Patent Application
20090263410
The present invention is included in the field of the preparation of immune response modulating agents.
TGF-β1 (transforming growth factor β1) is a potent immunomodulator that is present in all phases of the immune response generating different effects. It is currently known as a potent immune system cell regulator, including lymphocytes, macrophages and dendritic cells (Letterio J. J., 1998).
The biological activity of TGF-β1 varies a great deal depending on the type and state of cell 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 the dysfunction of the immune system. TGF-β1 regulates the immune response in a complex, context-dependent manner, which has been revealed by using experimental models of different diseases, as well as for the evaluation of genetically modified mice with regard to TGF-β1 expression, its receptors or regulating proteins. TGF-β1 regulates the function and interaction of immune system cells in the development of humoral, cytotoxic and immunotolerance responses and the pathological origin of many infectious and autoimmune diseases.
T lymphocytes are clearly regulated by TGF-β1 in all phases of their development (Fontana A. et al., 1992). The effect of TGF-β1 varies according to the state of differentiation of the lymphocyte and the type of activation signal that it has received. The first studies on the effect of TGF-β1 in human lymphocytes revealed their capacity to produce and secrete TGF-β1 as inhibitor of IL-2 dependent proliferation and cytolytic function (Pardoux C. et al., 1997).
Dendritic cells are a leukocyte population which is clearly differentiated due to their function as antigen presenting cells in the activation of T lymphocyte responses. They are a highly specialized cell population, which include epidermal Langerhans cells and follicular dendritic cells from the lymph nodes, and wherein TGF-β1 regulates both their differentiation and their activity (Strobl H, Knapp W., 1999).
It has been identified that TGF-β1 boosts the in vitro functional differentiation of dendritic cells, from CD34+ precursors, induced by the presence of other cytokines (TNF-α, SCF and GM-CSF). TGF-β1 also acts by increasing the viability of the dendritic cells in culture. On the other hand, the role of TGF-β1 in this cell type also seems to be related to a regulation mechanism which inhibits low specificity responses to avoid autoimmune processes.
In the differentiation, proliferation and production of B cell Ig (immunoglobins), TGF-β1 has a regulating role via the inhibition of the levels of certain surface molecules, including the major histocompatibility complex type II (MCH-II) both in pre-B lymphocytes and mature B cells. On the other hand, TGF-β1 inhibits Ig secretion in general, but clearly induces the production of IgA for which reason it performs an important role in the immune response associated to mucous membranes. Most studies on the effect of TGF-β1 as inhibitor of the production of all types of Ig, have been performed in vitro. However, the need for certain levels of TGF-β1 in lymphocyte culture, which act at an autocrine level, for the effective production of IgG and IgE has also been described. Thus, the function of TGF-β1 in the induction of antibodies is, as in many other processes, dual and opposing according to the context of the immune response (Lebman D. A., Edmiston J. S., 1999).
In the case of macrophages, the effect of TGF-β1 at a tissue level is generally suppressant and contributes to finalizing the inflammatory response.
Possibly, the most relevant effect of TGF-β1 on the inactivation of macrophages is due to its capacity for limiting the production of oxygen reactive species and metabolic intermediaries of nitrogen by cells activated with IFN-γ or LPS. The enzyme responsible for NO (nitric oxide) production by the activated macrophages is the inducible form of the nitric oxide synthase (iNOS) enzyme. The regulation of the activity of this enzyme by different cytokines, including TGF-β1, permits the regulation of the immune response in general, and in particular, the response of the macrophages to microorganisms and tumour cells. TGF-β1 inhibits the iNOS enzyme both at a transcriptional level, reducing mRNA levels, and suppressing protein activity. TGF-β1 also inhibits the production of intermediary oxygen reactive species and oxidative cytotoxicity, by the inactivation of macrophages and control of peripheral blood monocytes (Ashcroft G. S., 1999).
Additionally, the activation or production of TGF-β1, as well as the alternation of its signalling pathway, is described in many diseases produced by the infection of different microorganisms, including Leishmania, Trypanosoma cruzi, human immunodeficiency virus, hepatitis C virus, . . .
The documents in the state of the art closest to the present invention are the patent ES 2 146 552 and patent application ES200302020. The first document relates to the use of antagonist peptides of the TGF-β1 bond to its receptors in the organism, characterized in that it has partial sequences of amino acids which are similar or identical to those of TGF-β1 and/or its receptors; as well as its use to prepare a composition of application in liver diseases, in particular for hepatic fibrosis. This document protects peptide p144 (SEQ ID NO: 1) as well as its aforementioned use, although it does not mention its use in the preparation of an immune response modulating agent which constitutes the object of the present invention.
Patent application ES200302020 protects peptides inhibiting the biological activity of TGF-β1 which have been produced from a library of phages, and their use for the treatment of diseases which progress with a deregulated expression of TGF-β1, particularly fibrotic alterations. This document protects peptide p17 (SEQ ID NO: 2) and the use disclosed, but again it does not relate to its use in the preparation of an immunomodulating agent.
This immune system modulating effect is very important as it permits stimulating or inhibiting different aspects of the immune response according to requirements, and it may even have applications as a vaccination adjuvant.
Another relevant document is the patent application WO 2005/059133A2, which relates to a pharmaceutical composition which comprises at least one stimulator of immune cell function and at least one substance that inhibits cell proliferation and/or induces cell death. An antagonist of TGF-β1 is used as stimulator of the immune system function, selected from: oligonucleotides which hybridise with mRNA or with the DNA that encodes TGF-β1, TGF-β1-inhibiting proteins and peptides which have a molecular weight lower than 100 kDa which inhibit TGF-β1. Additionally, this document relates to the use of said pharmaceutical composition in the treatment of neoplasms. Nevertheless, this document does not use any peptide as TGF-β1 inhibitor, but instead an oligonucleotide, although it can be deduced that a peptide of these characteristics would have, in principle, a similar effect. This cannot be affirmed without considerable experimentation.
Below, a list of the bibliography cited in the present application is shown:
Letterio, J. J., Roberts, A. B. (1998). Regulation of immune responses by TGF-beta. Annu Rev Immunol 16, 137-761.
In order to facilitate understanding of the present text it is indicated that the term “peptide p144” refers to a peptide inhibiting TGF-β1 characterized in that its sequence of amino acids corresponds to that defined in SEQ ID NO:1. Likewise, the term “peptide p17” refers to a peptide inhibiting TGF-β1 activity, characterized in that its sequence of amino acids corresponds to that defined in SEQ ID NO: 2.
“Freund's incomplete adjuvant” refers to a composition that is very well known by a person skilled in the art, characterized in that it is composed of a water-in-oil emulsion, which acts as adjuvant delaying antigen release.
The present invention relates to an immune response modulating agent characterized in that it comprises a peptide inhibiting TGF-β1 selected from: peptide p144 whose sequence corresponds to SEQ ID NO: 1, peptide p17 whose sequence corresponds to SEQ ID NO: 2, a peptide which has at least 90% homology therewith, or fragments of the above. In a particular embodiment of the invention, said fragment of a peptide inhibiting TGF-β1 is selected from: fragment p17(1-11) defined in the sequence SEQ ID NO: 3, fragment p17(1-11)am which corresponds to SEQ ID NO: 4, and fragment Acp17(1-11)am defined by 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 or cellular immune responses, or both. In a preferred embodiment, the invention relates to the use of the modulating agent as vaccination adjuvant. In a particular embodiment of the invention, the immune response modulating agent is characterized in that it further comprises Freund's incomplete 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 which induce an immunosuppression mediated by TGF-β1 and cancer. Preferably, said microorganisms are selected from Leishmania, Trypanosoma cruzi, human immunodeficiency virus, the flu virus, and the herpes simple virus. Likewise, in a particular embodiment of the invention, the aforementioned composition is for the treatment of a cancer selected from: breast cancer, prostate cancer, colon carcinoma, pancreatic cancer, skin cancer, hepatocarcinoma, multiple myeloma and stomach cancer.
The present invention relates to the use of a peptide inhibiting of TGF-β1 selected from: peptide p144 whose sequence corresponds to SEQ ID NO: 1, peptide p17 whose sequence corresponds to SEQ ID NO: 2, a peptide which has at least 90% homology therewith, or fragments of the above, in the preparation of an immune response modulating agent.
Furthermore, the invention relates to a method for the use of a peptide inhibiting TGF-β1 selected from: peptide p144 whose sequence corresponds to SEQ ID NO: 1, peptide p17 whose sequence corresponds to SEQ ID NO: 2, a peptide which has at least 90% homology therewith, or fragments of the above, to prepare an immune response modulating agent.
Additionally, the invention relates to the use of a fragment of a peptide inhibiting TGF-β1 obtained from peptide p17 selected from: fragment p17(1-11) defined in the sequence SEQ ID NO: 3, fragment p17(1-11)am which corresponds to SEQ ID NO: 4, and fragment Acp17(1-11)am defined by sequence SEQ ID NO:5. To facilitate understanding of the text, it is indicated that peptide p17(1-11)am corresponds to a fragment which corresponds to amino acids 1 to 11 of peptide p17, wherein the amino acid in position 11 (tryptophan) is amidated; Acp17(1-11)am corresponds to the previous fragment which further has the amino acid in position 1 (lysine) acetylated.
The present invention also relates to peptides which have at least 70% homology with said peptides, and preferably that have at least 80% homology with them, provided that they maintain the capacity to inhibit the biological activity of TGF-β1. As well as any fragment of the above which maintains the capacity of inhibiting the biological activity of TGF-β1.
In a preferred embodiment, the present invention relates to the use of an aforementioned peptide inhibiting TGF-β1, characterized in that the aforementioned modulating agent regulates the humoral or cellular immune responses, or both. In a particular embodiment of the present invention, said modulating agent has a stimulating effect on the immune response, preferably as vaccination adjuvant.
On the other hand, a preferred embodiment of the present invention is characterized in that said modulating agent has an inhibiting effect on immune response.
Additionally, the invention relates to the use of a DNA sequence that encodes a peptide inhibiting TGF-β1 selected from: peptide p144 whose sequence corresponds to SEQ ID NO: 1, peptide p17 whose sequence corresponds to SEQ ID NO: 2, a peptide which has at least 90% homology therewith, or fragments of the above, to prepare an immune response modulating agent. The invention further relates to the use of a recombinant expression system that encodes peptide p144, peptide p17, a peptide which has at least 90% homology therewith, or fragments of the above, to prepare an immune response modulating agent. In a preferred embodiment of the invention, said immune response modulating agent has an effect selected from: immune response stimulator and inhibitor.
On the other hand, the invention relates to the use of a peptide inhibiting TGF-β1 whose sequence corresponds to SEQ ID NO: 1, a peptide which has at least 90% homology therewith, or fragments of one of the above in the preparation of a composition for the treatment of pathologies selected from: pathologies related to microorganisms which induce an immunosuppression mediated by TGF-β1 and cancer. In a particular embodiment of the present invention, said microorganisms are selected from: Leishmania, Trypanosoma cruzi, human immunodeficiency virus, the flu virus, and the herpes simple virus.
A particular embodiment of the present invention is characterized in that said composition would have an effect on the induction of immune responses to established tumours, inhibiting the immunosuppressant effect associated to the production and/or activation of TGF-β1 in various types of tumours (Teicher B. A., 2001): breast cancer, prostate cancer, colon carcinoma, pancreatic cancer, skin cancer, hepatocarcinoma, multiple myeloma and stomach cancer.
The modulating agent object of the present invention can be used in all kind of mammals, including rodents and primates. And, in a preferred embodiment, in human beings.
Below, some examples of functioning are shown of the invention with illustrative character, and in a way which by no means limits the scope thereof, are shown.
This example studies the effect of peptide p144 in a system wherein exogenous TGF-β1 is used as inducer of the differentiation of a population of splenocytes towards dendritic cells.
After sacrificing an 8-week old male C57 mouse, its spleen was extracted in sterile conditions and it was homogenised on a plate with clean medium to produce a cell suspension. The cells were centrifuged for 5 minutes at 1000 rpm and the cell sediment obtained was resuspended with 1 ml/spleen of ACK lysis solution (0.15M NH4Cl, 1 mM KHCO3, 0.1M sodium salt-EDTA solution, pH 7.2-7.4) for 1 minute at 37° C. Next, the cells were centrifuged and washed with 10 ml of cold R10 medium (RPMI-1640, 10% FBS, Glutamine, 2×10−5 M 2-Mercaptoethanol) to centrifuge and wash once more. Finally, the cells were resuspended in 50 ml of R10 medium. 6-well plates were prepared (Costar #3471) treating them with 1 ml per well of R10 medium to differentiate splenocytes from dendritic cells [R10+10 ng/ml of mouse GM-CSF (Peprotec, EC LTD, London, UK)+1 ng/ml of TGF-β1 (RD Systems, Minneapolis, USA)], for 15 minutes at room temperature. Next, 2 ml of the cell suspension were added to each well and they were incubated at 37° C. and 5% CO2. During the first weeks, the medium was changed twice, by the elimination of 1 ml of supernatant and the addition of 1 ml of fresh medium (R10+GM-CSF+TGF-β1) for dendritic cells. After these two weeks, the medium was separated. 1 ml per well of dissociation medium in enzyme-free BPS (GIBCO BRL) was added to the plates, and the medium was incubated at 37° C., then it was removed and 2 ml of tepid medium (R10+GM-CSF+TGF-β1) was added. Next, the cells were lifted with slow pipetting and the lifted cells were centrifuged at 1000 rpm and they were resuspended in fresh medium (R10+GM-CSF+TGF-β1). The cells thus obtained can be reamplified by seeding on a plate, repeating the initial process.
The following treatments were performed during 72 hours on an 18-day culture of dendritic cells derived from splenocytes.
After 48 hours, the media corresponding to each treatment were renewed and at the end of the treatment, the cells were collected by pipetting, after treatment with dissociation media (GIBCO BRL).
The determination of surface markers, from the splenocytes cultured with the different treatments, was performed using flow cytometry (FACScalibur, Becton-Dickinson, San Jose, Calif., USA). The cells were washed with 2 ml of saline solution per well and then 1 ml of dissociation medium in enzyme-free PBS (GIBCO BRL) was added, and it was incubated for 10 minutes at 37° C. The medium was then removed and 2 ml of PBS were added. The cells were lifted by slow pipetting and the lifted cells were centrifuged at 1000 rpm at a concentration of 2×106 cell/ml in PBS. 100 μl/well of the cell suspension obtained were incubated, with 1 μl of the phials of mouse anti-CD80, anti-CD11c and anti-MHC I monoclonal antibodies (Becton-Dickinson, Pharmingen), conjugated with FITC (1 mg/ml), in 96-well plates for 30 minutes at 4° C. and in the dark. Then, the cells were washed 3 times with PBS by centrifuging the plate at 1500 rpm (Centrifuge 5810R, eppendorf) for 5 minutes, eliminating the supernatant and resuspending the cells in 100 μl of PBS. As a negative control, a non-reactive monoclonal antibody was used, conjugated with FITC (Becton-Dickinson, Pharmingen).
During the maturation of splenocytes in vitro, the presence of certain factors and cytokines in the medium may differentiate the cells towards different leukocyte phenotypes. In this case, TGF-β1 is described as a necessary factor in vitro so that certain cell types express markers associated to the dendritic cells on the surface. When we study the effect of the incubation of dendritic cells during 72 hours in the presence of p144 on the markers of these cells, it was observed that p144 had a negative effect on MHC I, CD11c and CD80 expression. This effect will have the same sense and similar magnitude as that produced when the cells are incubated in the presence of anti-(TGF-β1) antibodies. Indeed, as can be observed in
This example studies the effect of peptide p144 in an in vivo system wherein TGF-β1 acts as supposed antagonist of the cytokines induced in this model. The production of mouse IL-12 by the expression of a transgen included in the recombinant adenovirus, induces an inflammatory state through the induction of a cascade of factors, including IFN-γ and nitric oxide. TGF-β1 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, 1×108 pfu of mouse RAd IL-12 were intraperitoneally administered in 500 μl of saline serum to groups of 3 BALB/c mice, from 4 to 8 weeks old (Harlan), distributing the animals in the following groups:
Blood samples were extracted from both groups on days 6 and 9 after immunization for the subsequent quantification of IFN-γ and NO levels in serum.
The quantity of IFN-γ was measured by a commercial ELISA (Mouse IFN-γ Duoset ELISA Development System, Genzyme, Cambridge and OPTEIA Mouse IFN-γ Ser, Pharmingen, San Diego, USA) in accordance with the manufacturer's instructions. The results were expressed as pg/ml of IFN-γ and using a standard curve of known quantities of IFN-γ.
The NO production levels are taken as an indirect measurement of the nitrite and nitrate levels in serum. The measurement was performed by a chemiluminescence assay using the Sievers NOA 280 nitric oxide detector, following the method recommended by the manufacturer (Sievers Instruments Inc. 1996)
The technique used permits measuring nitrates or nitrates+nitrites depending on the NO reduction process used. The nitrites present in the samples were reduced to NO by incubation with 350 mM of NaI in glacial acetic acid according to the following reaction:
I−+NO2−+2H+---------→NO+½I2+H2O
In the measurement of nitrites and nitrates, these were reduced to NO by incubation with 50 mM of VCl3 in 1N HCl at 90° C. according to the following reaction:
2NO3−+3V+3+2H2O-----→2 NO+3VO2+4H+
Reduction to NO occurs in the detector tank. The resulting NO of any of the two previous reactions is transported to the detector by a vacuum pump. In the detector, the chemiluminescence reaction is produced between the NO and ozone:
NO+O3---------------→O2+NO2*----------------→NO2+h
NO2* emission is in the red and infrared region of the light spectrum and is detected by a red-sensitive photomultiplier tube. This signal is quantified and the data obtained are collected and processed by a computer.
The administration to mice of a recombinant adenovirus (RAd) which expresses mouse IL-12, generates a cascade of responses amongst which an important increase in the serum levels of IFN-γ and NO stands out. As the IFN-γ and NO induction processes are affected by TGF-β1 levels (Schini V. B. et al., 1992), we also studied the effect of the administration of p144 on said levels.
The effect of p144 on this induction model of pro-inflammatory cytokines can be explained based on the action that TGF-β1 exerts on the regulation, expression and activity of IL-12 and on the processes that this cytokine activates. In fact, it is described that TGF-β1 exerts an antagonistic action on the expression and activity of IL-12 and IFN-γ. Therefore, if p144 neutralizes TGF-β1, it eliminates the antagonistic effect of this cytokine on the expression and activity of IL-12 and IFN-γ and they consequently increase the serum levels of IFN-γ (
With respect to the effect that p144 has on the increase in NO, we believe that it may also be explained based on the inhibition of TGF-β1 by p144. In fact, since TGF-β1 inhibits the expression and activation of the iNOS enzyme, responsible for NO production, it is logical to conclude that if the cytokine is eliminated, NO levels will tend to increase, as is observed in
Since IFN-γ induces the expression and activity of iNOS, the results of
To analyse the immunomodulating effect of peptide p144 on the humoral response, female BALB/c mice (Harlan, Barcelona) were used, of between 6 and 8 weeks old. For the induction of specific antibodies, recombinant adenovirus (RAd-LacZ) inactivated by heat in a bath at 100° C. for 10 minutes was inoculated.
Three mice were immunized per group, by the intraperitoneal of a mixture of 200 μl which contained 1×108 pfu of the inactivated RAd-LacZ adenovirus, physiological serum (PS) or with 50 pg of peptide p144, all emulsified in Freund's complete adjuvant (FCA) in a 1:1 volumetric ratio as indicated in table M4. Thirty days after the first immunization, the animals were reimmunized with the same mixture, but emulsified in Freund's incomplete adjuvant (FIA). Blood samples were taken from the retroorbital plexus on days 15 and 45 to quantify the anti-adenovirus antibodies generated in each animal.
In order to study the possible induction of antigen intolerance in the mice treated with p144, on day 50 after the Ist injection, the mice were inoculated intravenously with 4×108 pfu of active RAd-LacZ in 100 μl of RPMI-1640, including a new group of mice (cont iv), which had only received the intravenous dose of active LacZ adenovirus. Blood samples were taken after 7 days in all groups. Next, the animals were sacrificed to include liver samples in OCT® (Tissue-Tek®, SAKURA, the Netherlands) to the subsequent evaluation of LacZ expression in the liver.
The detection of antibodies in serum against RAd-LacZ was performed by ELISA assays using Maxisorp® flat-bottomed 96-well plates (Nunc, Roskilde, Denmark), based on the streptavidin-biotin system using 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) as developer. The plates were incubated throughout the night at 4° C. with 50 μl per well of a solution of 75 μl of RAd-LacZ 1010 pfu/ml in 10 ml 0.1 M Na2CO3 (pH=10.5). Next, 3 washes were performed with 200 μl per well of PBST wash buffer (pH=6 saline phosphate buffer with 0.1% Tween 20). The non-specific bonds were blocked by incubating the plates for one hour at room temperature with 400 μl per well of PBST with 1% powdered milk (PLT). The plates were emptied and three washes were performed with PBST. 4 μl of serum were added in 100 μl of PLT, making 8 double serial solutions and the plates were incubated for 1 hour in a stove 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 1/1000 dilution in PBST of biotinylated mouse anti-IgG antibody obtained from a goat (Amersham). It was washed three times with PBST and 50 μl per well were added of a 1/500 solution of streptavidin-peroxidase (Amersham). After 1 hour of incubation, three washes were performed with PBST and the plate was then developed. ABTS was used as a substrate of the developing reaction, which gives green colouring in the presence of hydrogen peroxide and the peroxidase enzyme. A solution was prepared with 10 ml of 0.6% acetic acid (pH=4.6), 7.5 μl of 33% H2O2 (v/v) and 100 μl of 40 mM ABTS. 100 μl per well were added and after one hour the plate was read at 405 nm in a Multiskan Plus MKII reader (Labsystem, Helsinki, Finland).
The cryostat cuts (6 μm) from the preparations of liver samples included in a compound for optimal cutting temperature compound (OCT) were dried at room temperature. Next, they were fixed with 0.5% glutaldehyde during 10 minutes, adding 200 μl per preparation. 3 washes were next performed in PBS, to then add 200 μl of the stain mixture: 30 mM K3Fe(CN)6, 30 mM K4Fe(CN)6, 20 ng/ml X-Gal and MgCl2 in PBS. The preparations were incubated for 12 hours at 37° C., after which, 3 washes were performed in PBS and, once dried, the preparations were mounted.
The use of certain recombinant virus as gene therapy tools has the drawback that they can be used few times due to the induction of antibodies to the virus. Indeed, if the virus is administered more than once, its effect will be notably (or completely) reduced due to the fact that the antibodies induced in the first administrations are capable of neutralizing the virus administered in subsequent treatments. For this reason, we decided to study the role of p144 in a process of the induction of antibodies against a recombinant virus. The basic idea behind this experiment was to study if the neutralization of TGF-β1 by p144 may or may not inhibit antibody induction, or even induce immunological tolerance to adenovirus in successive administrations. Thus, in a first experiment, we immunized mice with a heat-inactivated recombinant adenovirus for Lac Z (RAd LacZinact). This immunization was performed in the presence or absence of p144. As is shown on
Nevertheless, a very different effect was observed after a second immunization (
After these results, it was decided to study if the mice treated with p144 may have developed a degree of tolerance to the antigens shown in the form of inactivated adenovirus. To do this, the aforementioned groups of mice were inoculated by intravenous route 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 transgen was expressed in vivo, and both the presence of antibodies in serum and the expression of LacZ in livers were quantified. As can be observed in
The histological analysis on day 7 of the mouse livers of
This example studies the effect of the presence of peptide p144 in an immunization mixture together with a peptide (FIS), which acts as a T-helper determinant. This peptide induces a cytokine profile which favours the production of anti-bodies against different antigens.
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 analyse the effect of peptide p144 on the induction of a cytokine profile characterized among other things by the increase in IFN-γ and IL-12. In this model the following was administered by intravenous route to groups of 3 female BALB/c mice, 4 to 8 weeks old, (Harlan, Barcelona), distributing the animals in the following treatments:
10 days after immunization, the animals were sacrificed and the popliteal, inguinal and periaortic lymph nodes were extracted. The nodes were homogenized with a syringe and they were washed three times at 4° C. with washing medium (clean RPMI 1640 medium). Next, the cells were resuspended in complete medium (RPMI 1640 with 10% FBS, 2 mM of glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 5×10−5 M of β-mercaptoethanol, 25 mM of Hepes and sodium pyruvate), at a concentration of 5.3×106 cells/ml adding 150 μl in each well of a flat-bottomed 96-well plate. The different peptide concentrations (6 and 10 μM) were added in triplicate and at a volume of 100 μM per well. The cells were cultured in a stove at 37° C. and with 5% CO2 for two days. 24 hours later, 50 μl of the supernatant were collected on the 96-well plate to measure IL-2 produced in the cells, and after 48 hours, 50 μl of the supernatant were collected to measure IFN-γ. These supernatants were frozen at −20° C. until the quantification of the cytokine concentration.
The quantity of IFN-γ was measured using a commercial ELISA (Mouse IFN-γ Duoset ELISA Development System, Genzyme, Cambridge and OPTEIA Mouse IFN-γ Set, Pharmingen, San Diego, USA) in accordance with the manufacturer's instructions. The results were expressed as pg/ml of IFN-γ using a standard curve of known quantities of IFN-γ.
Measuring IL-2 levels
The quantity of IL-2 in each supernatant was measured by studying the proliferation of the CTL.L. cell line, whose growth is IL-2 dependent (Lai M. et al., 1987). This line 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 tritium labelled thymidine (Amersham) were added per well and the cells were collected 20 hours later on plates with a filter (Unifilter-96 GF/C®, Perkin Elmer) with a harvester (Filtermate 196 Harvester, Packard). The radioactivity was quantified in a scintillation counter (Top Count, Microplate Scintillation Counter, Packard) after the addition of 25 μl of scintillation liquid (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 which includes residues 106 to 118 of the sequence of sperm whale myoglobin. The immunization of BALB/c mice with FIS induces the activation of IFN-γ and IL-2 producing T-lymphocytes in response to the peptide. Since TGF-β1 plays a role in the induction of immune responses, the effect of p144 on cytokine production after immunization of mice with FIS was studied in the presence and absence of p144. To do this, BALB/c mice were immunized with 50 μg of FIS alone, or with 50 μg of FIS in the presence of 50 μg of p144.
As can be observed in
A DTc is a peptide which is presented by the MHC-II on the surface of the presenting cell, and a DTc is a peptide which is presented by MHC-I on the surface of the presenting cell and in tumour cells. It is known that the joint immunization of a DTc (AH1 peptide) and a DTh (LVQ peptide), both peptides coming from the gp70 protein of the tumour antigen expressed by CT26 cells, was capable of protecting against the subcutaneous growth of 500,000 CT26 tumour cells. Since TGF-β1 is important in the immune response induction process, we wanted to study the effect of peptide p144 on the induction of the response responsible for protection against the growth of CT26 cells. As is indicated in Table 1, three groups of BALB/c mice were immunized with the following mixtures of Freund's incomplete adjuvant: (i) with AH1 LVQ, (ii) with AH1+LVQ+p144 and (iii) just with Freund's incomplete adjuvant. It was observed that only the immunization with AH1+LVQ managed to protect the mice and that, therefore, the incorporation of p144 in the immune mixture, had a negative effect on protection against the growth with CT26 cells.
Since peptide p144 is capable of blocking TGF-β1 activity, the results obtained suggested that the cytokine plays a crucial role in the induction of an effective anti-tumour response and that its blocking at this stage has a negative effect on the induction of protective anti-tumour responses. This result is in harmony with another previous one (
It is speculated that the neutralization of TGF-β1, once the immune response has been induced, could have a beneficial effect on the evolution thereof. To prove this concept, the effect of administrating p144 was studied at different times and different immunization protocols, on the survival of mice in a pulmonary metastasis model induced by the administration of 5×105 CT26 cells by intravenous route.
It is known from previous experience that immunization with only DTc AH1 produced a certain delay in the appearance of tumours after the intravenous administration of CT26 cells. For this reason, in a survival experiment, the inclusion of p144 was compared at different times after immunization with AH1. Thus, the animals in group (i) just received the administration of CT26 cells, the animal groups (ii), (iii) and (iv) were immunized on day 0 by subcutaneous route with 50 μg of AH1 in FIA (Freund's incomplete adjuvant) and they were then administered 5×105 CT26 cells by intravenous route on day 10. Group (iii) further received 50 μg of p144 in 500 μl of PS (physiological serum) by intraperitoneal route on alternate days between days 4 and 20. Group (iv), as with group (iii), received 50 μg of p144 in 500 μl of PS by intraperitoneal route on alternate days, only between days 10 and 20. As can be observed in
Next, the effect of p144 was studied on a tumour progression model which was less aggressive than the intravenous administration of CT26. In this new model, mice were immunized with 50 μg of AH1 on day 0 and ten days later they were administered 5×105 CT26 cells by subcutaneous route. Furthermore, with the aim of testing the effect of blocking TGF-β1 on protection against CT26 tumour cells, another two groups of mice (groups 2 and 3) were treated on alternate days by intraperitoneal route between days 10-30 with 50 μg of p144.
As can be observed in Table 3, the blocking of TGF-β1 ten days after immunization with AH1 generates a protection against the tumour growth measured on day 50 after the subcutaneous administration of tumour cells. This protection reached 40% of the animals.
Given the increase in effectiveness of protection against the growth of tumour cells, due to the neutralization of TGF-β1, it is of great interest and can be a strategy to adopt in order to obtain better anti-tumour responses.
They were performed from spleen cells from RAG−/− mice, without T and B lymphocytes. In some cases, the total splenocytes was cultured on 6-well plates at 4×106 cells per ml of RPMI medium enriched with 10% SBF, L-glutamine, antibiotics, non-essential amino acids, β-mercaptoethanol and human recombinant interleukin-2 (Chiron) at 600 IU/ml. In half the wells, peptide p17 was added at a concentration of 150 μg/ml. After 48 hours, the medium was removed and the wells were washed with RPMI medium to discard the non-adherent cells. Next, fresh medium was added with/without peptide. On day +5, the medium was again changed, this time replacing all the cells and peptide p17 in the corresponding cultures. The cells counts with trypan blue were performed on days +5 and +6 of this culture. In other cases, NK cells from mouse splenocytes were purified by an immunomagnetic selection using the MiniMACS system, anti-DX5 beads and MS columns (Miltenyi Biotech) according to the manufacturer's instructions. The cells thus obtained were cultured on 48-well plates at 1.5×106/ml in the aforementioned medium with/without peptide p17 at 150 μg/ml. After 48 h, new peptide was added to the cells which had p17 and the counts were performed on days +2 and +4.
The following PE-labelled rat anti-mouse monoclonal antibodies were used: anti-CD25, anti-CD69 and an isotope control antibody, all from Pharmingen (BD). The acquisition and analysis of the samples were performed using a FACScalibur and the CellQuest program.
Proliferation Assay with Tritium Labelled Thymidine
For this assay, DX5+ cells were used on days 2 and 4 of culture. Briefly, triplicates of 10000 cells per well were plated with and without peptide, measuring their incorporation of tritium labelled thymidine in the typical culture medium with 6000 IU/ml IL-2, 6 hours after the addition of thymidine.
The cytotoxicity of NK cells was verified by standard 4.5 h 51Cr release assays. Briefly the targets were incubated with 50 μCi of 51Cr during 2 h, they were washed (3 times) and, next, the effector cells were added in different proportions, the maximum being 40:1 (effector:targets). Finally, the release of 51Cr due to lysis by NK cells was measured after 4.5 h in a TopCount Scintillation counter (Perkin Elmer). The cytotoxicity was measured as a percentage of Cr released with respect to the total acquired by the cells.
The following tumour lines were used as targets of the cytoxicity assays by NK cells: MC38 (colon carcinoma) and LLC (lung carcinoma), originated in C57BL/6 and CT26 mice (colon carcinoma) and RENCA (renal carcinoma) from BALB/c mice. LLC and RENCA were cultured with RPMI supplemented with foetal bovine serum, antibiotics and L-glutamine and MC38 and RENCA in DMEM supplemented in the same way.
Peptide p17 exerts a clear anti-proliferative effect on the population of NK cells obtained from RAG−/− mice and cultured in vitro (
Obtainment of Dendritic Cells (Dc) from Mouse Bone Marrow:
Firstly, the legs were separated and placed on a plate with 10% RPMI FBS on ice. To obtain the bone marrow, it is necessary to cut the heads off the femurs and pass medium through the interior of the bone to drag the marrow to a dish with 10% RPMI FBS. Next, the bone marrow was broken up with the aid of a syringe and the contents were collected in a Falcon tube which was centrifuged at 2000 rpm for 5 minutes; after the centrifugation, the supernatant was removed and the erythrocytes were lysated, which was performed with the ACK lysis buffer. Once the cells were lysated, the depletion of those cell populations not of interest was performed; to do this, both commercial antibodies and antibodies produced from ascites combined with rabbit complement were used. The depletion was performed at a cell concentration of 2×107 cells/to which the following mixture was added:
This mixture was incubated for 50 minutes at 37° C. stirring approximately every 15-20 minutes. After this time has elapsed, a wash was performed with clean RPMI medium 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 (3 ml/well) and 20 μg/ml of IL-4 and GM-CSF cytokines. On days and 3 and 5 after seeding, 2 ml was removed from each one of the cells which was replaced with fresh medium together with the cytokine concentration corresponding to that volume.
On day six the CD were collected, they were quantified and were placed on 12-well plates at a concentration of 1 million/ml (3 ml/well) and 20 μg/ml of the IL-4 and GM-CSF cytokines and the following treatment was performed:
The cells were left in the presence of the different stimuli for 48 hours; after this time had elapsed they were collected and quantified with the purpose of performing the mixed leukocyte response assay, which consists of an allogenic reaction where non-adherent spleen cells of a mouse belonging to a determined strain are opposed to dendritic cells of another different strain of mouse. The aim of this assay is to study the presenting capacity of the CD which, as it belongs to a different strain of mouse, shows a different HLA restriction which is recognised by the lymphocytes of the other mouse making them proliferate. The degree of proliferation is determined through the incorporation of tritium labelled thymidine. This parameter indicates the effectiveness with which the CD present antigen. In this case, the non-adherent cells were obtained from a BALB/c mouse and the dendritic cells derived from a C57 mouse.
Furthermore, the cells were incubated with different stimuli to see the effect of p17 in this context.
Peptide p17 is capable of increasing lymphocyte proliferation in mixed leukocyte response assays (MLR) as a consequence of an increase in the effectiveness with which the cells present antigen. This effect of peptide p17 is produced with dendritic cells, non-stimulated or stimulated with LPS or pIC (
1. OBTAINMENT OF TOTAL SPLENOCYTES: To obtain splenocytes from 6-week old, female Balb-c mice, 4 animals were sacrificed, and after extracting the spleen, they were transferred to a clean medium for their dispersion with crystals, the homogenate was filtered (70-micron filter) and transferred to 50-ml tubes, for their subsequent washing and centrifugation. The cells obtained were incubated for 1 minute in lysis buffer, for the elimination of erythrocytes and they were subsequently washed with clean culture medium. Finally, the cells obtained were resuspended in 1 ml of AUTOMACS medium and they were counted.
2. PURIFICATION OF CD25+ LYMPHOCYTES: the purification of CD25+ lymphocytes was performed by the use of magnetic columns labelling with CD25PE and after incubation, anti-PE magnetic microspheres (Phycoerythrin) were added. After incubation, washing and filtration (30-micron filter) the samples were passed through a magnetic column, obtaining the eluate containing the CD25− population by gravity. Once the column is extracted from the magnetic field it is washed under pressure, obtaining the CD25+ cells.
3. SUPPRESSANT ACTIVITY ASSAY: To verify the regulating nature of the CD25+ population, a total of 100,000 splenocytes was seeded per well on a U-bottomed 96-well plate, in a volume of 200 μl per well and anti-CD3 antibody (0.5 l/well) alone or opposing CD25+ or CD25−, placing 25,000 cells per well (CD25+) or 50,000 cells per well (CD25−), performing double dilutions of the concentration of these cells.
4. ASSAY FOR THE INHIBITION OF SUPPRESSANT ACTION BY PEPTIDES: On the U-bottomed 96-well plate, with 200 μl vol/well, 100,000 splenocytes were seeded per well and anti-CD3 antibody (0.5 μl/well) plus 25,000 CD25+ lymphocytes per well. Peptides were added to these mixtures (3 columns/peptide, 50 MicroM in the 1st row and double dilution in the following three rows). 3 peptides were assayed: P17(1-11) SEQ ID NO: 3, P17(1-11)am SEQ ID NO: 4 and AcP17(1-11)am SEQ ID NO: 5. Both this assay and that of the suppressant activity were incubated at 37° C. for 48 h, tritium labelled thymidine was added at 0.5 μCi/well and it is harvested after 8 h, subsequently counting the CPM emitted by the cells from each well.
5. VERIFICATION OF THE EFFECTIVE SEPARATION OF THE Treg: Labelling with fluorescent anti-CD4 and anti-CD25 antibodies and analysis by flow cytometry (89% of the population magnetically separated is DC4+CD25+).
THE CD25+SELECTED ARE T LYMPHOCYTES WITH REGULATING ACTIVITY:
The Inhibitory Peptides of TGFB are Capable of Blocking that SUPPRESSANT ACTIVITY OF regulatory T lymphocytes (CD25+): Based on the model established in
GROUPS: 4 groups of 7 female balb-c mice, of 6 weeks old/group.
AH1+FIA s.c.+p144 (50 μg i.p./dose/mouse)
AH1+FIA s.c.+p17(1-11) (50 μg i.p./dose/mouse)
DESIGN: AH1+FIA: day 10; Peptides: from day −6 on alternate days until day 10: Tumour model volume: Each 3 days from day 10 to day 41. Challenge on day 0 with CT26 (500000 cells/mouse s.c. in the flank).
REDUCTION IN THE MEAN TUMOUR VOLUME BY GROUPS TREATED WITH PEPTIDES: The effect of peptides p144 and p17(1-11)Am was studied in a tumour progression model. In this model, mice were immunized with 50 μg of AH1 10 days before the administration of 5×105 CT26 cells. In order to verify the effect of blocking the TGF-β1 on the protection against CT tumour cells, another two groups of mice were treated on alternate days by intraperitoneal route between days 6 and 10 (50 μg/mouse/48 h) with peptides p144 and p17(1-11)Am. These peptides are capable of generating a protection against tumour growth measured on day 42 after the subcutaneous administration of the tumour cells. This protection reached 100% of the animals in the case of p144 (
Since the increase in the effectiveness of protection against the growth of tumour cells, due to the neutralization of TGF-β1, the great interest and possible development of these peptides in strategies to adopt with the aim of improving anti-tumour therapies is reiterated.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/ES05/00569 | 10/24/2005 | WO | 00 | 9/15/2008 |
