Patent Application
20220193185
The invention concerns the combination of synthetic peptides that have affinity for different receptors present in cells of the immune system, more particularly the combination of synthetic peptides with affinity to the transforming growth factor beta (from the acronym in English: TGF-β) receptor, with synthetic peptides with affinity to the interleukin-10 (IL-10) receptor. Both receptors are known to play an important role in modulating immune, inflammatory, and allergic responses. In addition, the present invention also describes pharmaceutical compositions comprising the combination of at least one of each of these synthesized peptides and the use of said peptides to prepare drugs or immunogenic compositions for the treatment and/or prophylaxis of diseases in which there is an immunological disorder, more particularly in which the diseases related to the immune disorder are: chronic or acute inflammatory disease, allergic and/or autoimmune diseases.
Transforming growth factor (TGF)-β is a pleiotropic cytokine that acts on several biological processes, including embryonic development and cell proliferation, differentiation, adhesion, migration, and apoptosis. Its signaling has been proposed to control autoreactive peripheral T cells, present in patients with autoimmune diseases. Said cytokine also plays an important role in inducing auxiliary T cells capable of regulating cytokine production and pathway activation in order to repair the immune response.
TGF-β1 is produced by several cell types, such as platelets, neutrophils, malignant cells, dendritic cells, and macrophages. As a result, such cytokine has an important effect on inflammatory, allergic, and autoimmune diseases. Due to the diversity of TGF-β1, many factors are responsible for activating canonical or non-canonical pathways to induce multiple biological effects and interfere with different pro-inflammatory pathways. This cytokine can modulate different pathways directly related to TGF-β, receptors, as well as in response to crosstalk signaling.
TGF-β1 is a cytokine that plays an important role in allergic inflammation. It can suppress effector T cells and is involved in the development of conventional, innate, regulatory (Treg) T cells. T cells play a key role in maintaining peripheral tolerance against commensal, autologous, and environmental antigens. In mice with the TGF-β1 gene disrupted, and in mice without the (TORbRII) receptor II, severe inflammatory responses, tissue necrosis and early death were observed, confirming the regulatory role of TGF-β in peripheral tolerance.
IL-10 is a regulatory pleiotropic cytokine produced by different types of cells: monocytes, T cells, macrophages, dendritic cells (DC), natural killer cells (NK) and B cells. Since it influences different cell types, IL-10 has been shown to be an important molecule associated with a wide range of diseases, disorders, and conditions, including inflammatory and allergic conditions, immunity-related disorders, and cancer, due to its ability to regulate the humoral and cellular immune response. Thus, understanding how IL-10 regulates different cell types is crucial for the development of new intervention strategies for the treatment of various pathologies.
T lymphocytes are among the main factors involved in regulating the adaptive immune response in inflammatory diseases. In a healthy immune system, Type I helper T cells (Th1) are required for the elimination of many intracellular pathogens, whereas Th2 cells are required for the elimination of helminth infection. A disruption in the Th1/Th2 balance can lead to immunological disturbances with fatal outcomes. In an unbalanced immune system, Th1 cells are involved in delayed hypersensitivity immune reactions, while Th2 cells are involved in allergic diseases. Th1 lymphocytes produce IFN-g and IL-2 to support cell-mediated immunity, while Th2 lymphocytes produce cytokines such as IL-4, IL-5, and IL-13, which play important roles in exacerbating the allergic response. IL-4 is the main cytokine for the polarization of a Th2 response, which, together with IL-13, induces the class change of immunoglobulins production from B lymphocytes to IgE. Allergic reactions are initiated after subsequent exposure to the allergen, in which the allergen binds to specific IgE along with the allergen bound to the high-affinity FIERI receptor on mast cells or basophils, triggering cell degranulation. This event alerts the immune system to local infection and propagates the allergic inflammatory response. Thus, the modulation of cell cytokines found in a Th1/Th2 profile is essential for the maintenance or restoration of immune homeostasis.
Birch pollen (Verrucous birch) is the main cause of pollinosis in the temperate climate zone of the northern hemisphere. The vast majority of patients allergic to birch (over 90%) react to its main allergen, Bet v 1, which has the ability to promote the induction of a Th2 profile, and is used as a marker of allergy to birch pollen. Allergen-specific immunotherapy (ASIT) modulates the natural course of allergies, leading to improvement or even complete remission of allergic symptoms (Mobs, C. et al. Cellular and humoral mechanisms of immune tolerance in immediate-type allergy induced by specific immunotherapy. Int Arch Allergy Immunol. 2008; 147(3):171-8). However, although ASIT is the treatment of choice for many patients, local and systemic allergic side effects have been reported. Sublingual allergen immunotherapy has also been associated with side effects.
Transforming growth factor b1 (TGFβ) is a cytokine that has important immunoregulatory properties, as reflected by its ability to suppress Th1 and Th2 responses and by inducing the expression of transcription factor fork box protein 3 (Foxp3). Foxp3 is a master regulator involved in the development and function of regulatory T cells (Treg). Treg cells are essential for the maintenance of immune homeostasis and the induction of immune tolerance. Its dysfunction can lead to the development of autoimmune diseases, immunopathology, and allergy. On the other hand, Interleukin 10 (IL-10) is a cytokine associated with important immunoregulatory functions, including the survival and proliferation of several cells of the immune system. Thus, peptides that can mimic the activation domain of TGF-β1 and IL-10 may be promising candidates for suppressing the Th2 response.
To develop new molecules with pharmaceutical approaches, the Phage Display (PD) methodology may be a good option, as it allows us to select small molecules that can imitate specific amino acids from large proteins and induce the same or better response as expected with the natural molecule or in comparison with other recombinant molecules already described. Peptides selected by PD have low toxicity and high receptor/protein affinity.
Therapeutic mimetic peptides are potential drug candidates for many different diseases, as they have the potential to antagonize, stimulate, increase, or modulate the activity of natural ligands, binding to specific cell surface receptors and triggering intracellular pathways.
With the purpose of seeking therapeutic strategies, patent WO2012167143 A1 reports the use of the phage display technology to obtain a specific antibody binding to the TGF-β molecule that can be used in patients who have disorders related to TGF-β expression, such as cancer. Such results show the efficiency of the technique in the development of recombinant antibodies in the treatment of diseases.
Patent application BR 102015003903-4 describes 27 synthetic peptides and their inverse sequences, selected by Phage Display, which have affinity for the TGF-β receptor. Then, said patent application describes the use of such peptide sequences synthesized in pharmaceutical compositions in the regulation of the immune system, in order to obtain the inflammation control or allow the treatment of inflammatory and autoimmune diseases. Said patent application also describes the possibility of using such synthesized peptides in methods of diagnosis or prognosis of related diseases.
Recombinant IL-10, for example, is already shown to be safe, well tolerated, and effective in patients with Crohn's disease (Fedorak, R. N. et al. Gastroenterology 2000 December; 119 (6): 1473-82). Patent application BR 102017010787-6 results in 101 synthetic peptides, selected by Phage Display, which have affinity for the IL-10 receptor. Therefore, the aforementioned patent application shows knowledge of the use of these synthesized peptide sequences in the treatment and monitoring of an inflammatory process, allergic diseases and autoimmune diseases.
The present patent application aimed to select new mimetic peptides to TGF-β1 and IL-10, by PD technology, which had an improved capacity to modulate and attenuate immune response, particularly to control inflammatory and allergic responses.
Considering the studied peptides, eight (8) peptides with similar action to TGF-β1 were selected and synthesized and four (4) peptides with similar action to IL-10 were selected and synthesized, having their predicted three-dimensional structures.
The data obtained showed the ability of combining TGF-β1 mimetic peptides with IL-10 mimetic peptides to reduce the allergic response and inhibit inflammatory pathways in the assays performed. Such result shows the potential for pharmaceutical use of the combination of synthetic peptides presented here in the modulation of inflammatory allergic responses.
The present patent application describes synthetic peptides with affinity for the TGF-β receptor and synthetic peptides with affinity for the Interleukin-10 (IL-10) receptor. Such peptides were synthesized, and their three-dimensional structures were predicted. All of them were synthesized to present Histidine and Alanine as the first amino acid residue, in some cases Alanine as the second amino acid residue, and a sequence of three Glycine and a Serine as the last four amino acid residues. Such peptides showed important activity on immunological modulatory and regulatory pathways. Therefore, they can be used in the treatment and/or prophylaxis of diseases related to an immune disorder, such as: chronic or acute inflammatory, allergic and/or autoimmune diseases. Therefore, TGF-β1 and IL10 mimetic peptides may be promising candidates for attenuating inflammatory and allergic reactions when used in combination.
Another embodiment of the present application relates to methods of treatment and/or prophylactic of immune disorder related diseases, wherein the immune disorder related diseases are chronic or acute inflammatory, allergic and/or autoimmune diseases, comprising administering in a subject a pharmaceutically effective dose of a combination of at least one of the synthetic peptides mimetic to TGF-β1 and at least one of the synthetic peptides mimetic to IL-10.
Another embodiment of the present application are pharmaceutical compositions comprising a combination of at least one of the synthetic peptides mimetic to TGF-β1 and at least one of the synthetic peptides mimetic to IL-10, plus at least one pharmaceutically acceptable carrier or compound. Pharmaceutical Composition is for use in the treatment and/or prophylaxis of diseases related to an immune disorder, in which the diseases related to an immune disorder are chronic or acute inflammatory, allergic and/or autoimmune diseases.
Another embodiment of the present application is the use of the combination of synthetic peptides or pharmaceutical compositions described herein, for the preparation of a drug or immunogenic composition for use in the treatment and/or prophylaxis of diseases related to an immunological disorder, in which the immune disorder related diseases are chronic or acute inflammatory, allergic and/or autoimmune diseases.
The present invention will be better understood based on the description below, taken in conjunction with the attached figures, wherein:
Synthetic Peptides with Affinity for the TGF-β Receptor
The present patent application describes 8 synthetic peptides with affinity to the TGF-β receptor, which comprise or consist of the amino acid sequences: peptide 1-TGF-β1 (Pep 1-TGF-β1): SEQ ID N 01; peptide 2-TGF-β1 (Pep 2-TGF-β1): SEQ ID N 02; peptide 3-TGF-β1 (Pep 3-TGF-β1) SEQ ID N 03; peptide 4-TGF-β1 (Pep 4-TGF-β1) SEQ ID N 04; peptide 5-TGF-β1 (Pep 5-TGF-β1) SEQ ID N 05; peptide 6-TGF-β1 (Pep 6-TGF-β1) SEQ ID N 06; peptide 7-TGF-β1 (Pep 7-TGF-β1) SEQ ID N 07; and peptide 8-TGF-β1 (Pep 8-TGF-β1) SEQ ID N 08. Such peptides showed important activity on iμMunological modulatory and regulatory pathways. Therefore, they can be used in the treatment and/or prophylaxis of diseases related to an immune disorder, such as: chronic or acute inflammatory, allergic and/or autoimmune diseases.
The peptides described in this patent application were selected using the Phage Display (PD) methodology. The selection was performed by evaluating the ability of said peptides to recognize the TGF-β1 receptor on A549 cells, using an ELISA assay. MaxiSorp 96-well microtiter plates were coated with 1×106 cells diluted in PBS and incubated overnight at 4° C. Cells were blocked with 3% PBS-BSA for 1 h at 37° C., washed once with PBS and incubated with pg/ml of each TGF-β1 mimetic peptide or 5 ng/ml of recombinant TGF-β1 for 1 h at 37° C. After washing, the plate was incubated for 1 h at 37° C. with anti-TGF-β1 antibody. The plate was washed and incubated with HRP-labeled anti-human IgG antibody for 1 h at 37° C. After washing, TMB substrate solution was added to the plate; the reaction was stopped by adding the 2N H2SO4 solution and read at 492 nm in a microplate reader.
Peptides with high affinity for cellular receptors were then chemically synthesized following the phage selection manual (Barbas, C F et al. Phage Display: a laboratory manual. Cold Spring Harbor Laboratory Press (2001)) and all of them were synthesized in a similar way so as to present Histidine and Alanine as the first and second amino acid residues, respectively, and a sequence of three Glycine and a Serine as the last four amino acid residues, as well as some peptides were synthesized with an identity or similarity of at least 85% between them, with the replacement, for example, of serine residues by cysteine. Such specific substitution modified the three-dimensional structure, inducing the formation and conformation of disulfide bridges, or not. Its three-dimensional structures were predicted and demonstrated herein by the figures as follows.
Peptide 1-TGF-β1 (Pep 1-TGF-β1), SEQ ID N 01, was synthesized by inducing the formation of disulfide bridges between cystine residues, and has its three-dimensional structure predicted in
Peptide 2-TGF-β1 (Pep 2-TGF-β1), SEQ ID N 02, was synthesized by inducing the formation of disulfide bridges between cystine residues, and has its three-dimensional structure predicted in
Peptide 3-TGF-β1 (Pep 3-TGF-β1), SEQ ID N 03, is free of disulfide bridges and has its three-dimensional structure predicted in
Peptide 4-TGF-β1 (Pep 4-TGF-β1), SEQ ID N 04, was synthesized by inducing the formation of disulfide bridges between cystine residues and has its three-dimensional structure predicted in
Peptide 5-TGF-β1 (Pep 5-TGF-β1), SEQ ID N 05, is free of disulfide bridges and has its three-dimensional structure predicted in
Peptide 6-TGF-β1 (Pep 6-TGF-β1), SEQ ID N 06, is free of disulfide bridges and has its three-dimensional structure predicted in
Peptide 7-TGF-β1 (Pep 7-TGF-β1), SEQ ID N 07, is free of disulfide bridges and has its three-dimensional structure predicted in
Peptide 8-TGF-β1 (Pep 8-TGF-β1), SEQ ID N 08, is free of disulfide bridges and has its three-dimensional structure predicted in
Therefore, the synthesized peptides comprising or consisting of one of SEQ IDs No. 01, 02 or 04 may present three-dimensional formation with disulfide bridges. The synthesized peptides comprising or consisting of one of the SEQ IDs Nos. 03, 05, 06, 07 and 08 have linearized three-dimensional conformation, free from disulfide bridges. (
In addition, Pep 1 and Pep 5 peptides; Pep 2 and Pep 3 peptides; and Pep 4 and Pep 6 peptides; were synthesized with an identity or similarity of at least 85% between them, respectively. The amino acid residues at positions three (3) and eleven (11) were substituted from Serine in peptides Pep 3, Pep 5, and Pep 6, to Cysteine in peptides Pep 1, Pep 2 and Pep 4.
Peptide structures were predicted according to each modification made (
Although the peptides have different structures, the DLS result shows that all peptides are found as monomers with hydrodynamic radius between 0.58 nm (Pep1) and 2.94 nm (Pep7) (
Once the selected peptides were synthesized, several assays were carried out to determine the activity of such peptides. It is noteworthy here that both in the examples that we will demonstrate below and in the figures attached here, all peptides in general and in different concentrations showed surprising and positive results in modulating the immune, inflammatory and allergic response. However, we will highlight here, throughout the description, just as an example, those results that were statistically more significant. This does not exclude any other results that have not been expressly mentioned in the examples throughout the descriptive report, but which can be directly verified in the figures.
To investigate whether the synthesized peptides could bind to cell receptors, an ELISA assay was performed on cells of the A549 line using commercial anti-TGF-β1 antibody.
All eight tested peptides were able to recognize and bind to receptors on the cell surface of A549 cells. The synthesized peptides Pep 1-TGF-β1, Pep 2-TGF-β1 and Pep 3-TGF-β1, bound to cell receptors, were recognized by the anti-TGF-β1 antibody, in the same way that the antibody recognized the TGF-β1 recombinant molecule. However, the peptides Pep 4-TGF-β1, Pep 5-TGF-β1, Pep 6-TGF-β1, Pep 7-TGF-β1 and Pep 8-TGF-β1, also bound to cell receptors, were recognized as statistically different by the antibody, compared to recombinant TGF-β1 [Pep 4 (P<0.05), Pep 5 (P<0.005), Pep 6 (P<0.005), Pep 7 (P<0.005) or Pep 8 (P<0.00<0.05)] (
To investigate the ability of peptides to bind TFGβRII and activate the SMAD-dependent pathway, HEK-blue-TGFβ cells were treated with peptides at 0.1 μM, 1 μM, 10 μM and 100 μM for 12 hours (
The assay was performed in such a way that approximately 2.5×104 cells/well (100 μL) were treated with 100 μL of all synthetic peptides at different concentrations (100 μM, 10 μM, 1 μM and 0.1 μM) while rTGF-β1 (recombinant antibody) at 100 ng/ml was used as a positive control. After incubating the cells for 12, 24 and 48 hours at 37° C. in 5% CO2, 20 ml of supernatant was transferred to a Greiner plate to which 80 ml of Quanti-blue solution was added. The plate was incubated for 1 hour at 37° C. in 5% CO2 and the production of SEAP (secreted alkaline phosphatase) was detected at 655 nm. All tests were performed in triplicate.
Two different cell treatments were performed using Jurkat cells to analyze NFkB expression after 12, 24 and 48 hours. In the first test, Jurkat cells were treated only with peptides (no pathway activating molecule) at different concentrations to analyze the ability of each peptide to induce NFkB expression (Figure not shown). None of the tested concentrations induced any type of pathway activation. According to this result, cells were pretreated with peptides for 1 hour followed by TNF-α stimulation (
Peptide pretreatment was not able to reduce NFKB activation after 12 hours of treatment (
Finally, some peptides were able to reduce the NFKB pathway when cells were pretreated with TNF-α followed by the addition of the peptide after 12, 24 and/or 48 hours (
The assay was carried out in such a way that a total of 3×105 cells/well (100 pl) was pretreated with 100 ml of a composition comprising one of the synthetic peptides in concentrations of 100 μM, 10 μM, 1 μM, and 0.1 μM for 1 hour at 37° C. in 5% CO2, followed by stimulation with TNF-α (200 ng/ml) for 12, 24 and 48 hours. The same stimuli were applied to cells pretreated with TNF-α (200 ng/ml) for 1 hour at 37° C. in 5% CO2 followed by stimulation with peptides at 100 μM, 10 μM, 1 μM and 0.1 μM for 12, 24 and 48 hours. Then, 50 mI of supernatant was transferred to a white plate and Quanti-Luc reagent was used to measure the luciferase product. As a control, the peptides were tested at the same concentrations, without any pathway activating molecule, to investigate whether they could activate the NFkB pathway. TNF-α (200 ng/mL) was used as a positive control and rIL-10 (0.4 ng/mL) as a negative control. a (200 ng/mL).
Similar to the treatment using Jurkat cells, peptides in compositions with different concentrations were also tested in HEK cells to investigate their ability to interfere with the TLR4 pathway.
To ensure that the peptides could not induce any activation when tested alone (no pathway activation molecule) only the peptides were added into the cell line and SEAP production was measured after 12 hours of treatment (Figure not shown). In fact, peptides at all concentrations did not activate the signaling pathway.
All peptides were able to reduce the activation of the TLR4 pathway, particularly for some peptides at specific concentrations, and we obtained statistically significant results such as: Pep 1 (10 μM) (P<0.05), Pep 5 (1 μM and 10 μM) (P<0.05 and P<0.005), Pep 6 (1 μM) (P<0.005) and Pep 7 (0.1 μM; 1 μM and 10 μM) (P<0.005; P<0.05 and P<0.05) (
The assay was performed using HEK-Blue hTLR4 reporter cells (InvivoGen) to investigate the action of peptides in the TLR4 pathway. A total of 2.3×104 cells/well (100 ml) was pretreated with 100 ml of composition comprising at least one of the synthetic peptides at concentrations of 100 μM, 10 μM, 1 μM, and 0.1 μM for 1 hour at 37° C. ° C. in 5% CO2 followed by LPS stimulation at 100 ng/mL for 12 hours. The same stimuli were applied to cells pretreated with LPS at 100 ng/ml for 1 hour at 37° C. in 5% CO2, followed by stimuli with peptides at 100 μM, 10 μM, 1 μM and 0.1 μM for 12 hours. Stimuli were added in Quanti-blue medium and SEAP production was measured at 655 nm. As a control, peptides at the same concentrations were tested alone to investigate whether they could activate the TLR4 pathway and LPS was used as a positive control.
All synthetic peptides described in the present application, with affinity for the TGF-β receptor, exhibited an ability to modulate the autoimmune and inflammatory response, confirming that they can be used in the treatment of chronic or acute inflammatory, allergic and/or auto-inflammatory diseases. In addition, results presented herein demonstrate the potential for association of these peptides, since they presented distinct and, in several cases, complementary responses regarding the concentration and exposure time with the best response. Therefore, they can be used alone or in any combination thereof, in compositions to prepare drugs or immunogenic compositions for the treatment and/or prophylaxis of diseases in which there is an immunological disorder, more particularly in which diseases related to immunological disorders are: chronic or acute inflammatory, allergic and/or autoimmune diseases.
Since all peptides responded, we selected as an example Pep 2-TGF-β1 to show additional data that deepens our knowledge. Such results can be extrapolated to other synthetic peptides with affinity to the TGF-β receptor synthesized and demonstrated in the present patent application.
First, we tested the ability of the synthesized peptides to recognize TGF{circumflex over ( )}RII on the surface of Jurkat cells, immortalized human T lymphocytes, using ELISA. The recombinant TGFβ (rTGFβ1) was used as a positive control. In the example shown here, Pep 2 had higher levels of reactivity to TGF-βRII than rTGF-β1 (
Together, these results indicate that Pep 2-TGF-β1, as well as the other peptides synthesized in this application, are also able to modulate the allergic inflammatory microenvironment in vitro.
We then tested the ability of the synthesized TGF-β1 mimetic peptides to modulate the in vivo immune response during allergic sensitization to Phl p 5. We selected as an example Pep 2-TGF-β1 to show the results. Intradermal immunization with the recombinant Phl p5 allergen for 44 days (
To determine the number of splenic cells producing IFN-g, IL-4 and IL-10 in all groups of mice, an ELISPOT assay was performed. Immunization with Phl p 5 resulted in high induction of IFN-g and IL-4 release in allergen stimulation. In this example, in mice treated with Pep 2-TGF-β1 (in
To investigate the influence of synthesized peptides mimetic to TGF-β1 on Treg induction specific to Phl p5 during allergic sensitization, we analyzed splenocytes from BALB/c mice using flow cytometry. We sought to investigate CD4+CD25+Foxp3+cells expressing GATA3, CTLA4 or KI67, as these factors are necessary for the survival and function of Treg cells. Control and sensitized mice and Phl p5 had comparable levels of Treg cells expressing the factor GATA3 (16.1% and 16.2% of total T cells, respectively), whereas, in the example shown here, mice treated with the Pep 2-TGF-β1 (in
TH2 lymphocytes produce cytokines such as IL-4, IL-5 and IL-13 and lead to the class shift of immunoglobulin production from B cells to IgE, which triggers the allergic reaction. To investigate whether synthesized peptides mimetic to TGF-β1 can modulate TH2 polarization in the presence of grass pollen extract or rPhl p 5, we used an IL-4 reporter mouse. 4get mice express GFP as part of a bicistronic IL-4-IRES-GFP mRNA, allowing the identification of cells expressing IL-4 in situ during an allergen-induced TH2 response. In this experiment, we use skin-draining inguinal lymph node cells to monitor the TH2 response. In the example described here, flow cytometry analysis of GFP+CD4+ lymphocytes isolated from 4get mice 5 days after immunization (
Synthetic Peptides with Affinity for the IL-10 Receptor
The present patent application describes 4 synthetic peptides with affinity to the Interleukin-10 receptor which comprise or consist of the amino acid sequences: peptide 1-IL-10 (Pep 1-IL-10): SEQ ID 09; peptide 2-IL-10 (Pep 2-IL-10): SEQ ID NO: 10; peptide 3-IL-10 (Pep 3-IL-10) SEQ ID NO: 11; peptide 4-IL-10 (Pep 4-IL-10) SEQ ID NO: 12. Said peptides showed important activity on immunological modulatory and regulatory pathways. Therefore, they can be used in the treatment and/or prophylaxis of diseases related to an immune disorder, such as: chronic or acute inflammatory, allergic and/or autoimmune diseases.
The peptides described in this patent application were selected using the Phage Display (PD) methodology. They were randomly obtained after three rounds of selection using a PhD-7mer and PhD12 random peptide library. Selection was performed using the J774 cell line; for this, a competitive elution with rlL-10 (11-10 recombinant) was adopted to select peptides with affinity to the IL-10 receptor.
A pre-screening was performed on Peripheral Blood Mononuclear Cells (PBMC) using phage supernatant to select phage from the PhD-7mer and PhD12 libraries.
The most reactive phages were purified and tested against PBMC to confirm the ability of selected phages to bind to cell receptors.
Peptides with high affinity for cell receptors were then chemically synthesized following the phage selection manual (Barbas, C. F. et al. Phage Display: a laboratory manual. Cold Spring Harbor Laboratory Press (2001)) and their three-dimensional structures were predicted. All of them were synthesized to present Histidine as the first amino acid residue, and a sequence of three Glycine and a Serine as the last four amino acid residues.
Peptide 1-IL-10 (Pep 1-IL-10). SEQ ID NO 09, has its three-dimensional structure, with disulfide bridges, predicted in
Peptide 2-IL-10 (Pep 2-IL-10). SEQ ID NO 10, has its three-dimensional structure, without disulfide bridge, predicted in
Peptide 3-IL-10 (Pep 3-IL-10). SEQ ID NO 11, has its three-dimensional structure predicted in
Peptide 4-IL-10 (Pep 4-IL-10). SEQ ID NO 12, has its three-dimensional structure predicted in
Pep 1-IL-10 and Pep 2-IL-10 were synthesized with an identity or similarity of at least 85% between them. The amino acid residues at positions three (3) and eleven (11) were substituted from Serine in Pep 2 to Cysteine in Pep 1. Therefore, Pep 1-IL-10 is the only peptide that was synthesized with a disulfide bridge, being its three-dimensional structure distinct from the structure of Pep 2-IL-10 and more compact compared to Pep 3-IL-10 and Pep 4-IL-10. In
The aggregation behavior of Pep 1-IL-10, Pep 2-IL⋅10, Pep 3-IL-10 and Pep 4-IL-10 peptides in solution was determined by Dynamic Light Scattering (DLS), as can be seen in
Once the selected peptides were synthesized, several assays were carried out in order to determine the activity of these peptides. It is noteworthy that in the examples that we will demonstrate below and in the figures attached here, all peptides in general and in different concentrations showed surprising and positive results in modulating the immune, inflammatory and allergic response. However, we will highlight here, throughout the description, just as an example, those results that were statistically more significant. This does not exclude any other results that have not been expressly mentioned in the examples throughout the descriptive report, but which can be directly verified in the figures.
In order to investigate the ability of synthetic peptides with affinity to the Interleukin-10 receptor to interfere in the NFkB, IFN and TLR4 pathways, reporter cell lines were used. To analyze whether the isolated peptides (without activation molecules) could induce any type of activation, the isolated peptides were tested in cell lines during 12, 24 and 48 hours of treatment. In all reporter cells and at all times tested, synthetic peptides with affinity for the IL-10 receptor did not induce any type of activation when they were alone, without any inflammatory environment to induce activation of the NFKB, IFN or TLR4 pathways. As an example,
The assay was then carried out using TNF-α as activation molecule not only in the control, but after a pretreatment with the synthesized peptides Pep 1-IL10, Pep 2-IL10, Pep 3-IL10 and Pep 4-IL10, at different concentrations. All peptides were able to modulate NFKB activation after 12, 24 and 48 hours, with different results depending on the concentrations used. Pretreatment with Pep 1-IL10 at all concentrations tested was, with statistical significance, able to decrease NFKB activation after 12, 24 and 48 hours (P<0.0005) of stimulation with TNF-α (
Another assay was then performed in which cells were pretreated with TNF-α and the synthesized peptides Pep 1-IL10, Pep 2-IL10, Pep 3-IL10 and Pep 4-IL10, at different concentrations, were added later. All peptides were able to modulate NFKB activation after 12, 24 and 48 hours, with different results depending on the concentrations used. The most significant results will be described below. For example: Pep 1 at concentrations of 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM (P<0.05), 0.05 μM and 0.001 μM (P<0.0005) decreased pathway activation analyzed after 12 hours of treatment (
The analysis of the IFN signaling pathway demonstrated that the synthesized peptides Pep 1-IL10, Pep 2-IL10, Pep 3-IL10 and Pep 4-IL10 were able to modulate the activation of the IFN signaling pathway after 12, 24 and 48 hours, with different results depending on the concentrations used. The most significant results will be described below. For example, pathway activation was diminished when Jurkat cells were pretreated with 10 μM Pep 1 (P<0.005) for 12 hours (
HEK cells were used to investigate the ability of the synthesized peptides Pep 1-IL10, Pep 2-IL10, Pep 3-IL10 and Pep 4-IL10 to modulate the activation of the TLR4 signaling pathway after 12 hours of treatment. All peptides were able to modulate the activation of the TLR4 signaling pathway after 12 hours, with different results depending on the concentrations used. The most significant results will be described below. For example, pretreatment with the peptides demonstrated that Pep 1 at 0.005 μM (P<0.05); Pep 2 at 100 μM (P<0.05), 0.1 μM (P<0.0005); Pep 3 at 100 μM (P<0.05), 5 μM (P<0.05), 1 μM (P<0.005), 0.5 μM (P<0.005), 0.1 μM (P<0.005) and 0.005 μM (P<0.05); and Pep 4 at 100 μM (P<0.05), 10 μM (P<0.005), 5 μM (p<0.05) and 1 μM (P<0.05) significantly reduced pathway activation (
In the assay shown by
Murine bone marrow dendritic cells were isolated from mice to analyze the effect of synthesized peptides Pep 1-IL-10, Pep 2-IL-10, Pep 3-IL-10 and Pep-4-IL-10, at two concentrations, 1 μM and 10 μM, in the modulation of cell activation. After 24 hours of treatment with a composition comprising at least one of the peptides in the concentration After 24 hours of treatment with a composition comprising at least one of the peptides at the desired concentration, cell analysis showed that all four synthesized peptides showed responses at different levels and were able to interfere with the co-stimulatory molecules present on the surface of dendritic cells, being the Pep 1 and 2 peptides the ones that presented the most significant responses. For example, Pep 1 at 1 μM decreased CD86+ expression (P<0.005), and at 10 μM it also decreased CD86+ expression (P<0.05). Pep 2 at 1 μM reduced the expression of CD86+ and CD40+ on the cell surface (P<0.005; P<0.05, respectively) (
In the assays, CD11c+ CD86 cells (
Since all four synthetic peptides described in the present application, with affinity to the IL-10 receptor, exhibited an ability to modulate the autoimmune and inflammatory response, suggesting that they can be used in the treatment of chronic or acute inflammatory diseases, allergic and/or autoimmune, we selected as an example Pep 1-IL-10 to show the additional data that deepen the knowledge. Furthermore, the results presented here demonstrate the potential for association of these peptides, since they showed distinct and, in many cases, complementary responses regarding concentration and exposure time with better response. Therefore, they can be used alone or in any combination thereof, in compositions to prepare drugs or immunogenic compositions for the treatment and/or prophylaxis of diseases in which there is an immune disorder, more particularly wherein the diseases related to the immune disorder are: chronic or acute inflammatory, allergic and/or autoimmune diseases.
Such results can be extrapolated to other synthetic peptides with affinity to the IL-10 receptor synthesized and demonstrated in the present patent application.
In order to show the ability of synthesized peptides to modulate the activation of dendritic cells, Pep 1 to 10 μM was chosen and the proliferation of T cells from allergic patients was measured after 24 hours of treatment. Pep 1 at this concentration decreased T cell proliferation (P<0.005) (
The effect of IL-10 mimetic peptides on IgE-mediated release was examined using rat basophilic leukemia cells transfected with the human FIϵRI IgE receptor (hRBL). As an example, we selected as an example Pep 1-IL-10 to show the data obtained. The hRBL cells were sensitized with serum from seven patients allergic to birch pollen. Cells were stimulated with the antigen and its interaction with immobilized IgEs caused a release of b-hexosaminidase.
When cells were treated with a composition comprising Pep 1-IL-10 at 1 μM (
After 24 hours of treatment, the levels of IL-6, MCP-1 and TNF-α produced were measured. No interference with the production of IL-10, IFN-g or IL12p70 was observed (data not shown). In the example shown here, cells treated only with the synthetic peptide Pep 1-IL-10, without LPS stimulation to induce an inflammatory response, were unable to induce any response (data not shown). On the other hand, when cells were treated with a composition comprising Pep 1-IL-10 followed by incubation with LPS (1 mg/ml) for 24 hours to induce an inflammatory response, the peptide was able to decrease IL production-6, MCP-1 and TNF-α.
Pep1-IL-10 (1 μM, 10 μM and 100 μM, 1 μM P<0.05 and 10 μM and 100 μM P<0.0005) significantly reduced IL-6 levels (1 μM and 10 μM, P<0.05) (
Studies show consistent immunoregulatory actions of peptides mimetic to TGF-β and mimetic to IL-10 synthesized and described herein. In an inflammatory environment, such peptides were able to modulate several important pathways in regulatory actions to suppress an inflammatory response. In the case of TGF-β, mimetic peptides, it positively modulated IL-10. We therefore investigated the combination of TGF-β and IL-10 mimetic peptides.
The ability of synthesized TGF-β1 and IL-10 mimetic peptides to modulate the specific IgE immune response to Bet v 1 was investigated by mediator release assays using RBL-2H3 cells passively sensitized with IgE antibodies from mice immunized with extract of birch pollen. Bet v 1 was able to induce IgE production, as seen in the high levels of basophil degranulation in the group of mice immunized with birch pollen extract (
The combination of TGF-β1 and IL-10 mimetic peptides suppresses IgE-specific basophil degranulation.
For immunization, female BALB/c mice (6-10 weeks old) were used in this study. The immunization scheme was performed as described in
The ability of TGF-β1 and IL-10 mimetic peptides to interfere in the modulation of the number of IFN-g, IL-4 and IL-10 producing spleen cells in all groups of mice was investigated by ELISPOT. Immunization with birch pollen extract resulted in high induction of production of IFN-g (P<0.001) and IL-4 (P<0.001) after stimulation with Bet v 1. Mice treated with peptides, in this example with the combination of Pep 2-TGF-β1 with Pep 1-IL-10, had significantly lower levels of IFN-g (P<0.001;
Splenocytes restimulated with Bet v 1 and peptides produced significantly lower levels of IFN-g (
For this experiment, spleens were aseptically removed immediately after mice were sacrificed, lysed and centrifuged for 5 min in 1 ml of minimal essential culture medium (MEM). Lysis of erythrocytes was performed by adding 5 ml of lysis buffer (150 μM NH 4 Cl, 10 μM KFIC03, 0.1 μM Na2EDTA in ddFI20, pH 7.2) for 5 min at room temperature. White blood cells were separated by centrifugation at 300×g for 5 min at room temperature. Cells were resuspended in culture medium (MEM, 1% (v/v) of heat-inactivated fetal bovine serum, 2 μM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 20 μM HEPES, 1 μM sodium pyruvate, 2 M 2-mercaptoethanol and 1*non-essential amino acids) and used in subsequent experiments.
For detection of IFN-g, IL-4 and IL-10 after stimulation of splenocytes with Bet v 1; MultiScreen filter plates were activated with 70% ethanol for 10 min, washed three times with PBS, and 2 pg/ml of anti-mouse IFN-g, anti-mouse IL-4 or anti-mouse IL-10 was added during night at 4° C. in a humid chamber. Plates were washed three times with PBS and incubated for 2 h with blocking solution (culture medium supplemented with 5% fetal bovine serum) in a humid chamber at RT. Splenocytes were diluted to a density of 2×105 in culture medium, in the presence of Bet v 1 diluted to 20 pg/ml and incubated for 48 hours at 37° C. and 5% CO2. After three washes with PBS supplemented with 0.1% Tween-20; detection antibodies IFN-g, IL-4 and IL-10 were added to the plates and incubated for 2 h at room temperature. After three washes, HRP-conjugated streptavidin was added to the plates and incubated for 1 h at room temperature. After four washes, TMB substrate was added to each plate for 5 min before stopping by washing with ddhteO. Immunospot counting was performed using ImageJ software.
The ability of TGF-β and IL-10 mimetic peptides to modulate cytokine production was investigated using splenocytes and serum from mice immunized with only birch pollen extract (125 pg) (results were identified in
The ability of TGF-β and IL-10 mimetic peptides to modulate basophil degranulation was investigated using RBL-2H3 cells sensitized with serum from mice immunized only with birch pollen extract (125 pg) (results were identified in
The present patent application demonstrates the ability of new synthesized peptides mimetic to TGF-β1 and mimetic to IL-10 to modulate the response of several important pathways in the regulation of an autoimmune and/or inflammatory process.
In addition, although the potential of the new synthesized peptides described in this patent application in modulating an allergic or immune inflammatory response has already become clear, we deepened the studies and showed the regulatory role of synthesized peptides in modulating an allergic inflammatory response. The peptides were able to modulate basophil degranulation after antigen stimulation, to modulate TH1 and TH2 profile responses through the modulation of cytokines and antibodies, acted in the differentiation of induced Treg cells and affected other important cellular events that promote the exacerbation of the allergic or inflammatory microenvironment.
Allergen-specific immune modulation is an essential process to prevent the development of allergy, wherein IgE plays a key role. Thus, inhibition of IgE production is a crucial event to suppress allergic reactions. In our study, TGF-β and IL-10 mimetic peptides efficiently inhibited basophil degranulation. The ability of these two cytokines to modify the production of IgE-like antibodies in relation to non-inflammatory antibody isotypes has already been reported. However, we demonstrate that the new synthesized peptides described in the present application, which are small molecules, and which can mimic the action of specific large proteins that are able to induce the same or better response as expected with the natural molecule or compared to other synthetic molecules already described. Furthermore, we concluded that there is a surprising synergistic effect with the combination of TGF-β and IL-10 mimetic peptides described in the present invention. Additionally, even more surprising was the synergistic effect when the combination of peptides was associated with the allergen of interest. Therefore, we conclude that the treatment with the combination of TGF-β and IL-10 mimetic peptides had a synergistic effect in modulating an allergic or immune inflammatory response, as well as being able, when in association with an allergen of interest, to significantly reduce the basophil degranulation.
As IFN-g is a cytokine that contributes directly and indirectly to the differentiation of the Th1 profile, while IL-4 is a cytokine that promotes the differentiation of the Th2 profile, the downregulation of these two cytokines indicates a modulation of Th1 or Th2 polarization, essential event to control the inflammatory/allergic response. IL-4 is known to induce IgE class switching in mice (27), thus it may help to explain our previous result, showing that the peptides suppressed IgE-mediated degranulation of basophils. We hypothesize that inhibition of IL-4 consequently suppresses IgE production. On the other hand, the upregulation of IL-10 indicates that the peptides had an anti-inflammatory action, since this cytokine plays a crucial and often essential role in the prevention of inflammatory pathologies. Therefore, we conclude that treatment with TGF-β and IL-10 mimetic peptides modulated the production of cytokines that play a critical role in controlling the immune response.
Furthermore, what was observed in this study, and which is unexpected, was the fact that mice immunization with the combination of mimetic peptides to TGF-β and IL-10, when performed together with the antigen of interest (in the example herein, the birch pollen extract) has resulted in an improved effect both in modulating the allergic response (suppression of basophil degranulation) and in modulating the production of inflammatory cytokines.
Therefore, the present patent application describes synthetic peptides with affinity to the TGF-β receptor and synthetic peptides with affinity to IL-10, to be used in combination of at least one TGF-β mimetic peptide with at least one IL-10 mimetic peptide, in a pharmaceutical composition for use in the treatment and/or prophylaxis of diseases related to immunological disorder, more particularly, chronic or acute inflammatory and/or allergic, and/or autoimmune diseases.
The present application also describes pharmaceutical compositions comprising at least one TGF-β mimic peptide with at least one IL-10 mimetic peptide, plus at least one pharmaceutically acceptable carrier or compound. More particularly the composition may further comprise as a pharmaceutically acceptable compound an allergen of interest. The pharmaceutical composition is for use in the treatment and/or prophylaxis of diseases related to immunological disorder, more particularly, chronic or acute inflammatory and/or allergic, and/or autoimmune diseases.
The present application also describes the use of the synthetic peptides or pharmaceutical compositions described herein, for the preparation of a medicament or immunogenic composition for use in the treatment and/or prophylaxis of diseases related to immunological disorder, more particularly, chronic, or acute inflammatory diseases and/or allergic, and/or autoimmune.
Additionally, the present application also describes methods of treatment and/or prophylaxis of diseases related to immunological disorder which comprises administering to a subject a pharmaceutically effective dose of at least one recombinant peptide with affinity for the TGF-β receptor and at least one peptide recombinant with affinity to the Interleukin-10 receptor, or a pharmaceutical composition comprising the combination thereof. More particularly since it also comprises jointly administering an allergen of interest.
Although the present patent application has described the subject matter of the present invention with a certain degree of detail by way of illustration and example for purposes of clarity and understanding, it will be evident that certain changes and modifications may be practiced within the scope of the appended claims.
The examples described in this report are not limiting, allowing a person skilled in the art to change some aspects or components of the present invention, equivalent to the synthetic peptides, compositions or uses described herein, without departing from the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2019 007048 0 | Apr 2019 | BR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/BR2020/050118 | 4/6/2020 | WO | 00 |
