ES2394331B2 - USE OF AN ISOLATED ANTIGENIC PEPTIDE DERIVED FROM THE S100-BETA PROTEIN NOT UNDER A CLASS II MHC MOLECULE IN THE PREVENTION, TREATMENT, DIAGNOSIS AND / OR FOLLOW-UP OF TYPE DIABETES - Google Patents

Abstract

Use of an isolated antigenic peptide derived from the S100-beta protein not bound to an MHC class II molecule in the prevention, treatment, diagnosis and / or monitoring of type I diabetes. # The present invention relates to the use of a peptide isolated antigen derived from the S100-beta protein that is not bound to an MHC class II molecule in the manufacture of a drug for the prevention, treatment, diagnosis and / or monitoring of type I diabetes.

Description

Use of an isolated antigenic peptide derived from the S100-beta protein not bound to an MHC class II molecule in the prevention, treatment, diagnosis and / or monitoring of type I diabetes.

Field of the Invention

The present invention relates to an antigenic peptide derived from the S100-beta protein, as well as the method for its identification and its use in the prevention, treatment, diagnosis or monitoring of type I diabetes.

State of the art

Human type I (T1D) diabetes is an autoimmune disease characterized clinically by the lack of regulation of glucose levels and that in the medium-long term causes neurological and vascular complications. T1D is one of the most frequent autoimmune diseases among the Caucasian population. Only in Spain, between 10-20 new cases are diagnosed every 100,000 children under 14 years of age each year, a prevalence that is increasing, which shows that 1 in every 1,000 children at that age is diabetic, so there are About 10,000 diabetic children in our country.

T1D is characterized by an insulin deficiency, making patients depend on daily injections of exogenous insulin for survival. Before the onset of clinical symptoms such as hyperglycemia, there is an asymptomatic preclinical period where insulin-producing cells are progressively destroyed. The destruction of these insulin-producing cells, the beta cells present in the so-called pancreatic islets of Langerhans, is associated with the infiltration of these islets by lymphocytes and other cells of the patient's immune system. These cells respond against their own proteins expressed by beta cells, such as insulin itself or its precursor, pre-proinsulin.

The islets of Langerhans, where insulin-producing cells are found, form a differentiated structure within pancreatic tissue and are surrounded by a layer of nerve-type cells called peripheral Schwann cells (pSC) ("peri islet Schwann cells") whose functions are not clear but that do express a set of proteins or antigens that clearly differentiate them from the cells that make up the pancreatic islets, including beta cells. In the murine model of human T1D, the mouse NOD (“Non-Obese Diabetic mice”), which develops a metabolic syndrome that shares many characteristics with human T1D such as hyperglycemia and insulin deficiency, these pSC cells are destroyed by lymphocytes of the animal's immune system. Some of these lymphocytes respond against antigens expressed exclusively in these pSC cells such as glia acid fibrillary protein (GFAP) or neurotropic factor S100-beta. In human patients with T1D, the presence of autoantibodies directed against GFAP and S100-beta as well as specific self-reactive lymphocytes for these two proteins has been demonstrated, indicating that in these patients the islet pSC cells have also been destroyed by the immune system . In the NOD mouse it has also been shown that the lymphocytic infiltration of the islets begins with the so-called peripheral infiltration of the islets (“peri-islet infiltration”) during which destruction of the mantle of pSC cells surrounding the islets themselves occurs. . Then and once this mantle has been destroyed, the lymphocytes can access the islets, where they selectively destroy the insulin-producing beta cells.

The fact that pSC cells appear to be the initial objective of the autoimmune response against pancreatic islets and that their slowdown, regulation and / or absolute detention would keep the pancreatic islets intact, together with the fact that there is no therapy today that allows treating human T1D, point out the importance of discovering new methods and / or molecules that allow treating the disease at times that allow to keep the largest number of intact islets and for as long as possible.

There is a lot of previous experience that shows that CD4 + lymphocytes are involved in the development of T1D. Histological analyzes of islets obtained from affected individuals show infiltrations by CD4 + lymphocytes. In animal models of T1D the disease can be transferred to a healthy animal using CD4 + lymphocytes from a sick animal. In addition, there is a genetic association between the development of T1D and certain major histocompatibility system (MHC) class II molecules, critical for CD4 + lymphocytes to develop and be stimulated against their target. Finally, it is possible to detect the presence of activated CD4 + lymphocytes in peripheral blood of individuals who have developed the disease.

There is a growing interest in defining protein segments, called peptide epitopes, recognized by CD4 + lymphocytes involved in the autoimmune response against pSC cells. The identification of these epitopes is important to understand the mechanisms that trigger the disease, for the development of laboratory tests that allow to follow the damage to the islets and to design therapies that allow to slow down or stop the disease.

Peptide epitopes derived from exogenous antigens are presented to CD4 + T lymphocytes after a

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series of molecular events referred to as a whole as processing and natural presentation. For this, the exogenous antigens or proteins are captured by cells of the immune system called antigen presenting cells (APC). Once internalized, the corresponding antigen is fragmented by combining various chemical and enzymatic processes, generating peptides that bind to MHC class II molecules that are exported to the surface of the antigen presenting cell. The natural processing and presentation of exogenous antigens results in overlapping groups of peptides (Figure 1) that bind to the MHC class II molecule by the same "binding motif" but possess different amino and carboxyl ends. The amino acids at both ends, although not part of the reason for binding to the histocompatibility molecule, can have important effects both on the binding affinity to the histocompatibility molecule and on the activation of clones of CD4 + T cells.

In animal models such as the NOD mouse, APCs have been shown to capture proteins from the pancreas and carry them to lymph nodes that drain this organ to stimulate there autoreactive lymphocytes that recognize peptides derived from these proteins and bound to specific MHC molecules. Once stimulated, these autoreactive lymphocytes are directed to the pancreas, where they proceed to eliminate those cells that express the antigen from which that peptide epitope was derived. Although it is known that the S100-beta protein is an objective of the autoimmune process in human T1D from studies that demonstrate the presence of autoantibodies and autoreactive lymphocytes against the complete protein in diabetic patients, there are currently no data on epitopes peptides generated from this protein and that are recognized by CD4 + lymphocytes in patients with T1D.

Peptide epitopes generated in vivo by an APC from an exogenous protein and capable of stimulating a CD4 + lymphocyte therefore consist of several peptides of a variable length but that share a common area. This common zone or common amino acid sequence contains the amino acids that determine the so-called "binding motif" to the specific MHC molecule, or what is the same, the amino acids that allow said peptide to anchor to the MHC molecule. This set of peptides derived from a specific area of the protein and that share a common zone form a set of overlapping peptides (Figure 1), from which a consensus peptide epitope could be synthesized that includes that common area and that includes amino acids from the ends of some or all of the peptide epitopes generated from that area of the protein.

Different methods have been used to try to identify peptide epitopes presented by MHC class II molecules and recognized by CD4 + T lymphocytes. The following experimental approaches are among those commonly used:

one.

Prediction "in silico". MHC class II molecules tend to bind peptides that have certain amino acids at specific positions in the linear sequence, the so-called "binding motifs" cited above. There are computer applications available that allow searching in a protein for segments of the linear sequence of amino acids that have this "motif of binding" and therefore have theoretical possibilities of being presented to CD4 + T lymphocytes. However, this approach presents a series of drawbacks in identifying peptide epitopes bound to MHC class II molecules. First, it does not provide any information about whether said peptide is going to be actually generated in vivo in an APC. Secondly, from a protein many peptides can be generated that have possibilities of binding to a MHC class II molecule, generating many false positives. Third, the length of said peptide epitopes is variable, so that although a "binding motif" is identified, the length of the peptide generated in vivo in an APC is not known.

2.

Generation of CD4 + T lymphocytes. This approach involves the cloning of CD4 + T lymphocytes that respond against the protein and by using synthetic peptides overlapping each other, try to identify which epitope (s) it reacts against. This technique also has certain drawbacks, among which we can mention: a) the peptides synthesized in vitro again do not have to coincide with the epitope generated in vivo, and b) is a technique that has proven to be successful in those situations where it exists a high number of CD4 + T lymphocytes specific for said antigen, such as an acute viral infection, but it is much more difficult at the methodological level in autoimmunity or cancer, where the number of CD4 + T lymphocytes specific for a specific epitope is much lower.

3.

In vitro protein processing. A whole series of enzymes (proteases) capable of digesting proteins and generating peptides capable of binding to MHC class II molecules are known. However, the number of these enzymes involved in this process or their order of intervention in vivo in an APC on a particular antigen or protein is not known exactly and makes it very difficult to reproduce this process in vitro.

Four.

Synthesis of overlapping peptides. Another usual experimental approach when trying to identify peptides capable of stimulating CD4 + T lymphocytes is the synthesis of a library of overlapping peptides (Figure 2) in which the peptides have a specific length, overlap each other and cover the entire length. of protein. Once synthesized, these peptides are used as antigens to stimulate CD4 + T lymphocytes. This approach, however, does not identify naturally processed and presented peptide epitopes, since it does not determine which are the real ends of the peptides generated in the cell and which can influence in a way

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significant in the recognition of the epitope by CD4 + T lymphocytes. In addition, the response of CD4 + T cells against any of these overlapping peptides does not guarantee that said CD4 + T cell recognizes the natural epitope generated by an APC in vivo.

Therefore, to date there is no scientific study in humans that has identified S100-beta derived antigenic peptides, generated in vivo by a cell and that are subsequently bound to a MHC class II molecule such as the DRB1 * 0401 molecule ( DR4).

Human lymphocytes do not respond against the whole protein but against segments (peptides or peptide epitopes) generated from it; However, the determination of these segments is not obvious, nor can it be determined by simply reading the amino acid sequence of the protein or by generating libraries of arbitrary fragments of the protein.

The publication of Winer S. et al. (Nature Medicine, Vol. 9 (2): 198-205 (2003)) suggests treatment with S100-beta for diabetes, but uses the complete protein and no specific peptide epitopes. However, as mentioned in the previous paragraph, the lymphocytes of a diabetic patient do not respond against the complete protein but against peptides or peptide epitopes that must be generated from it and, again, the determination of said segments It is not trivial nor can it be determined by simply reading the amino acid sequence of the protein.

The S100-beta derived peptides described to date are based on mere fragmentation of the protein into segments of an arbitrary length and the determination of the presence of antibodies against said fragments. Patent application WO9801471, for example, describes epitopes that are used to generate monoclonal antibodies in mice, so that these antibodies subsequently determine the presence of the human S100-beta polypeptide in serum of patients with melanoma. To this end, S100-beta peptides are synthesized in this document, arbitrarily deciding their length with the intention of identifying those most useful for determining the presence of the human S100-beta polypeptide in a sample, but not those capable of generate an immune response in vivo. As stated above, it is impossible to predict by studying only the amino acid sequence of a protein which or what are the peptide epitopes capable of generating an immune response in vivo, that is, that they are generated by an antigen presenting cell, bind to a MHC class II molecule and be presented on the surface of said cell so that the MHC-peptide molecule assembly performs its function (for example, activating the immune response against said protein or generating a modulating response that reduces or eliminates said immune response against protein). This impossibility derives, in part, from the fact that MHC class II molecules have a cleft in which more or less large segments of a protein fit (for example, S100-beta), a segment that is then “filed or trimmed” by both ends by proteolytic enzymes present inside the cell. However, the number, order of action on the peptide (if such an order exists) and if a specific and specific group of these enzymes acts on each particular protein, is not known exactly today. For this reason, an antigen presenting cell does not generate a single peptide epitope (peptide, subfragment) of a region of a protein but instead generates several epitopes of the same region that can be grouped into "overlapping groups", mentioned above (Figure 1) . The peptide epitopes of an "overlapping group" share the same "common nucleus", which interacts directly with the cleft of the corresponding histocompatibility molecule, and variable ends, impossible to predict and which are generated, as mentioned, by the action of one or several proteases inside the cell. In patent application WO9801471, a peptide library is manufactured by segmenting S100-beta into arbitrary fragments of 31 amino acids, providing two fragments obtained from said library (SEQ. ID. NO. 4 and 6 in WO9801471; see Figure 18 for homology to the sequences of the present invention), on the basis of which subfragments are proposed (SEQ. ID. NO. 5 in WO9801471) or larger fragments encompassing those sequences (SEQ. ID. NO. 2 and 3 in WO9801471; see Figure 18 for homology with the sequences of the present invention), completely arbitrarily, but without determining its immunogenicity. Therefore, WO9801471 provides arbitrary fragments of S100-beta that are used to determine the presence of antibodies against this protein in a sample of melanoma patients, but it does not determine which parts of the protein are actually involved in the immune response in a subject. , nor does it provide information about the involvement of these peptides in the immune response to T1D, so it is necessary to identify those peptides derived from S100-beta that trigger said response, so that it is possible to develop diagnostic and treatment systems for T1D much more sensitive and precise.

The document Tsui H. et al. (Diabetes, Vol. 57: 918-928 (2008)) identifies epitopes of GFAP and performs an immunotherapy treatment that significantly inhibits type I diabetes, emphasizing the importance of epitope identification in the development of tetramers. Tetramers made in vitro using a histocompatibility molecule and a peptide are not equivalent to each other because they serve to detect groups of immune cells that can have completely different roles within the same pathology, so that even if there were tetramers made with a molecule of determined histocompatibility and GFAP derived peptides, tetramers manufactured using S100-beta derived peptides would detect lymphocytes with completely different functions than the lymphocytes that would be detected by tetramers manufactured with GFAP derived peptides (Figure 3). In this document, GFAP peptide epitopes are predicted by using an algorithm, obtaining different epitopes (amino acid positions 79-87 and 253-261 in GFAP), which are then used to

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immunize mice. Of these, only one of the peptide epitopes (79-87) but not the other (253-261) produces an inhibition of type I diabetes, although both have been generated in the same way. Therefore, although it describes the use of one of the multiple algorithms used in the prediction of peptide epitopes, it does not provide useful epitopes to generate an immune response since some of the predicted epitopes do not work. This document also mentions that there are no algorithms to predict epitopes bound to one of the MHC class II molecules that possesses the animal model used in the described experiments, which is a problem in the identification of peptide epitopes using this method. In addition, although peptide epitopes are predicted for both MHC classes (class II through libraries and class I using algorithms), positive results are only presented in vivo when immunizing mice with peptide epitopes bound to MHC class I molecules, but not with those bound to MHC class II molecules, which highlights the difficulty of predicting or obtaining specific peptide epitopes presented by MHC class II molecules.

Finally, US patent application 2003/0054414 A1 also uses the GFAP protein for the diagnosis and treatment of type I diabetes, but it also does not identify specific peptide epitopes derived from the S100-beta protein that are presented to CD4 + lymphocytes and that generate an immune response from said lymphocytes.

Therefore, it is necessary to identify those specific peptide epitopes of the S100-beta protein that really intervene in the in vivo immune response that occurs in type I diabetes and that are potentially useful in the prevention, diagnosis, monitoring and treatment of this pathology .

Summary Description of the Invention

The present inventors have specifically identified the antigenic peptides (also referred to herein as peptide epitopes) generated in vivo after S100-beta is processed by an antigen presenting cell (APC) and which are present on the surface of said APCs. bound to an MHC class II molecule, isolating the peptides that really intervene in the immune response in vivo. Therefore, the present invention provides the specific fragments of the S100-beta protein that are generated during an immune response and that are capable of activating lymphocytes, thereby identifying which specific peptide epitopes are those that can be used for prevention, treatment , diagnosis and monitoring of type I diabetes.

The present invention also relates to a process for obtaining said antigenic peptide.

The present invention also relates to the use of said antigenic peptide in diagnosis systems of type I diabetes and in the manufacture of a medicament for the prevention, monitoring, diagnosis and / or treatment of type I diabetes.

Additionally, the present inventors have also observed that when the antigenic peptide of the present invention is not bound to the MHC class II, said peptide is useful in the manufacture of a medicament for the prevention, monitoring, diagnosis and / or treatment of type I diabetes.

Brief description of the figures

Figure 1. Overlapping groups of peptides resulting from intracellular processing of exogenous antigens. Class II histocompatibility molecules present on the cell surface peptides derived from the same region of the antigen, which have in common a group of amino acids that interact directly with amino acids of the histocompatibility molecule ("binding motif") (amino acids within the box) but which have different amino-and / or carboxy-terminal ends.

Figure 2. Libraries of overlapping peptides. They consist of the synthesis of peptides of a specific and identical length for all, which overlap each other in a specific number of amino acids and that cover the entire length of the antigen. A library consisting of four peptides of 15 amino acids in length overlapping each other in 12 amino acids and covering the entire amino acid sequence of the protein (upper sequence) is shown in the figure.

Figure 3. MHC tetramers made with different peptides are recognized by different T lymphocytes. An MHC tetramer made with a specific MHC molecule (1) but containing different peptides (2, squares or circles) are reagents that specifically detect different T lymphocytes (3), which may have opposite functions in one pathology. In addition, MHC tetramers can carry other components coupled as magnetic nanoparticles, for the detection of lymphocytes in vivo, or toxins for the specific elimination of those autoreactive lymphocytes that recognize the MHC-peptide complex, such as those that recognize tetramers containing S100 derived peptides. -beta. In addition, these MHC tetramers can be conjugated to other molecules or compounds to generate new diagnostic reagents (for example, attached to magnetic nanoparticles) or for disease treatment (for example, bound to cytokines or toxins).

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Figure 4. General scheme of the ELISPOT methodology. In this analysis, cell culture plates are coated with an antibody specific for the cytokine to be detected (1). Once the T lymphocytes have been activated using the corresponding antigen, they are added to the plate coated with anti-cytokine antibody so that in those places where there is a specific T lymphocyte and stimulated by the antigen, the cytokine to be detected will be captured. (2, dark cell). Once the cells have been removed, a second detection antibody coupled to an enzyme is added so that when a substrate is added, a color is generated in the place where the cytokine-specific antigen-producing cell that was to be detected was located (3). The presence of said cell is seen as a spot ("spot") on the plate (4), so that each spot represents a specific cell for the antigen used in the stimulation.

Figure 5. System used in the internalization of S100-beta in Priess cells. The Priess lymphoblast line (1) was sequentially incubated with a lectin obtained from Phytolacca americana (2) biotinylated in vitro (biotin = black circles), with avidin (3) and finally with purified S100-beta and biotinylated in vitro (4 ) (biotin = black circles). After incubation with S100-beta, the cells were incubated at 37 ° C so that they could internalize and process the antigen and present peptide epitopes on the cell surface bound to MHC DR4 molecules (5). The cells used as control were incubated with all elements except S100-beta.

Figure 6. Fractionation by high performance liquid chromatography (HPLC) of the peptides present in MHC class II DR4 molecules in Priess cells incubated or not with S100-beta. Peptides obtained from Priess cells incubated with S100-beta (upper chromatogram) or priess cells not incubated with S100-beta (lower chromatogram) were fractionated by C18 reverse phase column HPLC collecting fractions every approximately 30 seconds. Each fraction was subsequently analyzed by mass spectrometry. mAU: absorbance units.

Figure 7. Groups of overlapping peptide epitopes derived from S100-beta identified by mass spectrometry (MS). After comparing the MS spectra of each of the fractions obtained in the HPLC of peptides obtained from cells incubated with S100-beta with their corresponding fraction of control Priess cells (without incubating with S100-beta), 9 unique corresponding masses were identified with 3 overlapping peptide groups (AC groups) after the search for the amino acid sequence in Findpept (http://expasy.org/tools/findpept.html).

Figure 8. S100-beta consensus peptide epitopes synthesized with the information obtained from the three overlapping groups identified by mass spectrometry (MS). The consensus epitopes synthesized contain at least one reason for binding to the class II histocompatibility molecule DR4. As demonstrated in the examples of the present invention, these consensus peptides can be used in the in vitro identification of autoreactive CD4 + T cells in diabetic patients.

Figure 9. ELISPOT results for IL-10 of a healthy non-diabetic individual. A photograph of the results of IL-10 synthesis obtained from a healthy individual whose lymphocytes have been incubated with dimethyl sulfoxide (DMSO, solvent used in the dissolution of the peptides) is shown (upper row); the EYLNKIQNSLSTEWSPCSVT (SEQ ID NO: 17) peptide derived from a Plasmodium falciparum protein (Pf, used as a negative control) (second row); forbol 12-myristate 13-acetate (PMA, used as a positive control) (third row) and consensus peptide epitope S100-beta 68-92 (SEQ ID NO: 15) (last row). Some "spot" is detected in those cells incubated with PMA and a huge amount of "spots" in those cells incubated in the presence of the consensus peptide epitope.

Figure 10. Amino acid sequences of the peptide epitopes corresponding to Region 1 of S100-beta and of the library of overlapping peptides of 15 amino acids in length synthesized from the consensus peptide epitope SEQ ID NO: 12.

Figure 11. Results of the class II DR4 molecule binding assay of the consensus peptide epitope SEQ ID NO: 12 and the peptide epitopes of Region 1 of S100-beta. For each peptide the binding is shown at 0 hours of assay (blank columns) and the amount of peptide that remains bound to the DR4 molecule after 24 hours of incubation at 37 ° C (black part of the columns). As a comparison, the results of binding of an epitope with high affinity (positive control) and intermediate affinity (intermediate control) are shown.

Figure 12. Amino acid sequences of the peptide epitopes corresponding to Region 3 of S100-beta and the library of overlapping peptides of 15 amino acids in length synthesized from the consensus peptide epitope SEQ ID NO: 13 and SEQ ID NO: 14.

Figure 13. Results of the class II DR4 molecule binding assay of the consensus peptide epitope SEQ ID NO: 13 and of the peptide epitopes of Region 3 of S100-beta. For each peptide the binding is shown at 0 hours of assay (blank columns) and the amount of peptide that remains bound to the DR4 molecule after 24 hours of incubation at 37 ° C (black part of the columns). As a comparison, the results of binding of an epitope with high affinity (positive control) and intermediate affinity (intermediate control) are shown.

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Figure 14. Results of the class II DR4 molecule binding assay of the consensus peptide epitope SEQ ID NO: 14 and the peptide epitopes of Region 3 of S100-beta. For each peptide the binding is shown at 0 hours of assay (blank columns) and the amount of peptide that remains bound to the DR4 molecule after 24 hours of incubation at 37 ° C (black part of the columns). As a comparison, the results of binding of an epitope with high affinity (positive control) and intermediate affinity (intermediate control) are shown.

Figure 15. Amino acid sequences of the peptide epitopes corresponding to Region 2 of S100-beta and the library of overlapping peptides of 15 amino acids in length synthesized from the consensus peptide epitope SEQ ID NO: 15.

Figure 16. Results of the binding assays to the class II DR4 molecule of the consensus peptide epitope SEQ ID NO: 15 and the peptide epitopes of Region 2 of S100-beta. For each peptide the binding is shown at 0 hours of assay (blank columns) and the amount of peptide that remains bound to the DR4 molecule after 24 hours of incubation at 37 ° C (black part of the columns). As a comparison, the results of binding of an epitope with high affinity (positive control) and intermediate affinity (intermediate control) are shown.

Figure 17. A Table showing the homologies with respect to the epitopes described in the present invention, of sequences containing changes in 1, 2 or 3 amino acids, is shown. The asterisk (*) indicated in the heading of the fourth to sixth columns indicates that it has been calculated as the number of identical amino acids / number of total amino acids * 100.

Figure 18. A Table showing reference to homologies of the sequences of the present invention with the sequences of WO 98101471 is shown.

Description of the invention

The present inventors have specifically identified the antigenic peptides (also referred to herein as peptide epitopes) generated in vivo after S100-beta is processed by an antigen presenting cell (APC) and which are present on the surface of said APCs. attached to an MHC class II molecule, isolating the peptides that really intervene in the immune response in vivo. Therefore, the present invention provides the specific fragments of the S100-beta protein that are generated during an immune response and that are capable of activating lymphocytes, thereby identifying which specific peptide epitopes are those that can be used for prevention, treatment , diagnosis and monitoring of type I diabetes.

In a first aspect, the present invention relates to an antigenic peptide (peptide epitope) isolated and derived from the S100-beta protein, wherein said peptide is bound to an MHC class II molecule.

In the present invention, "antigenic peptide" means a linear sequence of amino acids present in the cleft of a histocompatibility molecule and therefore possesses the ability to behave like an antigen.

In the present invention, "MHC class II" means a histocompatibility molecule formed by two a (alpha) and 1 (beta) chains present on the cell surface, which has a cleft capable of housing an antigenic peptide.

In a preferred embodiment, said peptide is located in region 1 of the S100-beta protein, and comprises any of the sequences SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In another preferred embodiment, said peptide is located in region 2 of the S100-beta protein, and comprises any of the sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 .

In another preferred embodiment, said peptide is located in region 3 of the S100-beta protein, and comprises any of the sequences SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

Region 1 of the S100-beta protein is understood as that between amino acids 2 and 25, region 2 to that between amino acids 60-92 and region 3 as between amino acids 19-46 of the S100 protein -beta (the sequence of the S100-beta protein is available in the accession number NP_06263 of the NCBI protein database), both ends included.

In another preferred embodiment, said peptide is a consensus peptide for each of regions 1, 2 and 3, selected from the group comprising:

a) sequence peptide SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, b) peptide that at least shows 80% homology with the peptides described in a).

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Preferably, the peptide homology with the peptides described in a) is 85%, more preferably 90%, more preferably 95%, and even more preferably 98%.

In the present invention, "consensus peptide" means a linear amino acid sequence that overlaps the amino acid sequences of the peptides identified from an S100-beta region and that has at least one possible region of MHC class II molecule binding. .

In a more preferred embodiment, the MHC class II molecule attached to the peptide according to any of the above embodiments is DR4.

In the present invention, MHC class II DR4 molecules are understood as those formed by chains a and 1 encoded by the DRA1 * 0101 and DRB1 * 0401 alleles respectively.

In the present invention, "homology" means the percentage of amino acid residues in the candidate sequence that are identical to the residues of the peptide sequences SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, after aligning the sequences and entering the spaces that are necessary to achieve the maximum percentage of identity with said sequences. Peptides that maintain at least 80% homology with the sequences SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, are those that differ by 1 , 2 or 3 amino acids with respect to said sequences (Figure 17). Preferably, the homology of the candidate peptide with the peptides of the invention is 80%, more preferably 85%, more preferably 90%, more preferably 95%, and even more preferably 98%.

The present invention refers, for the first time, to isolated peptides located in three regions of the S100-beta protein, of a variable length, and which have been generated in vivo by an APC to which the S100- protein has been provided. purified beta so that the APC could process it, generate peptide epitopes and present said epitopes on its surface attached to the MHC class II molecule.

More specifically, the present invention discloses the sequence of a series of isolated peptides whose length varies between 11-27 amino acids and which are grouped into three regions of the complete S100-beta protein, forming three groups of overlapping peptides. These peptides are the following:

a) Region 1: three peptides whose core is the sequence KAMVALIDVFHQYSG SEQ ID NO: 1: KAMVALIDVFHQYSGREGD SEQ ID NO: 2: AMVALIDVFHQYSGREGDK SEQ ID NO: 3: SELEKAMVALIDVFHQYSG

b) Region 2: four peptides whose nucleus is the sequence FQEFMAFFVAMVTTAC SEQ ID NO: 4: TLDNDGDGECDFQEFMAFVAMVTTACH SEQ ID NO: 5: FQEFMAFVAMVTTACHEFFEHE SEQ ID NO: 6: EFMAFVAMVTTAC SEQ ID NO: 7: FMA

c) Region 3: four peptides whose nucleus is the sequence KHKLKKSELKELINN SEQ ID NO: 8: REGDKHKLKKS SEQ ID NO: 9: HKLKKSELKEL SEQ ID NO: 10: KHKLKKSELKELINNELSHFLE SEQ ID NO: 11: SGREGDKHKLKKSELKELIN

For each of the three regions described above, one or more consensus peptide epitopes that include all or part of the sequences described above can be synthesized. These "consensus peptide epitopes" include one or more possible "binding motifs" to the MHC class II molecule, preferably DR4. The amino acid sequences of said "consensus peptide epitopes" are:

a) KAMVALIDVFHQYSGREGDK (SEQ ID NO: 12) b) REGDKHKLKKSELKEL (SEQ ID NO: 13) c) KHKLKKSELKELINNELSHFLE (SEQ ID NO: 14) d) ECDFQEFMAFVAMVTTACHEFFEV (SEQ ID NOEF) (SEQ ID NOEF) (SEQ ID NOEF)

From the sequences described above and their derivatives (altered peptide ligands) different molecules or reagents useful in the prevention, treatment, diagnosis or monitoring of diseases can be generated.

Among these molecules, the present invention relates to MHC tetramers of class II-antigenic peptide.

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Such tetramers can also be fused to one or more other molecules. Preferably, for therapeutic purposes, said molecules are cytokines, for example interleukin-10, interleukin-5, interleukin-4 or interleukin-13, toxins, for example, saporin, or some isotopes, for example, actinium -225, which regulate the activity of CD4 + lymphocytes specific for S100-beta or eliminate them specifically (Figure 3). For diagnostic purposes, said tetramers can also be fused to magnetic nanoparticles or fluorescent compounds for the detection of CD4 + lymphocytes specific for S100-beta, for example for in vivo detection by axial tomography (Figure 3) when fused to magnetic nanoparticles, or for Ex vivo detection by flow cytometry when fused to fluorescent compounds. The diagnosis is made so that when a subject has a number of CD4 + lymphocytes specific for S100-beta above a certain value, it is diagnosed as diabetic, considering those subjects showing levels below it as healthy. Likewise, tetramers can be fused to different combinations of toxins, cytokines, magnetic nanoparticles and / or fluorescent compounds.

In a second aspect, the present invention also relates to a method that allows the direct identification and isolation of peptide epitopes naturally processed from a specific antigen and presented by class II histocompatibility molecules to CD4 + lymphocytes. Said procedure comprises the steps of:

a) purification of the class II histocompatibility molecule, b) elution of the peptides bound to the class II histocompatibility molecule; and c) identification of the peptides eluted in step b).

The purification of the class II histocompatibility molecule in step a) can be carried out by various procedures, such as precipitation with ammonium sulfate, molecular exclusion chromatography, anion exchange chromatography or preferably by affinity chromatography using specific antibody Elution of the peptides bound to the histocompatibility molecule in step b) can be carried out by various procedures, such as incubation in citrate phosphate buffer pH: 3.3 at room temperature or preferably by incubation in acetic acid at 10% at 70 ° C. The identification of the peptides according to step c) can be carried out by different procedures, preferably by HPLC and mass spectrometry.

In a third aspect, the present invention also relates to an isolated antigenic peptide derived from the S100-beta protein, wherein said peptide is bound to an MHC class II molecule, for use in prevention, treatment, diagnosis and / or follow-up of type I diabetes. Said antigenic peptide may be that described in any of the above embodiments.

The present invention also relates to the use of an isolated antigenic peptide derived from the S100-beta protein, wherein said peptide is bound to an MHC class II molecule, for the manufacture of a medicament for prevention, treatment, diagnosis and / or follow-up of type I diabetes. Said antigenic peptide may be that described in any of the above embodiments.

In the present invention, "prevention" is understood to prevent the occurrence of a disease, in this case type I diabetes.

In the present invention, "treatment" means clinical intervention in an attempt to alter the natural evolution of the disease and is performed during the evolution of clinical pathology. Desirable effects of treatment include prevention of disease recurrence, relief of symptoms, decrease in any direct or indirect pathological consequences of the disease, decrease in the rate of disease progression, improvement or partial cure of the disease status and Improved remission or prognosis.

In the present invention, "diagnosis" means the ability to identify a certain disease or pathological condition, in this case type I diabetes.

In the present invention, "follow-up" means the continuous and dynamic process of collecting data on the pathological state of the patient, in this case with respect to type I diabetes in order to establish the stage in which the disease is and consequently apply the appropriate therapeutic action.

In a preferred embodiment, the MHC-bound class II antigenic peptide according to any of the above embodiments is used as a reagent to identify and quantify specific CD4 + lymphocytes for S100-beta peptide epitopes, in diagnostic or follow-up systems of type diabetes I. In a preferred embodiment, the detection of CD4 + lymphocytes specific for S-100 beta is carried out in vivo by axial tomography or ex vivo by flow cytometry, fusing the tetramers to magnetic nanoparticles or fluorescent compounds, respectively.

The present invention also relates to a method of prevention or treatment of type I diabetes which

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it comprises the administration of a therapeutically effective amount of a class II MHC-bound antigenic peptide according to the present invention in its different embodiments to a subject in need thereof. The administration of a therapeutically effective amount would preferably be done intradermally or subcutaneously, and may alternatively be done orally, parenterally or nasally. The administration of a therapeutic amount of peptide would be preferably carried out by mixing it in "alum" (hydrated potassium aluminum sulfate) or in saline solution, preferably 0.9%.

Additionally, MHC class II bound antigenic peptides according to any of the embodiments of the present invention can be used for the treatment, prevention, monitoring and specific diagnosis of diseases involving both S100-beta and DR4, for example, rheumatoid arthritis.

In a fourth aspect, the present invention also relates to the use of an isolated antigenic peptide derived from the S100-beta protein as defined above, but without being bound to a MHC class II, for the manufacture of a medicament for the prevention, treatment, diagnosis or monitoring of type I diabetes, as well as said isolated antigenic peptide derived from the S100-beta protein for use in the prevention, treatment, diagnosis or monitoring of type I diabetes. Such prevention or treatment of type I diabetes comprises the administration of a therapeutically effective amount of an antigenic peptide according to the present invention in its different embodiments to a subject in need thereof. The administration of a therapeutically effective amount would preferably be done intradermally or subcutaneously, and may alternatively be done orally, parenterally or nasally. The administration of a therapeutic amount of peptide would be preferably carried out by mixing it in "alum" (hydrated potassium aluminum sulfate) or in 0.9% saline solution, being able to be done in another preferred embodiment with the peptide encapsulated in a vehicle, or attached to the external surface of a vehicle, where said vehicle is selected from the group comprising: a liposome, a micelle, a vesicle, a microparticle, a microcapsule, a microsphere, a nanoparticle, a nanocapsule and a nanosphere.

Diagnosis or monitoring of type I diabetes using the peptides of the invention alone, without being bound to an MHC, is carried out by quantifying the amount of CD4 + lymphocytes producing different molecules. In a preferred embodiment, the diagnostic or monitoring system consists of detecting the synthesis of interferon-gamma, interleukin-10, interleukin-17, or combinations thereof by CD4 + lymphocytes in response to said antigenic peptides (Figure 4, Example 2), preferably by ELISPOT.

In a preferred embodiment of this fourth aspect, the peptide is located in region 1 of the S100-beta protein, and comprises any of the sequences SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In another preferred embodiment of this fourth aspect, the peptide is located in region 2 of the S100-beta protein and comprises any of the sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

In another preferred embodiment of this fourth aspect, the peptide is located in region 3 of the S100-beta protein and comprises any of the sequences SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO : eleven.

In another preferred embodiment of this fourth aspect, the peptide is a consensus peptide for each of regions 1, 2 and 3, and is selected from the group comprising:

a) sequence peptide SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, b) peptide showing at least 80%, preferably 85% , more preferably 90%, more preferably 95% and even more preferably 98% homology with the peptides described in a).

The terms "consensus peptide" and "homology" in this fourth aspect of the invention mean as defined above considering that the peptide is not bound to the MHC.

The present inventors have observed that consensus peptide epitopes (not bound to an MHC class II molecule) designed for each of the three regions, from the identified peptides (see Example 1, where identification is done by mass spectrometry ), have the same binding characteristics to the MHC class II DR4 molecule as shown in the tests carried out, in addition to that in these tests it is possible to define exactly what the amino acid sequence that serves as the “reason for union ”(Example 3).

The validity of these peptide epitopes (not bound to an MHC class II molecule), both of SEQ ID NO: 1 to SEQ ID NO: 11 and of consensus peptide epitopes, in the diagnosis and monitoring of type I diabetes, It has been demonstrated using the latter (SEQ ID NO: 12 to SEQ ID NO: 16) in clinical samples from patients with T1D. By ELISPOT analysis (Figure 4), the frequency of interferon-gamma-producing CD4 + lymphocytes (IFN-y), interleukin-17 (IL-17) and interleukin-10 (IL-10) was analyzed in response to these peptides in patients healthy and diabetic (Example 2), demonstrating the ability of the peptides to discriminate effectively between both groups and therefore highlighting the diagnostic capacity of the peptides in type I diabetes, both to diagnose the presence or absence of the disease, as to determine the evolution of it

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in a subject In addition, the inventors have demonstrated that the sequences SEQ ID NO: 1-11 are also useful in the prevention, treatment, diagnosis or monitoring of type I diabetes, since in the binding assays (Figures 11, 13, 14 and 16 ), both consensus and SEQ ID NO: 1-11 peptides have similar binding patterns to MHC molecules and therefore have similar antigenic abilities.

Peptides not bound to an MHC class II molecule can be used directly or can be encapsulated in a vehicle, or attached to the outer surface thereof. Therefore, in another preferred embodiment, the peptide is either encapsulated in a vehicle, or attached to the outer surface of a vehicle, where said vehicle is selected from the group comprising: a liposome, a micelle, a vesicle, a microparticle, a microcapsule, a microsphere, a nanoparticle, a nanocapsule and a nanosphere, preferably liposomes, which allow to improve the immunogenicity of the peptides against type I diabetes.

A series of examples are shown below which are intended to illustrate the present invention but which in no case should be construed as limiting it.

EXAMPLES

Example 1: Isolation and identification of peptide epitopes derived from S100-beta bound to MHC class II

Methodology

The system used for the identification of peptide epitopes derived from S100-beta is schematized in Figure 5. In summary, the human S100-beta protein was expressed in bacteria containing a biotinylation sequence at its carboxyl-terminal end, a sequence used to biotinylate in S100-beta vitro using the BirA enzyme or biotin ligase (EC 6.3.4.15), and a sequence of 6 histidines at the amino-terminal end to be able to purify it using nickel resins. Once purified and biotinylated, S100-beta was incubated with Priess cells, a homozygous lymphoblast line for the MHC class II DR4 molecule, which had been pre-incubated with the other elements of the internalization system (first, a lectin obtained from from Phytolacca americana (pokeweed mitogen, name in English) biotinylated and secondly, avidin). Once incubated with S100-beta, the cells were incubated at 37 ° C to allow the entry of S100-beta, its processing and the presentation of epitopes on the cell surface bound to MHC class II DR4 molecules. The same Priess cells that had been incubated with all system elements excluding S100beta were used as control cells.

After the incubation period, the Priess cells incubated with S100-beta or control without S100-beta, were collected and specifically purified by MHC class II DR4 molecules, using Sepharose columns containing the antibody monoclonal conjugated L243. The peptide epitopes bound to these MHC molecules were eluted by acidic pH, filtered using filters with a cut-off point of 10,000 daltons, dried to reduce their volume and fractionated by high efficiency liquid chromatography (HPLC) using a C-reverse phase column. 18 (Figure 6). Each of the fractions obtained in the HPLC was analyzed by MALDI-TOF mass spectrometry and the mass spectra of the peptide repertoire obtained from the purified MHC DR4 molecules of Priess cells incubated with S100beta was compared with the mass spectra of the repertoire peptide obtained from control Priess cells, not incubated with S100-beta, to identify unique m / z values corresponding to peptides derived from S100-beta. The unique m / z values were compared with the linear sequence of S100-beta using the FindPept program (http://www.expasy.ch/tools/findpept.html) to identify the corresponding amino acid sequence in the protein.

Results

From this analysis, 9 m / z values were identified as unique, which correspond to three overlapping sets of peptides, sets where there is at least one reason for binding to the DR4 molecule (Figure 7). Once these peptide epitopes were identified, 4 consensus peptide epitopes were synthesized (Figure 8) that represented each of the three sets of overlapping peptide epitopes and that could be used in the specific identification of autoreactive CD4 + T lymphocytes in diabetic patients and healthy individuals.

Example 2: Diagnostic capacity of peptides / MHC-peptide complexes in type I diabetes

Methodology

The basis of the ELISPOT technique is described schematically in Figure 4. For the identification of CD4 + lymphocytes in peripheral blood of patients with T1D (28 patients) and healthy individuals (24 people), it was added for 1 hour at 37 ° C to different wells of plates Maxisorp a solution of monoclonal antibody specific for three cytokines: interferon-gamma (IFN-y), interleukin-17 (IL-17) and interleukin-10 (IL-10). Once washed, a solution of bovine albumin was added to the plates to block the places where there was no

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antibody and, after 1 hour of incubation at 37 ° C and having removed the albumin solution, peripheral lymphocytes obtained from patients with T1D or healthy subjects were added in triplicate by adding the corresponding antigens and controls. The cells were incubated 37 ° C for 72 hours and, after the incubation period, the plates were washed and incubated at 37 ° C for 1 hour with a corresponding specific monoclonal antibody for each of the three biotin-conjugated cytokines (developing antibody) . Washing the excess of developing antibody, the presence of IFN-y, IL-17 or IL-10 was revealed using an antibody directed against biotin (GABA) and conjugated to an enzyme, which once added the corresponding substrate, transforms it into an insoluble substance that is deposited on the spot-like plate ("spot").

Results

The frequency of CD4 + lymphocytes producing IFN-y, IL-17 and IL-10 measured as Stimulation Index (number of "spots" in the presence of antigen / number of "spots" in the presence of DMSO) in response to peptide epitopes Consensus are shown in Tables 1, 2 and 3, respectively. A stimulation index greater than 3 is considered significant and in the Tables where a stimulation index> 3 is indicated, it is because there is a positive response but in DMSO the number of spots is zero. As can be seen from the results shown in these Tables, CD4 + lymphocytes of patients with T1D respond more frequently by synthesizing IFN-y (Table 1) and IL-17 (Table 2) against any of the consensus peptide epitopes, which discriminate against effective way between healthy individuals and diabetic patients, highlighting its usefulness in the diagnosis of human type I diabetes. It is noteworthy that while some diabetic patients respond against any of the consensus peptide epitopes secreting IL-10 (Table 3), especially against the consensus peptide epitope S100 6892 (SEQ ID NO: 15), all healthy individuals analyzed They respond against this epitope by synthesizing large amounts of this cytokine (Table 3 and Figure 9) whose immunoregulatory role is widely studied. In addition, the Table includes a whole series of antigens that serve both as positive controls (forbol 12-myristate 13acetate (PMA)) to check cell viability and as negative controls (dimethyl sulfoxide (DMSO) or a peptide corresponding to a protein of Plasmodium falciparum (Pf), the latter provided that sufficient cells had been isolated) to check the specificity of the response.

Example 3. Binding assays to the MHC class II DR4 molecule of the peptide epitopes and delimitation of the "binding motif"

Methodology

Binding tests have been performed by ProImmune Ltd (http://www.proimmune.com) using company-owned technology. The experiments carried out consist of mixing a peptide with the purified MHC class II DR4 molecule so that, depending on its affinity, said peptide can bind to the DR4 molecule and this peptide-DR4 binding is detected by the use of a specific antibody . The affinity and binding stability of each peptide was measured as the amount of signal generated at 0 hours after mixing the components and after incubating the mixtures for 24 hours at 37 ° C, subsequently measuring the amount of peptide-DR4 remaining. As controls to compare the company uses two peptide epitopes of affinity known by the MHC class II DR4 molecule, one with high affinity and one with medium affinity.

In an attempt to narrow down the possible "reason for binding" to the MHC class II DR4 molecule of the consensus peptides used in the ELISPOT experiments, new peptides of 15 amino acids in length (15mer) were synthesized and displaced from each other a single amino acid to cover the length of the corresponding consensus peptide epitope. Once synthesized, the binding affinity for DR4 of each of the peptides in the library was determined.

Results

Region 1 includes three peptide epitopes identified by mass spectrometry (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) and a consensus peptide epitope (SEQ ID NO: 12) (Figure 10). From SEQ ID NO: 12 six peptides of 15 amino acids in length were synthesized and a single amino acid displaced from each other (15-mer # 1, 15-mer # 2, 15-mer # 3, 15-mer # 4, 15 -mer # 5 and 15-mer # 6) (Figure 10). Binding assays for each of the peptide epitopes of this Region 1 are shown in Figure 11. As can be seen in Figure 11, the peptide epitopes of this region possess a binding affinity to the intermediate MHC class II DR4 molecule. , similar to the intermediate affinity epitope used as a control. In addition, the consensus peptide epitope (SEQ ID NO: 12) has an affinity similar to the three peptide epitopes identified by mass spectrometry (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3).

Region 3 includes four peptide epitopes identified by mass spectrometry (SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11) from which two consensus peptide epitopes were synthesized (SEQ ID NO: 13, SEQ ID NO: 14). From SEQ ID NO: 13 two peptides of 15 amino acids in length were synthesized and a single amino acid (15-mer # 1 and 15-mer # 2) displaced from each other (Figure 12) while from SEQ ID NO: 14 eight peptides of 15 amino acids in length were synthesized and a single amino acid displaced from each other (15-mer # 3, 15-mer # 4, 15-mer # 5, 15-mer # 6, 15-mer # 7, 15- mer # 8, 15-mer # 9, 15-mer # 10)

ES 2 394 331 A1

(Figure 12).

The binding assay of the consensus peptide epitope SEQ ID NO: 13 shows that it has no affinity for the MHC class II histocompatibility molecule DR4, a circumstance that occurs with some epitopes involved in autoimmunity (Figure 13). In the same way, neither of the two 15 amino acid length peptides synthesized from SEQ ID NO: 13 has an affinity for said DR4 molecule (Figure 13). The consensus peptide epitope binding assays SEQ ID NO: 14 show that, in the same way as SEQ ID NO: 13 derived from the same region of S100-beta, it also has no affinity for the MHC class II histocompatibility molecule DR4 ( Figure 14). In the same way, none of the eight 15 amino acid length peptides synthesized from SEQ ID NO: 14 has an affinity for said DR4 molecule (Figure 14).

Region 2 includes four peptide epitopes identified by mass spectrometry (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7) and a consensus peptide epitope (SEQ ID NO: 15) ( Figure 15). From SEQ ID NO: 15, eleven peptides of 15 amino acids in length were synthesized and a single amino acid displaced from each other (15-mer # 1, 15-mer # 2, 15-mer # 3, 15-mer # 4, 15 -mer # 5, 15-mer # 6, 15-mer # 7, 15-mer # 8, 15-mer # 9 15-mer # 10 and 15-mer # 11) (Figure 15). Binding assays for each of the peptide epitopes of this Region 2 are shown in Figure 16. As can be seen in this Figure, the peptide epitopes described for this region are those with the highest binding affinity for the MHC class molecule. II DR4, a feature that is replicated by the consensus peptide epitope (SEQ ID NO: 15) designed for this region and used in ELISPOT assays to quantify autoreactive lymphocytes. From the binding assays to the MHC class II DR4 molecule using the library of eleven 15 amino acid length peptides synthesized from SEQ ID NO: 15 it can be deduced that the "binding motif" of SEQ ID NO: 15 to the Class II DR4 molecule appears to be in the amino acid sequence MAFVAMVTTACHEFFEH (SEQ ID NO: 16) since the 15 amino acid length peptides possessing this sequence in whole or in part (15-mer # 8, 15-mer # 9, 15-mer # 10) are those with the highest affinity and stability of binding to the class II DR4 molecule.

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Table 1. Frequency of CD4 + lymphocytes producing IFN-y against peptide epitopes derived from S100-beta

S100 25-46

S100 6-25 S100 21-36 S100 68-92

Subject DMSO * PMA ** (SEQ ID

(SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 15)

NO: 14) Healthy individuals

C14 3> 10 17.67 18.33 C15 147> 10 C16 7> 10 3.64 C17 300> 10 C18 26> 10 C19 220> 10 C20 96> 10 C21 11> 10 C22 102> 10 C23 145> 10 C24 81> 10

Answers 100 18.2 0 0 9.1 positive (%)

Type 1 diabetic patients

D17 2> 10 8.50 D18 3> 10 D19 2> 10 4.50 5.00 D20 1> 10 11.00 D21 2> 10 D22 2> 10 D23> 10 D24> 10 D25 0> 10> 3 D26 0 > 10> 3> 3 D27 0> 10> 3> 3 D28 0> 10> 3> 3

Answers 100 44.44 0.00 30.00 44.44 slides (%)

* In DMSO the results are indicated as number of spots per million cells ** Results for PMA and peptide epitopes are indicated as Stimulation Index (number of spots sample / number of DMSO spots). A stimulation index greater than 3 is considered positive. A stimulation index> 3 indicates that there is a positive response but the number of spots in DMSO is zero.

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Table 2. Frequency of CD4 + lymphocytes producing IL-17 against peptide epitopes derived from S100beta

S100 6-25 S100 21-36 S100 25-46 S100 68-92 Subject DMSO * Pf * PMA ** (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID NO: 12) NO: 13) NO: 14) fifteen)

Healthy individuals

C1 0> 10 C2 2> 10 C3 0 0> 10 C4 0> 10 C5 1 1> 10 C6 0 0> 10 C7 2 4> 10 C8 2 2> 10 C9 1 0> 10 C10 0 1> 10 C11 1 0 > 10 C12 0 0> 10 C13 0 0> 10

Positive responses (%) 100 0 0 0 0

Type 1 diabetic patients

D1 0> 10> 3> 3> 3 D2 0> 10> 3> 3> 3> 3 D3 1 0> 10 3.1 D4 2> 10 D5 4> 10 D6 1> 10 D7 6> 10 D8 0 1> 10> 3 D9 0 0> 10> 3> 3> 3 D10 0 0> 10> 3 D11 0 8> 10 D12 1 4> 10 D13 0> 10 D14 1 0> 10 D15 0 0> 10

Positive responses (%) 100 13.3 26.7 20 26.7

* In DMSO the results are indicated as number of spots per million cells ** Results for PMA and peptide epitopes are indicated as Stimulation Index (number of spots sample / number of DMSO spots). A stimulation index greater than 3 is considered positive. A stimulation index> 3 indicates that there is a positive response but the number of spots in DMSO is zero.

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Table 3. Frequency of CD4 + IL-10 producing lymphocytes against peptide epitopes derived from S100beta

S100 6-25 S100 68-92

DMS S100 21-36 S100 25-46

Subject PMA ** (SEQ ID NO: (SEQ ID NO:

OR * (SEQ ID NO: 13) (SEQ ID NO: 14)

12) 15) Healthy individuals

> 10

C14 8> 10> 10> 10

C15 18> 10

C16

> 10> 10

C17 7.5> 10> 10> 10

C18 6> 10> 10

C19 8 8.1> 10> 10

C20 4> 10> 10> 10

C21 8 4.4> 10> 10

C22 9> 10> 10

C23 16> 10> 10

C24 1 Answers

10040 0 0 100

positive (%)

Type 1 diabetic patients

> 10

D17 2> 10

D18

> 10

D19 7 6.9> 10

D20 3> 10

D21 5 6.6> 10

D22 29> 10

D23 0> 3> 10

D24 0> 3> 3> 10

D25

> 10

D26 24> 10

D27 1> 10

D28 2> 10

Answers

10011 11 22 50

positive (%)

* In DMSO the results are indicated as number of spots per million cells ** Results for PMA and peptide epitopes are indicated as Stimulation Index (number of spots sample / number of DMSO spots). A stimulation index greater than 3 is considered positive. A stimulation index> 3 indicates that there is a positive response but the number of spots in DMSO is zero.

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Claims (9)

one.

Use of an isolated antigenic peptide derived from the S100-beta protein that is not bound to an MHC class II molecule in the manufacture of a medicament for the prevention, treatment, diagnosis and / or monitoring of type I diabetes.

2.

Use of a peptide according to claim 1 wherein the peptide is located in region 1 of the S100beta protein and comprises any of the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, in the manufacture of a medicine for the prevention, treatment, diagnosis and / or monitoring of type I diabetes.

3.

Use of a peptide according to claim 1 wherein the peptide is located in region 2 of the S100beta protein and comprises any of the sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, in the manufacture of a medicine for the prevention, treatment, diagnosis and / or monitoring of type I diabetes.

Four.

Use of a peptide according to claim 1 wherein the peptide is located in region 3 of the S100beta protein and comprises any of the sequences SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 , in the manufacture of a medicine for the prevention, treatment, diagnosis and / or monitoring of type I diabetes.

5.

Use of a peptide according to claim 1 wherein the peptide is a consensus peptide for each of the regions 1, 2 and 3, and is selected from the group comprising: a) sequence peptide SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, b) peptide that at least shows 80% homology with the peptides described in a). in the manufacture of a medicine for the prevention, treatment, diagnosis and / or monitoring of type diabetes

I.

6.

Use of a peptide according to any one of claims 1 to 5 wherein the peptide is either encapsulated in a vehicle, or attached to the outer surface of a vehicle, wherein said vehicle is selected from the group comprising: a liposome, a micelle , a vesicle, a microparticle, a microcapsule, a microsphere, a nanoparticle, a nanocapsule and a nanosphere.

7.

Use of a peptide according to any one of claims 1 to 6 wherein the diagnosis is based on the detection of interferon-gamma, interleukin-10, interleukin-17, or combinations thereof.

8.

Use of a peptide according to claim 7 wherein the detection of interferon-gamma, interleukin-10, interleukin-17 or combinations thereof is carried out by ELISPOT.

ES 2 394 331 A1 Figure 1 ES 2 394 331 A1 Figure 4 ES 2 394 331 A1 Figure 6 ES 2 394 331 A1 Figure 7 ES 2 394 331 A1 Figure 9 ES 2 394 331 A1 Figure 10 ES 2 394 331 A1 ES 2 394 331 A1 Figure 12 ES 2 394 331 A1 ES 2 394 331 A1 Figure 15 Figure 16 ES 2 394 331 A1 Figure 17 ES 2 394 331 A1 Figure 18 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201230456 SPAIN Date of submission of the application: 06.17.2011 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

WO 9801471 A1 (AB SANGTEC MEDICAL [SE / SE]) 15.01.98, page 3, line 11 - page 5, line 1-8

6; claims 1-6; SEQ ID NO: 2-6.

TO

WINER S. et al. Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell 1-8

exclusive. Nature Medicine 2003, Vol. 9 (2), pages: 198-205, the whole document.

TO

US 2003054414 A1 (JACKOWSKI ET AL.) 20.03.2003, the whole document. 1-8

TO

TSUI H. et al. Targeting of pancreatic glia in type 1 diabetes. Diabetes. 2008, Vol. 57 (4), pages: 1-8

918-928, the whole document.

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 23.07.2012

Examiner M. D. García Grávalos Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201230456 CLASSIFICATION OBJECT OF THE APPLICATION  G01N33 / 543 (2006.01) G01N33 / 564 (2006.01) G01N33 / 68 (2006.01) Minimum documentation sought (classification system followed by classification symbols) G01N Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, NPL, BIOSIS, MEDLINE, EMBASE, USPTO PATENT DATABASE, PUBMED, GOOGLE PATENTS, GOOGLE SCHOLAR, EBI. State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201230456 Date of Written Opinion: 23.07.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-8 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-8 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201230456  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

WO 9801471 A1 01.01.0098

D02

WINER S. et al. Nature Medicine 2003, Vol. 9 (2), pages: 198205. 2003

D03

US 2003054414 A1 03.20.2003

D04

TSUI H. et al. Diabetes. 2008, Vol. 57 (4), pages: 918-928. 2008

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present invention discloses the use of an antigenic peptide derived from the S100-beta protein in the preparation of a medicament for diagnosis, prevention and treatment of type 1 diabetes (claim 1). Said peptide may be located in regions 1, 2 or 3 of the protein, or be a consensus peptide for each of the regions and any of the sequences SEQ ID NO: 1-16 (claims 2-5); It can be encapsulated in a vehicle (claim 6) and the diagnosis based on the detection of interferon gamma, IL-10 or IL-17 by ELISPOT (claims 7 and 8). Document D01 discloses useful epitopes for generating antibodies to detect the presence of the S100-beta protein in samples of melanoma patients (see page 3, line 11-page 5, line 6; claims 1-6; SEQ ID NO : 2-6). Document D02 discloses a study on the effect of immunotherapy treatment with GFAP or S100-beta on the development of type 1 diabetes, which shows a delay and a significant reduction in the development of the disease (see the whole document). Document D03 discloses the use of GFAP proteins for the diagnosis and treatment of type 1 diabetes; on the other hand, in claim 5 includes a reference that the diagnostic marker can also be S100-beta (see the whole document). Document D04 discloses the development of epitopes of GFAP, emphasizing the importance of the identification of epitopes in the development of tetramers; as well as its usefulness for the treatment of type 1 diabetes (see the whole document).  1. NEW AND INVENTIVE ACTIVITY (Art. 6.1 and Art. 8.1 LP 11/1986) The present invention discloses the use of an antigenic peptide derived from the S100-beta protein in the preparation of a medicament for diagnosis, prevention and treatment of type I diabetes. 1.1. CLAIMS 1-8 Document D01 is considered the closest to the state of the art as it anticipates antigenic peptides derived from the S100-beta protein whose sequences, corresponding to SEQ. ID NO: 2-6, have a high homology with those of the peptides claimed in the present invention. The difference between document D01 and the present invention lies in the use of these molecules for diagnosis, prevention and treatment of type 1 diabetes. The use of the S100-beta protein for immunotherapy treatment in the development of type 1 diabetes, it is anticipated in document D02; however, this document refers to the use of the complete protein and not that of the claimed peptides. Consequently, as disclosed in D01 and D02, claims 1-8 meet the requirement of novelty and inventive activity (Art. 6.1 and Art. 8.1 LP11 / 1986). Documents D03 -D04 refer to the state of the art and are not considered relevant in relation to the object of the invention. State of the Art Report Page 4/4