ug / ml) - 1668 (luM) - without pl7 - with pl7 (150ug / ml) - 3T3-CD40L - without pl7 - with pl7 (150ug / ml) LPS: Lipopolysaccharide; Poly (I: C): Synthetic bicatenapo RNA (poly mosinmicopolicitidílico acid). 1668: oligodeoxynucleotide (ODN). 3T3-CD40L: cell line that produces CD40 ligand.

The cells were left in the presence of the different stimuli for 48 hours, after which time they were collected and quantified in order to perform the mixed leukocyte response test, which consists of an allogeneic reaction where non-adherent cells of a mouse spleen belonging to a Certain strain faces dendritic cells from another different mouse strain. The objective of this test is to study the presenter capacity of CDs, which, when belonging to a different strain of mouse, present a different HLA restriction that is recognized by the lmfocytes of the other mouse, causing them to proliferate. The degree of proliferation is determined through the incorporation of tritiated thymidine. This parameter indicates the efficacy with which CDs present antigen. In this case the non-adherent cells were obtained from a Balb / c mouse and the dendritic cells are derived from a C57 mouse. In addition the cells were incubated with different stimuli to see the effect of pl7 in this context.

RESULTS The peptide pl7 is able to increase the proliferation of lmfocipapa in mixed response assays leucocitapa (MLR) as a consequence of an increase in the efficiency with which the dendritic cells present antigen. This effect of peptide pl7 is produced with dendritic cells not stimulated or stimulated with LPS or pIC (F? G.13). However other stimuli (1668 and 3T-CD40L) do not allow peptide p7 to mediate a difference in the efficacy of antigenic presentation and therefore in the proliferative response lmfocipa. These results reveal the potential of a TGF-β1 inhibitory peptide in the stimulation of antigen-presenting cells (CD) and the effectiveness of antigen presentation.

EXAMPLE 8 Modulation of regulatory T lymphocytes MATERIAL AND METHODS 1. OBTAINING TOTAL SPLENOCYTES: To obtain splenocytes from 6-week-old female Balb-c mice, 4 animals were sacrificed and after the extraction of the spleen they were transferred to clean medium for disintegration with crystals, the homogenate obtained was filtering (70 Mieras filter) and transferred to 50 ml tubes, for further washing and centrifugation. The cells obtained were incubated for 1 minute in lysis buffer, for the elimination of erythrocytes and subsequently washed with clean culture medium. Finally, the cells obtained were resuspended in 1 ml of medium-AUTOMACS and their counting was carried out. 2. PURIFICATION OF CD25 + LYMPHOCYTES: the purification of CD25 + lymphocytes was carried out using magnetic columns by labeling with CD25PE and after an incubation magnetic anti-PE microspheres (Phycoerithrin) were added. After incubation, washing and filtering (30 Miter filter) the samples were passed through the magnetic column, obtaining by gravity the eluate containing the CD25- population. Once the column is extracted from the magnetic field, it is washed under pressure obtaining the CD25 + cells. 3. SUPPRESSIVE ACTIVITY TEST: To verify the regulatory character of the CD25 + population, a total of 100,000 splenocytes were plated per well in a 96-well U-bottom plate in a volume of 200 μl. per well, and Anti-CD3 antibody (0.5 μl / poc.) alone or compared with CD25 + or CD25- placing 25,000 cells per well (CD25 +) or 50,000 cells per well (CD25-) making double dilutions of the concentration of these cells. 4. INHIBITION TEST OF THE SUPPRESSIVE ACTIVITY BY THE PEPTIDES: In a 96-well U bottom plate, with 200 μl vol. / poc. , 100,000 splenocytes per well and Anti-CD3 antibody (0.5 μl / poc.) plus 25,000 CD25 + lymphocytes per well were seeded. To these mixtures were added peptides (3 columns / Peptide, 50 MicroM in the row and in the following three double dilutions.) 3 Peptides were tested: P17 (1-11) SEQ ID NO: 3, P17 (1-11) a SEQ. ID NO: 4 and AcP17 (l-11) a SEQ ID NO: 5. Both this assay and the suppressor activity are incubated at 37 ° C for 48 h, tritiated thymidine is added at 0.5 μCi / poc. it is harvested at 8 o'clock, afterwards counting the CPMs that emit the cells of each well 5. VERIFICATION OF THE EFFECTIVE SEPARATION OF TREGs: Labeling with fluorescent anti-CD4 and anti-CD25 antibodies and analysis by flow cytometry (89% of the magnetically separated population is CD4 + CD25 +).

RESULTS The CD25 + SELECTED ARE T-LYMPHOCYTES WITH REGULATORY ACTIVITY: Fig.14 shows how the splenocyte population proliferates in the presence of the appropriate stimulus (AntiCD3) and in the absence of regulatory cells. The presence of CD25 + lymphocytes produces a complete inhibition of the proliferation of total splenocytes. TGFB INHIBITOR PEPTIDES ARE CAPABLE OF BLOCKING THAT SUPPRESSIVE ACTIVITY OF T-regulatory lymphocytes (CD25 +): Based on the model established in Figure 14, the peptides derived from peptide pl7 are able to block the antiproliferative effect in a dose-dependent manner of the CD25 + lymphocyte population. At a concentration of 50 μM the peptide AcP17 (1-11) Am and P17 (1-11) am are capable of totally inhibiting the effect of regulatory T lymphocytes (Fig.15).

EXAMPLE 8 Effect on tumor growth MATERIAL AND METHODS GROUPS: 4 groups of 7 female mice balb-c of 6 weeks / group. Control AHÍ + IFA s.c. AHÍ + IFA s.c. + P144 (50μg i. / Dose / mouse) THERE + IFA s.c. + P17 (l-ll) am (50μg i.p./ dose / mouse) DESIGN: AHÍ + IFA: day -10; Peptides: from day -6 to alternate days until day 10. Tumor volume measurement: Every 3 days from day 10 to day 41. Challenge at day 0 with CT26 (500000 cells / mouse s.c. on the spine) RESULTS DECREASE IN THE AVERAGE TUMOR VOLUME BY GROUPS IN TREATMENTS WITH PEPTIDES: The effect of peptides p144 and P17 (1-11) Am in a model of tumor progression was studied. In this model, mice were immunized with 50 μg of AHI 10 days before the administration of 5 x 10 5 CT26 cells. In order to verify the effect of blocking TGF-ßl on protection against CT26 tumor cells, two other groups of mice were treated every other day intraperitoneally between days 6 and 10 (50 μg / mouse / 48h) with the peptides p144 and P17 (1-11) Am. These peptides are capable of generating protection against tumor growth measured on day 42 after subcutaneous administration of the tumor cells. This protection reached 100% of the animals in the case of pl44 (Fig.16). Given the increased efficacy of protection against the growth of tumor cells, due to the neutralization of TGF-β1, the great interest and possible development of these peptides in strategies to be adopted in order to improve anti-tumor therapies is reiterated.

Claims (19)

1. - An immune response modulating agent characterized in that it comprises a TGF-β inhibitor peptide selected from: peptide pl44 whose sequence corresponds to SEQ ID NO: 1, peptide pl7 whose sequence corresponds to SEQ ID NO: 2, a peptide which It has at least 90% homology with them, or fragments of the previous ones.

2. - An immune response modulating agent according to claim 1, characterized in that it comprises a fragment of a TGF-β1 inhibitor peptide selected from: the pl7 (l-ll) fragment defined in the sequence SEQ ID NO: 3, the fragment pl7 (1-11) am corresponding to SEQ ID NO: 4, and the Acpl7 (1-11) am fragment defined by the sequence SEQ ID NO: 5.

3. - Use of a modulating agent defined in one of claims 1 or 2, in the regulation of humoral, cellular or both immune responses.

4. - Use of a modulating agent according to claim 3 as a vaccination adjuvant.

5. - Use of a modulating agent according to claim 3 in the preparation of a pharmaceutical composition for the treatment of pathologies selected from: pathologies related to microorganisms that induce an immunosuppression mediated by TGF-β1 and cancer.

6. - Use of a people modulating the immune response according to claim 5, characterized in that said microorganisms are selected from: Lei sh ania, Trypanosus a cruzii, the human immunodeficiency virus, the hepatitis C virus, the influenza virus , and the herpes simplex virus.

7. - Use of a modulating agent according to claim 5, characterized in that said composition is for the treatment of a cancer selected from: breast cancer, prostate cancer, colon carcinoma, pancreatic cancer, skin cancer, hepatocarcoma, multiple myeloma and stomach cancer.

8. - Use of a TGF-β inhibitor peptide selected from: peptide pl44 whose sequence corresponds to SEQ ID NO: 1, peptide pl7 whose sequence corresponds to SEQ ID NO: 2, a peptide having at least 90% homology with them, or fragments of the foregoing, in the preparation of an immune response modulating agent.

9. - Use of a fragment of a TGF-β inhibitor peptide according to claim 8, selected from among the fragment pl7 (1-11) defined in the sequence SEQ ID NO: 3, the fragment pl7 (1-11) am corresponding to SEQ ID NO 4, and the Acpl7 (1-11) am fragment defined by the sequence SEQ ID NO: 5.

10. - Use of a TGF-β inhibitor peptide of TGF-β1 according to one of claims 8 or 9, characterized in that said modulating agent regulates humoral, cellular immune responses, or both.

11. - Use of a peptide inhibitor of TGF-ßl according to any one of claims 8 to 10, characterized in that said modulating agent has an immune response stimulating effect.

12. - Use of a TGF-β1 inhibitor peptide according to any one of claims 8 to 11, as a vaccination adjuvant.

13. - Use of a TGF-β inhibitor peptide according to one of claims 8 to 10, characterized in that said modulating agent has an inhibitory effect on the immune response.

14. - Use of a DNA sequence coding for a TGF-β inhibitor peptide selected from: peptide pl44 whose sequence corresponds to SEQ ID NO: 1, peptide pl7 whose sequence corresponds to SEQ ID NO: 2, a peptide possessing at least 90% homology with the same, or fragments of the above, to make an immune response modulating agent

15. - Use of a recombinant expression system coding for a TGF-β inhibitor peptide selected from: peptide pl44 whose sequence corresponds to SEQ ID NO: 1, peptide pl7 whose sequence corresponds to SEQ ID NO: 2, a peptide which it possesses at least 90% homology with them, or fragments of the foregoing, to manufacture an immune response modulating agent.

16. - Use of a DNA sequence according to claim 14 or of a recombinant expression system according to claim 15, characterized in that said immune response modulating agent has an effect selected from: stimulator and inhibitor of the immune response.

17. - Use of a peptide inhibitor of TGF-β1 whose sequence corresponds to SEQ ID NO: 1, SEQ ID NO: 2, a peptide having at least 90% homology with them, or fragments of one of the above, in the preparation of a composition for the treatment of pathologies selected from: pathologies related to microorganisms that induce a mediated immunosuppression TGF-ßl and cancer.

18 -. 18 - Use of a TGF-β1 inhibitor peptide according to claim 17, characterized in that said microorganisms are selected from Lei shmama, Trypanosoma cruzn, the human immunodeficiency virus, the hepatitis C virus, the influenza virus, and the herpes simplex virus.

19. - Use of a TGF-β1 inhibitor peptide according to claim 17, characterized in that said composition is for the treatment of a cancer selected from: breast cancer, prostate cancer, colon carcinoma, pancreatic cancer, skin cancer, hepatocarcoma , multiple myeloma and stomach cancer.