Immunodominant proteins and fragments in multiple sclerosis

Abstract

The present disclosure relates to the treatment, diagnosis and / or prevention of multiple sclerosis (MS) by the use of immunodominant proteins or peptides. More specifically, the present invention relates to the field of antigen-specific immunotherapy, such as the induction of tolerance.

Description

[0001] This application is a divisional application. The corresponding parent application has application number 201980050348.2, application date June 28, 2019, and the name of the invention is “Immunodominant proteins and fragments in multiple sclerosis”. Technical Field

[0002] The present invention relates to the treatment, diagnosis and / or prevention of multiple sclerosis by using immunodominant proteins or peptides. More specifically, the present invention relates to the field of antigen-specific immunotherapy, such as the induction of tolerance. Background Art

[0003] Multiple sclerosis (MS) is a devastating autoimmune inflammatory disease that primarily affects young people. MS is the prototypical example of an organ-specific autoimmune disease (AID) because the autoimmune response targets only the central nervous system (CNS), which consists of the brain and spinal cord. Organ-specific AIDs are when the patient's immune system destroys specific tissues or cell types through autoreactive T cells and / or antibodies.

[0004] MS preferentially affects young people between the ages of 20 and 40, but children and older adults may also develop MS. The disease is about 2-3 times more common in women than in men. MS usually presents clinically with temporary problems with vision (acute optic neuritis), sensation, or motor and autonomic function, but can cause a wide range of neurological symptoms.

[0005] At first presentation, if alternative diagnoses have been ruled out, the disease is called clinically isolated syndrome (CIS) as long as the cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI) findings are consistent with the diagnosis. MRI shows lesions in typical locations for MS (i.e., paracortical, paraventricular, in the brainstem or spinal cord). If specific criteria are met that can be summarized as spatial spread (more than one lesion or clinical symptom / sign) and temporal spread (more than one event), then a diagnosis of relapsing-remitting multiple sclerosis (RRMS) is made. A special case is an incidental MRI lesion consistent with clinically asymptomatic MS. This is called radiologically isolated syndrome (RIS) and can be considered a pre-stage of CIS and RRMS. More than 80% of patients have one of these, and most patients later develop the so-called secondary progressive MS (SPMS). At this point, relapses / exacerbations become less frequent or stop completely, and neurological deficits steadily increase between relapses or when there are no relapses.

[0006] A special form of MS is primary progressive MS (PPMS), which never relapses but instead begins with a steady worsening of neurological symptoms, such as a steady worsening of the ability to walk. PPMS affects about 10% of people with MS and affects men and women equally. Its onset is usually later than CIS or RRMS. With regard to etiology and disease mechanisms, PPMS is thought to be similar to the RIS-CIS-RRMS-SPMS described above.

[0007] Typically, MS is diagnosed based on the revised McDonald or more recently Lublin criteria. These criteria also allow differentiation of different forms of MS and disease activity (Thompson et al., 2018, Lancet Neurol, 17(2): 162-173).

[0008] MS is a disease with a complex genetic background. In the past decade, more than 200 MS risk alleles or quantitative traits (common variants of genes detected as single nucleotide polymorphisms (SNPs)) have been identified, however, the most important to date is the human leukocyte antigen (HLA)-DR15 haplotype. In addition, several environmental / lifestyle risk factors have been discovered. These include infection with Epstein-Barr virus (EBV), smoking, low vitamin D3 levels, and obesity as the most important factors.

[0009] All genetic and environmental risk factors are shared by many individuals in the healthy population. The exact reason why the disease starts in individuals with certain genetic and environmental risk factors is not known, but it has been hypothesized that viral and bacterial infections, such as changes in the gut microbiota, may be predisposing factors. The concordance rate among identical twins is 10-30%, the risk to first-degree relatives of MS patients is about 2-4%, and the risk in the general population is 1 / 1000, which provides an estimate of genetic risk versus environmental risk, although the interaction between the two is also complex.

[0010] In order to determine the components of the CNS that are targeted by the autoimmune response in MS, researchers have focused on the cells and structures affected in MS, especially myelin and axons / neurons and proteins specific to these cells / structures. In the past three decades, in animal models (experimental autoimmune encephalomyelitis; EAE), some myelin proteins such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) have been identified as brain-derived, that is, injection into susceptible rodent strains causes diseases similar to MS, but can also be tested by immune cells from MS patients (Sospedra and Martin, 2005, Annu Rev Immunol, 23: 683-747). The above autoantigens are CNS-specific and are expressed only in the brain (PLP and MOG) or almost only in the brain (MBP). In MS, some non-CNS-specific autoantigens such as α-B crystallin and transaldolase-H have also been described as potential targets.

[0011] Current evidence suggests that CD4+ autoreactive T cells are important factors in the autoimmune pathogenesis of MS and may be involved not only in the induction and maintenance of autoimmune responses, but also during tissue damage (Sospedra and Martin, 2005). In MS patients, the frequency of high-avidity CD4+ T cells that react to major components of myelin, such as MBP, PLP, and MOG, is increased (Bielekova et al., 2004, J Immunol, 172: 3893-3904). Due to their involvement in the pathogenesis of the disease, CD4+ T cells have become a target for therapeutic intervention.

[0012] Detailed studies of the immune response to CNS-specific proteins have shown that certain peptides thereof are recognized by a large proportion of patients and are recognized in the context of disease-associated HLA-DR molecules. Such peptides are called immunodominant (Bielekova et al., 2004).

[0013] The following characteristics indicate that certain peptides of a protein are immunogenic with respect to MS:

[0014] a) the peptide is frequently recognized by T cells, i.e., by approximately 10% or more of MS patients, often in the context of disease-associated HLA alleles or haplotypes (Sospedra and Martin, 2005), and

[0015] b) Disease-associated T cells recognize this peptide, such as those that respond to low concentrations of the peptide (high-avidity T cells) (Bielekova et al., 2004) and are therefore considered particularly dangerous, and / or have a pro-inflammatory phenotype, and / or are isolated from the target organ or compartment (CNS), in the case of MS, brain, spinal cord or CSF-infiltrating T cells.

[0016] However, high-avidity recognition is not a prerequisite, as low-avidity myelin-specific T cells have also been shown to be pathogenic in a humanized transgenic mouse model (Quandt et al., 2012, J Immunol, 189(6): 2897-2908).

[0017] It has recently been demonstrated that T cells from MS patients show increased in vitro proliferation in the absence of exogenous antigens (Mohme et al., 2013, Brain, 136: 1783-1798). These "self-proliferating" T cells are enriched in cells that home to the CNS compartment of MS patients and can therefore be considered a peripheral blood source of brain / CSF-infiltrating T cells (Jelcic et al., 2018, Cell, 175(1): 85-100.e23).

[0018] If data from in vitro T cell tests are not available, or in addition to such tests, the immune recognition of peptides can also be predicted / inferred from those peptides that bind well to the individual's HLA class I or class II alleles and are used for CD8+ and CD4+ T cells, respectively. Peptide binding prediction is well known to the skilled person. It can be performed by well-established prediction algorithms (NetMHCII - www.cbs.dtu.dk / services / NetMHCII / ; IEDB - www.iedb.org / ) and analysis of HLA binding motifs (SYFPEITHI - www.syfpeithi).

[0019] Immunodominant peptides can be used for antigen-specific immunotherapy, such as tolerance induction. An example is EP 2 205 273 B1, which discloses immunodominant peptides of MBP, PLP and MOG and their use in the treatment of MS. In the method disclosed therein, the peptides are coupled to leukocytes or erythrocytes.

[0020] Tolerance induction is antigen-specific and renders autoreactive T cells nonfunctional or anergic or induces regulatory T (Treg) cells that specifically suppress adverse autoimmunity to the target antigen. Inducing tolerance to target self-antigens is a very important therapeutic goal in autoimmune diseases. It provides an opportunity to specifically attenuate pathogenic autoimmune responses in an effective manner with almost no side effects. Tolerance induction can also be achieved by using the entire protein instead of or in addition to the immunodominant peptide as a fragment of the protein (Kennedy MK et al., 1990, J Immunol, 144(3): 909-15).

[0021] Some pathological features of MS are reflected in the EAE model, which is a model animal model of autoimmune diseases driven by Th1 / Th17 cells. Studies on recurrent EAE (R-EAE) in SJL mice clearly show that chronic demyelination involves activation of T cell responses to immunodominant myelin peptides (i.e., PLP 139-154), with initial disease exacerbations directed to immunodominant myelin peptides. Subsequently, the immune response extends to other myelin peptides of PLP, MBP, and MOG, a process known as epitope expansion. For example, when antigen presenting cells (APCs) loaded with antigenic peptides are treated, for example, with the cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI; also referred to as EDC), T cell unresponsiveness, i.e., tolerance, can be induced.

[0022] Preclinical experiments have demonstrated that a single intravenous (iv) injection of naive mouse spleen cells loaded with a mixture of brain-pathogenic myelin peptides and fixed with the cross-linking agent EDC is highly effective in inducing peptide-specific tolerance in vivo. In EAE, this regimen not only prevented the animals from becoming ill, but even when given after disease induction, it effectively reduced the onset and severity of all subsequent relapses, suggesting that specific tolerance can downregulate ongoing autoimmune responses (Miller et al., 1991, AcadSci, 636: 79-94). More relevant to the treatment of MS, studies in EAE have shown that the use of a mixture of brain-pathogenic myelin peptides can induce tolerance to multiple epitopes simultaneously, thereby providing the ability to target autoreactive T cells with multiple specificities.

[0023] Tolerization of human T cells by autoantigen-coupled cells, such as EDC-treated APCs (Vandenbark et al., 2000, Int Immunol, 12:57-66) or anucleated cells (i.e., red blood cells (RBCs)), is effective in vitro, as shown by the inability of tolerized T cells to proliferate or produce Th1 cytokines and the reduction in the expression of co-stimulatory molecules on these cells.

[0024] There is evidence that at least two different mechanisms are involved in the induction of antigen-specific tolerance:

[0025] 1) direct tolerance, in which Th1 clones encountering the nominal antigen / MHC complex on antigen-coupled APCs become anergized due to an inability to receive adequate CD28-mediated co-stimulation, and

[0026] 2) Indirect mechanisms, such as cross-tolerance, where tolerance is induced by reprocessing and re-presentation of antigen by tolerogenic host APCs and / or expansion of Treg cells.

[0027] The latter cross-tolerance may involve the induction and / or expansion of antigen-specific Treg cells, a hypothesis also supported by the data obtained in the Phase Ib trial disclosed herein. In addition, treatment of cells with EDC can induce apoptosis in a significant proportion of treated cells. Therefore, an indirect mechanism involving fixed APCs undergoing apoptosis that are then processed and presented by host APCs is feasible. This is further supported by the effective induction of tolerance in MHC-deficient and allogeneic mice. In vitro bone marrow-derived dendritic cells effectively engulf and process antigen-loaded, fixed APCs.

[0028] Currently approved MS therapies involve various antigen-nonspecific immunomodulatory or immunosuppressive strategies that are only partially effective. All current therapeutic agents require daily oral administration or injection / infusion at various intervals and for long periods of time. In addition, they are associated with numerous side effects, sometimes severe side effects.

[0029] A therapy for the pathogenesis of MS, the fundamental purpose should be to specifically delete or functionally inhibit pathogenic autoreactive cells without changing the "normal" immune system. This is important because comprehensive immunomodulation and / or immunosuppression is at the expense of suppressing beneficial regulatory cells and immune cells that play a protective function against pathogens. Ideally, peptide-specific immune tolerance, i.e., specific correction of the misdirected autoimmune response of brain / spinal cord tissue, should be achieved as early as possible in the inflammatory stage of the disease, when the blocking of the autoreactive immune response can inhibit the spread and propagation of the disease, and can prevent irreversible obstacles. Therefore, the preferred target patient group is a relapsing-remitting MS patient in the early stage of the disease process, or even a patient with the first clinical event (i.e., CIS) suggesting MS, or a patient who finds the disease earlier in the RIS stage. At this point in time, the degree of neurological impairment in MS patients is usually low, which enables them to participate in all activities of daily life and work without causing significant damage. Summary of the invention

[0030] One object of the present invention is to identify MS-associated antigens suitable for use in the treatment, diagnosis and / or prevention of MS, in particular for use in tolerization methods. Another aspect of the present invention is to identify human subjects suitable for tolerization.

[0031] The present invention is based on a novel approach for the identification of MS-associated antigens: T cells clonally expanded in the brain of an MS patient (homozygous for the HLA DR15 haplotype, known to be the main genetic risk factor in MS) who died of a very aggressive form of MS were examined. Thus, for the first time, T cells originating from and clonally expanded in a target organ were analyzed for the purpose of antigen identification. In all previous approaches, peripheral blood lymphocytes were analyzed to identify immunodominant antigens.

[0032] The clonal expansion of T cell clones (TCCs) in MS brain lesions indicates that the cells are associated with MS. Here, the target antigen of a specific TCC previously described in Planas et al. (Planas et al., 2015, Ann Clin Transl Neurol, 2(9): 875-893), namely TCC21.1, has been identified. In addition, the target antigen of another TCC (TCC14) from the same patient has been identified. In the case of TCC14, the clone was isolated from a peripheral blood T cell population that was identified as being associated with the disease by showing increased spontaneous proliferation (self-proliferation) and enrichment of brain-homing autoreactive T cells. Specifically, even though TCC14 had been isolated from peripheral blood, it was found to be clonally expanded in MS brain lesions. The isolation and identification of disease-associated T cells (such as TCC21.1 and TCC14) is schematically shown in Figure 1 middle.

[0033] Target epitopes recognized by biologically relevant (e.g., tissue-infiltrating) T cells can then be identified ( Figure 2 ).

[0034] This novel approach revealed that the proteins GDP-L-fucose synthase (gene abbreviation: TSTA3; also known as: GDP-L-fucose:NADP+4-oxidoreductase (3,5-epimerization) or GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase 4-reductase) and proteins of the RASGRP (RAS guanosine releasing protein) family, including RASGRP1, RASGRP2, RASGRP3 and RASGRP4 as well as splice variants and isoforms thereof, are particularly relevant in MS. RASGRP2 is particularly preferred. Thus, it has been found that these proteins are immunodominant and autoantigens in MS.

[0035] The association has also been tested for GDP-L-fucose synthase in CSF-infiltrating CD4+ T cells from CIS / MS patients and for RASGRP2 in peripheral blood-derived monocytes. In a further analysis, both GDP-L-fucose synthase and RASGRP2 have been tested in CSF-infiltrating CD4+ T cells from CIS / MS patients.

[0036] Thus, the antigen described in the examples herein is the first immunodominant antigen in MS, which has been discovered by examining the specificity of T cells that clonally expand in MS brain lesions and is therefore hypothesized to be involved in the destruction of autoimmune reactions in the brain. The method leading to its identification, namely a combinatorial peptide library, does not involve the above-mentioned focus on myelin / brain proteins, but is completely unbiased. This antigen has not been previously described to be implicated in MS. In addition, RNA sequencing and proteomics have shown that the autoantigens GDP-L-fucose synthase and RASGRP2, as well as the related proteins RASGRP1 and -3, are expressed in MS brain tissue.

[0037] The identified antigens can be used for the treatment, diagnosis and / or prevention of MS, in particular for tolerization methods, and for identifying human subjects suitable for tolerization. By identifying human subjects suitable for tolerization, MS can be diagnosed in vitro in human subjects. In other words, the identified autoantigens can be used for in vitro diagnosis of MS. Such in vitro tests can be supplemented by clinical and imaging findings (i.e., MS diagnosis according to the prior art, in particular according to the revised McDonald criteria). Therefore, the identified autoantigens can be used to diagnose MS in human subjects with or without other diagnostic tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Isolation of disease-associated T cells.

[0039] Figure 2 Target epitope discovery.

[0040] Figure 3. Single- and dual-defined decapeptide positional scan mixtures combined with biometric analysis to characterize the specificity of TCC21.1.

[0041] Figure 4 Summary of human decapeptides predicted by biostatistical methods, synthesized and tested for stimulatory capacity.

[0042] Figure 5 GDP-L-fucose synthase transcripts and peptides identified in brain tissue.

[0043] Figure 6 GDP-L-fucose synthase, myelin and CEF peptides (control peptides from cytomegalovirus, Epstein-Barr virus and influenza virus).

[0044] Figure 7 Recognition of GDP-L-fucose synthase peptide by CSF-infiltrating CD4+ T cells from CIS / MS patients.

[0045] Figure 8 Isolation of autophagic and self-proliferating T cells found in MS brain lesions.

[0046] FIG. 9 Screening procedure for identification of peptide ligands using a positional scanning library for brain-homing TCC14 isolated from proliferating peripheral blood.

[0047] Fig.10 RASGRP2 reactivity of peripheral blood-derived memory T cells.

[0048] Fig.11 Expression of RASGRP in peripheral blood B cells and brain.

[0049] Fig.12 Peptide identification of RASGRP1, 2, and 3 proteins in brain tissue.

[0050] Fig.13 Staining of cells for biotin-PLP1 conjugate.

[0051] Figure 14 CSF infiltrating CD4 from 105 MS patients + T cell recognition of GDP-L-fucose synthase and myelin peptides.

[0052] Figure 15 CSF infiltrating CD4 from 57 MS patients + T cell recognition of RASGRP2 and myelin peptides.

[0053] Figure 16 In vivo tolerance induction using erythrocytes coupled to myelin peptides. DETAILED DESCRIPTION

[0054] Ideally, the T cells used to identify immunodominant peptides and corresponding proteins are those that are etiologically relevant to the disease. With regard to the latter characteristics, those T cells that are clonally expanded in the target tissues of MS (brain, spinal cord and CSF) are of greatest interest for identifying target antigens associated with the disease. According to the method described by Planas et al. (2015), next-generation sequencing of T cell receptor (TCR) β chain complement or genomic DNA sequences has been used to identify clonally expanded T cells in autopsy lesions of brains of MS patients and to isolate these T cells as TCCs from autologous CSF and / or tissues (including living cells and obtained by, for example, biopsy or early autopsy) and characterize them with respect to functional phenotype and antigenic specificity. TCCs associated with the disease have thus been isolated. Specifically, TCC21.1 has been identified and characterized: TCC21.1 displays a Th2 phenotype, releases mainly Th2 cytokines, and is able to provide B cell help for antibody production. This strategy has led to the identification of the relevance of the GDP-L-fucose synthase protein.

[0055] The above strategy, deep TCR sequencing of brain / spinal cord / CSF-infiltrating T cells, has also been used to isolate disease-associated T cells from peripheral blood and clone identified T cells from proliferating peripheral blood mononuclear cells (PBMCs). This strategy has led to the identification of the relevance of the RASGRP family of proteins.

[0056] This study used CSF infiltrating T cells (for GDP-L-fucose synthase, and in further analysis for both GDP-L-fucose synthase and RASGRP2) as well as peripheral blood T cells (for RASGRP2) to show that these are immunodominant targets in the autoimmune response in MS, and that both GDP-L-fucose synthase and RASGRP2 (as well as other members of the RASGRP family) are recognized by brain infiltrating T cells in MS (including CIS, RRMS and SPMS). The in silico peptide binding prediction algorithms of NetMHCII and IEDB have also been used to identify immunodominant regions within the corresponding proteins (as described in the Examples).

[0057] The cytosolic enzyme GDP-L-fucose synthase converts GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose, which is then used by fucosyltransferases to fucosylate all oligosaccharides. In mammals, fucosylated glycans play important roles in many biological processes, including transfusion reactions, host-microbe interactions, cancer pathogenesis, and the maintenance of a non-inflammatory environment in the brain.

[0058] RASGR protein is present in at least four variants RASGRP1, RASGRP2, RASGRP3 and RASGRP4. In particular, RASGRP1, RASGRP2, RASGRP3 are relevant to the present invention. This protein family is characterized by the presence of a Ras superfamily guanine nucleotide exchange factor (GEF) domain, which acts as a nucleotide exchange factor regulated by diacylglycerol (DAG), and specifically activates Ras by exchanging bound GDP with GTP. The proteins of this protein family activate the Erk / MAP kinase cascade. They are involved in reducing apoptosis and tumorigenesis of EBV-infected B cells, B and T cell signaling, -adhesion, -movement, and are crucial for maintaining BT cell homeostasis. There are at least four isoforms of RASGRP2, i.e., splice variants.

[0059] Previously, the identified antigens GDP-L-fucose synthase and RASGRP2 (as well as RASGRP1, RASGRP3 and RASGRP4) have not been implicated in the etiology or pathogenesis of MS or its animal model EAE. The finding that both proteins or their fragments, derivatives or splice variants are immunodominant targets of the autoimmune response in MS allows their use in the treatment, diagnosis and / or prevention of MS.

[0060] Protein is intended to represent oligopeptides, polypeptides and protein itself. Protein sequence can be defined by GenBank entries. Protein sequence can also be defined by UniProtKB / Swiss-Prot entries and / or GenPept entries. Entry can be defined by numbers, such as accession numbers. If applicable, database entries include corresponding accession numbers (i.e. entry numbers) and version numbers. Protein can also be defined by any other database known to the technician. Different isoforms, derivatives and / or splice variants can be present, which are also encompassed in the present invention. Therefore, the sequence can be different from the known sequences from, for example, GenBank or UniProtKB / Swiss-Prot entries.

[0061] Unless otherwise specifically stated, "a" protein or "the" protein according to the present invention refers to a GDP-L-fucose synthase protein or a protein of the RASGRP protein family, preferably RASGRP2, or refers to both a GDP-L-fucose synthase protein or a protein of the RASGRP protein family (preferably RASGRP2).

[0062] Splice variants result from alternative splicing during gene expression. The splice variants according to the invention are preferably immunodominant.

[0063] A fragment is preferably any part of a protein which is shorter than the parent protein, i.e. has fewer amino acids. A fragment may be a peptide. In one embodiment, the fragment comprises 5 to 50, preferably 5 to 20. More preferably 10 to 15 amino acids, even more preferably 15 amino acids. The fragment according to the invention is preferably immunodominant.

[0064] It is also possible to use more than one fragment according to the invention. Preferably, more than three, more than five, more than ten, more than fifteen, or even more than twenty different fragments are used. In a preferred embodiment, 5 to 20, preferably 5 to 15 different fragments are used. A fragment is different from another fragment if it does not consist of the same amino acid sequence.

[0065] In another embodiment, at least one fragment of each protein according to the present invention (GDP-L-fucose synthase protein or protein of the RASGRP protein family, preferably RASGRP2) is used in combination. It is particularly advantageous to combine at least one fragment of each protein according to the present invention with at least one known peptide from the state of the art, in particular with at least one myelin peptide, in particular with at least one or all of the myelin peptides defined by SEQ ID NOs: 261 to 267.

[0066] A derivative of a sequence is preferably defined as an amino acid sequence that has at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homology or identity with the corresponding part of the reference amino acid sequence over its entire length. In the sense of the present invention, "corresponding part" preferably refers to the same amino acid segment of the parent sequence. For example, if a derivative of 100 amino acids in length differs from the amino acid segment of SEQ ID NO: 1 (amino acids 1 to 100 of SEQ ID NO: 1) by 20 amino acids, then this particular derivative has 80% identity with its corresponding part, i.e. 1 to 100 amino acids of the reference amino acid sequence (SEQ ID NO: 1), over its entire length. The derivatives according to the invention are preferably immunodominant.

[0067] According to the present invention, the "homology" or "identity" of an amino acid sequence is preferably determined over the entire length of a reference amino acid sequence or over the entire length of the corresponding part of a reference amino acid sequence, wherein the reference amino acid sequence corresponds to the sequence for which the homology or identity is defined.

[0068] "Identity" is defined as identical amino acids, and "homology" includes identical amino acids as well as conservative substitutions. Conservative substitutions are known to those skilled in the art, e.g.

[0069] - Aromatic and aromatic F and W / Y

[0070] - Positively charged and positively charged R and K / H

[0071] - Negatively charged E and D or

[0072] - Aliphatic V and L / M / I, or A and S / T.

[0073] The nucleotide sequence encoding any protein of the present invention or its fragment, derivative or splice variant refers to any coding nucleotide sequence, such as RNA or DNA, particularly mRNA or cDNA. In one embodiment, the nucleotide sequence is a plasmid or any type of vector known to those skilled in the art. In a preferred embodiment, the nucleotide sequence does not contain introns, and the gene sequence contains exons and introns.

[0074] In one aspect of the invention, the protein used for the treatment, diagnosis and / or prevention of multiple sclerosis (MS) is GDP-L-fucose synthase or a fragment, derivative or splice variant thereof. In another aspect of the invention, the protein is a member of the RASGRP family or a fragment, derivative or splice variant thereof. The present invention also relates to a nucleotide sequence encoding any one of the proteins or fragments, derivatives or splice variants thereof for the treatment, diagnosis and / or prevention of multiple sclerosis (MS).

[0075] In a preferred embodiment, the protein is a human protein, and / or the nucleotide sequence and / or gene sequence is a human sequence.

[0076] In one embodiment, the GDP-L-fucose synthase exhibits enzymatic activity that converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose.

[0077] In another embodiment, members of the RASGRP family activate Ras by exchanging bound GDP for GTP. Additionally or alternatively, the protein activates the Erk / MAP kinase cascade.

[0078] In a preferred embodiment, the GDP-L-fucose synthase protein

[0079] a) having the amino acid sequence shown in SEQ ID NO: 1, or

[0080] b) having an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence shown in SEQ ID NO: 1, or

[0081] c) having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the amino acid sequence shown in SEQ ID NO: 1, or

[0082] d) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence shown in SEQ ID NO: 1 and the protein or its fragment or splice variant binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the amino acid sequence shown in SEQ ID NO: 1 or its fragment, or

[0083] e) encoded by the TSTA3 gene, in particular encoded by the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11, or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11.

[0084] In another preferred embodiment, a member of the RASGRP protein family

[0085] f) having an amino acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or

[0086] g) having an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or

[0087] h) having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or

[0088] i) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, and the protein or its fragment or splice variant binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the corresponding amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or a fragment thereof, or

[0089] j) is encoded by the RASGRP gene, in particular by the following gene sequence:

[0090] - nucleotides 38488101 to 38565575 of NC_000015.10,

[0091] - nucleotides 64726911 to 64745456 of NC_000011.10,

[0092] - nucleotides 33436324 to 33564750 of NC_000002.12, or

[0093] - nucleotides 38409051 to 38426305 of NC_000019.10,

[0094] or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of:

[0095] - nucleotides 38488101 to 38565575 of NC_000015.10,

[0096] - nucleotides 64726911 to 64745456 of NC_000011.10,

[0097] - nucleotides 33436324 to 33564750 of NC_000002.12, or

[0098] -nucleotides 38409051 to 38426305 of NC_000019.10.

[0099] Binding to self-HLA alleles, recognition by T cells and / or recognition by antibodies may indicate that the protein or its fragment or splice variant is immunodominant. Immunodominance can also be tested as described below.

[0100] In a particularly preferred embodiment, the present invention uses a GDP-L-fucose synthase or RASGRP2 protein or a splice variant thereof, preferably a GDP-L-fucose synthase or RASGRP2 protein. The GDP-L-fucose synthase protein has a sequence such as that shown in SEQ ID NO: 1, and the RASGRP2 protein has a sequence such as that shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0101] In one embodiment, the fragment comprises 5 to 50, preferably 5 to 20, more preferably 10 to 15 amino acids, even more preferably 15 amino acids.

[0102] In another embodiment, the fragment

[0103] a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to the respective corresponding amino acid sequence, or

[0104] b) is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the respective corresponding amino acid sequence, or

[0105] c) is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the respective corresponding amino acid sequence and binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize said respective amino acid sequence.

[0106] By "each corresponding amino acid sequence" is meant each fragment of the corresponding amino acid sequence (i.e. SEQ ID NO: 1) which has the same length as the homologous fragment (see also the definition of "corresponding part" above). Fragments having these identities and / or homologies may comprise 5 to 50, preferably 5 to 20, more preferably 10 to 15, even more preferably 15 amino acids. The identities and / or homologies are determined over the entire length of the respective fragment. In other words, a "corresponding amino acid sequence" refers to the unaltered sequence, i.e. when a fragment of the sequence shown in SEQ ID NO: 1 is 85% identical to the respective corresponding amino acid, the fragment is 85% identical (over the entire length of the fragment) to an unaltered fragment "cut out" from SEQ ID NO: 1 (i.e. taken directly from or copied from SEQ ID NO: 1).

[0107] Therefore, in one embodiment, the protein (GDP-L-fucose synthase or a member of the RASGRP protein family) has an amino acid sequence that has a certain homology (at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%) with each of the depicted sequences shown in SEQ ID NO, and it is also required that the protein or its fragment or splice variant binds to self-HLA alleles, is recognized by T cells or is recognized by antibodies that bind to or recognize the amino acid sequence shown in each SEQ ID NO or its fragment. In another embodiment, the protein or its fragment or splice variant binds to self-HLA alleles and is recognized by T cells, which bind to or recognize the amino acid sequence shown in each SEQ ID NO or its fragment.

[0108] The determination method for measuring and / or predicting the combination with autologous HLA allele, the recognition by T cell or the recognition by antibody is well known to those skilled in the art. For example, the combination of peptide and HLA allele can be predicted by using the recognized NetMHCII (http: / / www.cbs.dtu.dk / services / NetMHCII / ) or IEDB (http: / / www.iedb.com) computer peptide binding prediction algorithm. T cell recognition can be measured, for example, by T cell proliferation determination method, for example, by measuring the radioactivity incorporated. The combination of peptide and / or protein with antibody can be measured by standard determination methods known to those skilled in the art, for example, by ELISA. The combination with autologous HLA allele, the recognition by T cell or the recognition by antibody may indicate the immunodominance of peptide or protein. Immunoantigenicity can also be tested as described below.

[0109] In one embodiment, the fragment represents a stretch of contiguous amino acids (eg, 20 to 30 amino acids) that is at least 90% identical or homologous between proteins of the RASGRP family.

[0110] In a preferred embodiment, the peptide for use in treatment according to the invention is a fragment of GDP-L-fucose synthase or a fragment of a protein of the RASGRP protein family and comprises a sequence selected from the group consisting of SEQ ID NOs: 10 to 98. In a particularly preferred embodiment, the peptide consists of an amino acid sequence as shown in one of SEQ ID NOs: 10 to 98. The sequences of SEQ ID NOs: 10 to 35 are preferred.

[0111] Sequences according to SEQ ID NOs: 10 to 35 have been identified as immunodominant peptides due to recognition by disease-associated T cells and subsequent validation of recognition by CSF-infiltrating bulk T cells for GDP-L-fucose synthase and PBMC for RASGRP2 (see "Examples"). The amino acid sequences according to SEQ ID NOs: 36 and 37 represent a segment of identical sequence between RASGRP2 and RASGRP3 (alignment of UniProtKB / Swiss-Prot Q7LDG7-1 and UniProtKB / Swiss-Prot Q8IV61). The amino acids according to SEQ ID NOs: 38 to 98 are included in a peptide library that elicits memory T cell responses from MS patients (see Examples 9 and Fig.10 ). Sequences with SEQ ID NOs: 12, 21, 23, 28 and 32 (GDP-L-fucose synthase) and SEQ ID NO: 46 (RASGRP2) have been used in CSF-infiltrating CD4 + This was confirmed in further analysis of T cells (see 13 of "Examples" and Figures 14 and 15).

[0112] The sequences of SEQ ID NOs: 1-98 are also listed in Table 1 below:

[0113] Table 1: Protein sequences, locations and database entries. Sequence Q7LDG7-1 is the canonical sequence of the RASGRP2 protein, also referred to as RASGRP2 isoform 1. The proteins and peptides described below are particularly preferred embodiments of the present invention.

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] The following gene sequences (Table 2) represent the gene sequences of the proteins GDP-L-fucose synthase (gene: TSTA3), RASGRP1, RASGRP2, RASGRP3 and RASGRP4 (Table 2). The proteins or their derivatives or splice variants are preferably encoded by the respective genes.

[0128] Table 2: Gene sequences and database entries

[0129]

[0130] The following nucleotide sequences (Table 3) represent preferred nucleotide sequences encoding any one of the proteins of the present invention or fragments, derivatives or splice variants thereof. The table also includes coding sequences (CDS), i.e. proteins or polypeptides, which can also be used for the treatment, diagnosis and / or prevention of multiple sclerosis (MS).

[0131] Table 3: Nucleotide and protein sequences and corresponding database entries (Genbank accession numbers)

[0132]

[0133]

[0134]

[0135]

[0136] All sequences were retrieved from various online databases on June 22, 2018.

[0137] In one embodiment, the protein, fragment, derivative or splice variant according to the invention can be used to identify a human subject suitable for tolerization to an autoantigen in MS, preferably in early MS. Identifying a human subject suitable for tolerization to an autoantigen in MS, preferably in early MS, preferably comprises measuring the positive reactivity of T cells and / or antibodies to the autoantigen in the human subject. Thus, MS can also be diagnosed in vitro in a human subject. In other words, the identified autoantigen can also be used for in vitro diagnosis of MS.

[0138] The in vitro diagnosis of MS preferably comprises the following steps: separating T cells (preferably CD4+T cells) and / or antibodies from the subject's blood, CSF or other body fluids, and measuring the reactivity of the T cells and / or antibodies to the protein according to the present invention or its fragment, derivative or splice variant. Those skilled in the art are aware of methods for separating T cells and / or antibodies from the subject's blood, CSF or other body fluids and measuring the reactivity of T cells and / or antibodies to the protein, fragment, derivative or splice variant. The reactivity of T cells (preferably CD4+T cells) and / or antibodies to the tested protein or its fragment, derivative or splice variant can indicate that the subject suffers from MS. Diagnosis can also be combined with clinical and imaging findings (i.e., according to the prior art, in particular, according to the revised McDonald criteria for MS diagnosis).

[0139] In another embodiment, the protein, fragment, derivative or splice variant according to the present invention may be used to distinguish MS subgroups. In particular, the protein, fragment, derivative or splice variant according to the present invention may be used to diagnose pattern II MS in human subjects.

[0140] The following characteristics indicate that certain peptides of a protein are immunodominant with respect to MS:

[0141] a) frequent recognition of this peptide by T cells, i.e., by approximately 10% or more of MS patients, often in the context of disease-associated HLA alleles or haplotypes (Sospedra and Martin, 2005), and

[0142] b) Disease-associated T cells that recognize this peptide, such as those that respond to low concentrations of the peptide (high-avidity T cells) (Bielekova et al., 2004) and are therefore considered particularly dangerous, and / or have a pro-inflammatory phenotype, and / or are isolated from the target organ or compartment (CNS), in the case of MS, brain, spinal cord or CSF-infiltrating T cells.

[0143] However, high-avidity recognition is not a prerequisite, as low-avidity myelin-specific T cells have also been shown to be pathogenic in a humanized transgenic mouse model (Quandt et al., 2012).

[0144] Thus, it is possible to test whether a protein or a fragment, derivative or splice variant thereof is immunodominant in the context of MS. Such a test is preferably an in vitro test. Particularly suitable are in vitro tests that allow the measurement of T cells and / or antibodies (preferably CSF-infiltrating CD4 T cells) obtained from the blood, CSF or other body fluids of a human subject diagnosed with MS. +T cells) to the tested protein or fragment, derivative or splice variant. Those skilled in the art will know how to test T cells (preferably CD4 + T cells) and / or antibody reactivity. For example, CD4 + Proliferation of T cells and / or their IFN-γ secretion or reactivity in ELISPOT / FLUOROSPOT assays or reactivity to HLA-peptide tetramers. If the tested protein or its fragment, derivative or splice variant induces reactivity in a human subject diagnosed with MS, then in the case of T cell reactivity, especially a stimulation index (SI) higher than 2 and / or IFN-γ secretion higher than 20pg / ml, the tested protein or fragment, derivative or splice variant can be referred to as immunodominant. 10 patients diagnosed with MS can also be selected for such testing. If reactivity is induced in at least 2 patients, the tested protein or fragment, derivative or splice variant can be referred to as immunodominant. Preferably, according to the established revised McDonald criteria, 10 patients have been diagnosed as suffering from RRMS.

[0145] It has recently been demonstrated that T cells from MS patients show increased proliferation in vitro in the absence of exogenous antigens (Mohme et al., 2013; Jelcic et al., 2018). These "self-proliferating" T cells are enriched in cells that home to the CNS compartment of MS patients and can therefore be considered a peripheral blood source of brain / CSF-infiltrating T cells.

[0146] If the data of in vitro T cell test cannot be obtained or in addition to such test, the immune recognition of peptides can also be predicted / inferred from those peptides that are well combined with individual HLA class I or class II alleles and used for CD8+ and CD4+T cells respectively. Peptide binding prediction is well known to technicians. Can be performed by perfect prediction algorithm (NetMHCII-www.cbs.dtu.dk / services / NetMHCII / ; IEDB-www.iedb.org / ) and analysis of HLA binding motifs (SYFPEITHI-www.syfpeithi), see 11 of "Examples".

[0147] According to the present invention, the proteins GDP-L-fucose synthase and members of the RASGRP protein family, in particular RASGRP2, have been identified as being immunodominant in MS and have therefore been identified as autoantigens.

[0148] It is not necessary that the binding to the HLA allele be particularly strong. In fact, peptides that bind poorly to HLA alleles may also be immunodominant (Muraro et al., 1997, J Clin Invest; 100(2): 339-349).

[0149] Tolerance induction is antigen-specific and renders autoreactive T cells nonfunctional or non-allergic, or induces Treg cells that specifically suppress adverse autoimmunity to the target antigen. Inducing tolerance to target autoantigens is a very important therapeutic goal in autoimmune diseases. It provides an opportunity to specifically weaken pathogenic autoimmune responses in an effective manner with almost no side effects. Tolerance induction can also be achieved by replacing or supplementing immunodominant peptides that are fragments of the protein with complete proteins (Kennedy MK et al., 1990). It has been shown herein that immunodominant peptides coupled to erythrocytes via intravenous (iv) injection can induce immunological changes consistent with tolerance in human patients (Example 14). Therefore, immunodominant peptides can be used for tolerance induction.

[0150] The immunodominance of the protein and / or fragments thus allows the use of said protein and / or fragments, derivatives or splice variants thereof for antigen-specific immunotherapy, such as tolerance induction.

[0151] According to the present invention, antigen-specific tolerization treatment can be used for all forms of MS: at the time of first presentation, when differential diagnoses have been excluded, the disease is called CIS as long as the CSF and MRI results are consistent with the diagnosis. MRI reveals lesions located in typical locations of MS (i.e. paracortical, paraventricular, in the brainstem or spinal cord). If specific criteria that can be summarized as spatial spread (more than one lesion or clinical symptom / sign) and temporal spread (more than one event) are met, a diagnosis of RRMS can be made. A special case is the accidental discovery of MRI lesions consistent with asymptomatic MS. This is called RIS and can be considered a pre-stage of CIS and RRMS. More than 80% of patients suffer from one of these, and most patients later develop so-called SPMS. At this point, relapses / aggravations become less frequent or stop completely, and neurological dysfunction increases steadily between relapses or when there are no relapses.

[0152] A special form of MS is PPMS, which never relapses but instead begins with a steady worsening of neurological symptoms, such as a steady worsening of the ability to walk. PPMS affects about 10% of people with MS and affects both sexes equally. Its onset is usually later than CIS or RRMS. With regard to causes and disease mechanisms, PPMS is thought to be similar to the above-mentioned RIS-CIS-RRMS-SPMS.

[0153] Typically, MS is diagnosed according to the modified McDonald criteria. These criteria also allow differentiation between different forms of MS and disease activity (Thompson et al., 2018, Lancet Neurol, 17(2): 162-173).

[0154] Preferably, the tolerization method is applied in the early stages (i.e., RIS, CIS, and early RRMS), since it is assumed that the immune process at this stage is mainly mediated by autoreactive T lymphocytes, while tissue damage (i.e., so-called degenerative changes) gradually becomes more and more important as the disease progresses. However, tolerization is meaningful as long as there is an autoreactive T cell response to the antigen used for tolerization (this is also the case during SPMS and PPMS).

[0155] In a particularly preferred embodiment, in the early stages, i.e., RIS, CIS and early RRMS, GDP-L-fucose synthase or RASGRP2 protein or its splice variant, preferably GDP-L-fucose synthase or RASGRP2 protein is used in the tolerization treatment method. The GDP-L-fucose synthase protein has, for example, the sequence shown in SEQ ID NO: 1, and the RASGRP2 protein has, for example, the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0156] In one aspect of the present invention, a method for inducing antigen-specific tolerance to self-antigens in a human subject suffering from MS or at risk of developing MS is provided. The method comprises the following steps: applying at least one protein selected from the group consisting of: GDP-L-fucose synthase and a member of the RASGRP protein family, a fragment (peptide), derivative and / or splice variant thereof, a nucleotide sequence encoding any one of the protein or its fragment, derivative or splice variant and / or a gene sequence described herein, or applying at least one carrier comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence described herein to a patient in need thereof (i.e., a human subject).

[0157] In one embodiment, the nucleotide sequence or gene sequence is applied to the patient via a carrier (e.g., a cell). The antigen is then expressed by the carrier (e.g., a cell). Transferring autoantigen encoding RNA / DNA to a carrier (e.g., a cell) and thus encoding the autoantigen in this way is similar to tumor vaccination methods using antigen encoding RNA.

[0158] Particularly preferably, the complete protein of GDP-L-fucose synthase (SEQ ID NO: 1) or the complete protein of RASGRP2 (SEQ ID NO: 2 or SEQ ID NO: 6, 7, 8 or 9) is used for inducing antigen-specific tolerance. In another preferred embodiment, a fragment of any of these proteins is used. Particularly preferably, a fragment as shown in any one of SEQ ID NO: 10 to 98 is used. Even more preferably, a peptide as shown in any one of SEQ ID NO: 10 to 35 is used.

[0159] The at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence can be administered nasally, by inhalation, orally, subcutaneously (sc), intracavitary (ic), intramuscularly (im), intradermally (id), transdermally (td) or intravenously (iv), preferably by an administration route considered to be tolerogenic, such as by iv, sc, id, td, orally, by inhalation, nasally or coupled to a tolerogenic carrier, preferably RBC.

[0160] In particular, this approach can be used to induce antigen-specific tolerance to self-antigens in early MS or even in preclinical stages of the disease.

[0161] The antigen-specific tolerance regimens provided herein can selectively target activated and naive autoreactive T cells specific for multiple potentially encephalogenic epitopes that perpetuate the disease.

[0162] Tolerization methods can also be used to prevent MS. The method can include identifying individuals who have a high risk of developing MS (e.g., in a family with an MS patient). For example, it is possible tolerize, for example, children of a mother with MS or identical twins of a patient with MS who are at particularly high risk of developing MS.

[0163] The diagnosis of MS or one of its forms is made by demonstrating the spatial and temporal spread of neurological deficits and / or MRI lesions consistent with MS. Positive laboratory tests for autoimmune responses to novel target proteins GDP-L-fucose synthase and the RASGRP family and / or their fragments, derivatives and / or splice variants can be used to identify patients who are particularly likely to benefit from antigen-specific tolerance induction, i.e., allow personalized antigen-specific tolerance methods. By identifying patients who are particularly likely to benefit from antigen-specific tolerance induction, patients with MS can be diagnosed in vitro. In other words, the identified autoantigens GDP-L-fucose synthase and the RASGRP family (particularly RASGRP2) and / or their fragments and / or derivatives and / or splice variants can be used for in vitro diagnosis of MS. Therefore, this in vitro test can be supplemented by clinical and imaging results (i.e., MS diagnosis according to the prior art, in particular according to the revised McDonald criteria).

[0164] In another aspect of the present invention, a method for identifying human subjects suitable for tolerization of autoantigens in MS, preferably early MS, is provided. Thereby determining individual patients who will benefit from or are suitable for the treatment. The method can also be used for in vitro diagnosis of MS. Patients with CIS (and possibly RIS) and RRMS are most suitable for tolerization, although as long as the patient responds to at least one antigenic peptide included in the tolerization treatment, the tolerization treatment seems to be meaningful for any form of MS (including SPMS and PPMS). The steps of the method include isolating T cells (preferably CD4+T cells) and / or antibodies from the subject's blood, CSF or other body fluids, and measuring the reactivity of the T cells and / or antibodies to the protein according to the present invention or its fragments, derivatives or splice variants. Those skilled in the art are aware of methods for isolating T cells and / or antibodies from the subject's blood, CSF or other body fluids and measuring the reactivity of T cells and / or antibodies to proteins, fragments, derivatives or splice variants.

[0165] If an individual responds to one of the one or more proteins or fragments, he / she can thereby be diagnosed in vitro as having MS and is suitable for treatment and a good candidate. A further selection step may be HLA class II typing and the presence of HLA-DR alleles associated with MS.

[0166] Therefore, proteins derived from disease-related antigens, especially GDP-L-fucose synthase proteins or proteins of the RASGRP family, especially RASGRP2, or fragments, derivatives or splice variants thereof, can be used to identify patients or patient subgroups with existing and / or particularly strong proinflammatory (potentially harmful) T cells or antibody responses to respective autoantigens. By identifying those patients or patient subgroups with existing and / or particularly strong proinflammatory (potentially harmful) T cells or antibody responses to respective autoantigens, patients with MS can therefore be diagnosed in vitro. In this case, patients are pre-tested with appropriate tests to assess reactivity to autoantigens, and tolerization treatments (e.g., compositions of peptides / proteins for tolerization treatment) can also be tailored for individual patients or patient subgroups, with the goal of making tolerization treatment as specific as possible and avoiding potential adverse effects. However, antigen-specific tolerization treatments can also be performed in patients who have not yet shown T cell responses to tolerization treatment antigens. Thus, in one embodiment, the proteins, fragments, derivatives and / or splice variants according to the invention may be used for in vitro pre-testing of human subjects diagnosed with MS or at risk of developing MS.

[0167] In one aspect of the invention, a carrier is provided, which comprises at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as described herein. The carrier can be coupled with at least one protein, fragment, derivative and / or splice variant, and / or the carrier can comprise at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence. In one embodiment, the term "comprising" refers to that the protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence is inside the carrier rather than on its surface. Protein, fragment, derivative and / or splice variant can also be coupled with the carrier and contained in the carrier, which means that a part of the protein, fragment, derivative and / or splice variant is coupled with the carrier and another part of the protein, fragment, derivative and / or splice variant is contained in the carrier.

[0168] Those skilled in the art are familiar with possible carriers.For example, the carrier can be any cell, protein, lipid, glycolipid, bead, nanoparticle, virus-like particle (VLP) or molecule (for example sugar molecule), or any combination thereof, which is suitable for use in humans and can be coupled with one or more proteins and / or fragments by coupling methods, for example by chemical coupling methods, preferably by EDC coupling. The carrier can be derived from a naturally occurring carrier or be a synthetic carrier. Preferably, the cell, molecule, bead, nanoparticle or VLP is biodegradable in vivo, or at least suitable for living humans, and decomposes in vivo, or eliminates from the body of the carrier. The term cell also includes cell precursors, for example RBC precursors. Preferably, the carrier is a hemocyte, even more preferably a red blood cell or a leukocyte. The leukocyte can be a splenocyte or a PBMC or is typically an APC.

[0169] In one embodiment, the protein, fragment, derivative and / or splice variant is expressed by a cell (preferably a blood cell). Thus, before the cell expresses the protein, fragment, derivative and / or splice variant, the genetic information encoding the protein, fragment, derivative and / or splice variant is introduced into the cell.

[0170] Any coupling agent or method for coupling the protein and / or its fragment to the carrier can be used. For example, a synthetic or natural joint can be used for coupling. An example of such a joint is glycoprotein A present on the surface of RBC. In one embodiment, chemical crosslinking is performed. In a preferred embodiment, a chemical crosslinking agent EDC that catalyzes the formation of peptide bonds between free amino groups and carboxyl groups is used. In particular, in the presence of EDC, a variety of peptides can be coupled to the surface of the carrier, thereby allowing simultaneous targeting of a variety of T cell specificities. Preferably, more than 3, more than 5, more than 10, more than 15 or even more than 20 different peptides are coupled to the surface of the carrier. In a preferred embodiment, 5 to 20, preferably 5 to 15 different peptides are used. If a peptide is not composed of the same amino acid sequence, it is different from another peptide. The carrier is preferably a cell but not necessarily a cell. As long as there is a free amino group, EDC can be used for coupling to any carrier.

[0171] In another embodiment, at least one peptide of each protein according to the present invention (GDP-L-fucose synthase protein or protein of the RASGRP protein family, preferably RASGRP2) is used in combination and coupled to a carrier. It is particularly advantageous to combine at least one peptide of each protein according to the present invention with at least one known peptide from the prior art, in particular with at least one myelin peptide, in particular with at least one or all of the myelin peptides defined by SEQ ID NOs: 261 to 267.

[0172] In a preferred embodiment, the carrier is a blood cell, and the blood cell is chemically coupled to at least one protein, fragment, derivative and / or splice variant via a coupling agent, preferably EDC. A method for producing such chemically coupled (i.e., antigen-coupled) blood cells is also provided, comprising isolating blood cells from a human subject, adding at least one protein, fragment, derivative and / or splice variant, i.e., antigen, and then adding a coupling agent, preferably EDC.

[0173] The mechanism of action of cells coupled to peptides by EDCs is not fully understood, but involves covalent attachment of the amino and carboxyl groups of the peptide to cell surface molecules, subsequent programmed cell death (apoptosis for nucleated cells and erythropoiesis for RBCs) of the peptide-coupled (i.e., antigen-coupled) cells, and then tolerogenic presentation of the dying cells in vivo.

[0174] The EDC dosage for the coupling reaction can be titrated to obtain maximum safety and optimal efficacy. At high concentrations, EDC may cause lysis of cells, particularly RBCs. In order to provide RBCs with optimal stability, a final EDC concentration of less than 15 mg / ml, preferably less than 10 mg / ml, even more preferably less than 5 mg / ml, even more preferably about 3 mg / ml can be used. The optimal dose can also vary. Those skilled in the art know how to determine the optimal stability of RBCs and the optimal dose of EDC.

[0175] The protein, fragment, derivative and / or splice variant to be coupled can be added in an amount that can be easily determined by a person skilled in the art. A person skilled in the art is aware of measures to determine the optimal amount in the interaction with the optimal amount of EDC.

[0176] Incubation time can be changed and customized (for example, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 120 minutes) for each specific coupling reaction. Along with the extension of incubation time, coupling efficiency can be better. In one embodiment, maximum value is reached after 60 minutes.

[0177] The incubation temperature may also vary. For example, 15-25°C or 2-8°C may be used. In one embodiment, the coupling efficiency is higher when the coupling reaction is performed at 15-25°C.

[0178] Any excipient that allows the coupling reaction can be used. In one embodiment, the excipient is sterile and endotoxin-free. In a preferred embodiment, the excipient is sterile, endotoxin-free saline (NaCl 0.9%). Saline is approved for use in humans and provides the greatest safety.

[0179] A person skilled in the art knows how to determine the optimal incubation time and temperature as well as possible excipients.

[0180] In one aspect of the present invention, a pharmaceutical composition is provided, comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence described herein and a pharmaceutically acceptable carrier.

[0181] In another aspect of the present invention, a method for the therapeutic treatment, prevention or diagnosis of MS in a human subject is provided, comprising administering to the subject a therapeutically effective amount of a protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence as described herein and / or a carrier as described herein. Thus, a carrier comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to the present invention, in particular a carrier coupled to at least one protein, fragment, derivative and / or splice variant according to the present invention, and / or a carrier containing at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to the present invention can be used for the treatment, diagnosis and / or prevention of MS.

[0182] In another aspect of the invention, the peptide according to the invention, preferably a peptide comprising 5 to 50, preferably 5 to 20, more preferably 10 to 15, even more preferably 15 amino acids, is for use as a medicament.

[0183] Preferred peptides for use as drugs are

[0184] a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to the respective corresponding amino acid sequence, or

[0185] b) is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the respective corresponding amino acid sequence, or

[0186] c) is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the respective corresponding amino acid sequence, binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the respective amino acid sequence.

[0187] Even more preferred for use as a drug are peptides having a sequence selected from the group comprising SEQ ID NOs: 10 to 98, preferably SEQ ID NO: 10 to 35, and even more preferred are peptides consisting of a sequence selected from the group comprising SEQ ID NO: 10 to 98, preferably SEQ ID NO: 10 to 35.

[0188] On the other hand, the peptide motif of Figure 3(ii)E (SEQ ID NO: 99), such as Figure 5 The GDP-L-fucose synthase peptides (SEQ ID NOs: 199-215) shown in FIG. 6 (I and II) (SEQ ID NOs: 10-14, 19-20, 22-23, 25-32 and 216-290) and Fig.12 The RASGRP1, RASGRP2 and RASGRP3 peptides in the table (SEQ ID NOs: 291-309) are all subject of the present invention and can be used according to the present invention.

[0189] Example

[0190] GDP-L-fucose synthase specificity

[0191] 1. Characterization of TCC21.1

[0192] Unbiased target antigen identification using positional scanning synthetic combinatorial libraries (ps-SCL) Figure 2Schematic representation. Clone TCC21.1 has been identified as described by Planas et al. (2015). TCC21.1 is a CD4+TCC isolated from CSF-infiltrating cells and clonally expanded in two active white matter demyelinating MS lesions (LI and LIII) from an SPMS patient with pattern II demyelination (1154SA). TCC21.1 releases Th2 cytokines and helps the proliferation and antibody production of autologous B cells after nonspecific activation. In order to study the peptides recognized by this TCC of unknown specificity using a ten-peptide position scanning library, the HLA class II molecules used by TCC21.1 to recognize peptide mixtures were first identified. This is a prerequisite for unbiased identification of target antigens using peptide library / biostatistical analysis methods (Zhao et al., 2001, J Immunol, 167: 2130-2141; Sospedra et al., 2010, J Immunol Methods, 353(1-2): 93-101). Since patient 1154SA was homozygous for the DR15 haplotype, TCC21.1 was initially tested with a mixture of amino acids (AA) defined at position 5 presented by EBV immortalized bare lymphocyte syndrome (BLS) B cell line (BCL) cells transfected with different single autologous HLA DR / DQ molecules (DRA*01:01 / DRB5*01:01=DR2a, DRA*01:01 / DRB1*15:01=DR2b and DQA1*01:02 / DQB1*06:02). Since TCC21.1 releases GM-CSF cytokine after non-specific stimulation with anti-CD3 and PMA, a readout of GM-CSF production by T cell activation was used (Planas et al., 2015). Only the mixture presented by BCL expressing DRB1*15:01(DR2b) class II molecules was stimulatory (data not shown).

[0193] 2. Positional Scanning Synthetic Combinatorial Libraries as a Method for Identifying Antigens / Identifying GDP-L-fucose Synthase

[0194] TCC21.1 was then tested with a complete ten-peptide position scanning library (200 mixtures; i.e., mixtures of 10 positions representing 10-mers and one of the 20 L-amino acids at each fixed position) presented by BCL expressing DRB1*15:01 class II molecules. GM-CSF release in response to the complete library is shown in Figure 3A. A similar response pattern was obtained for IL-10 release. Next, a biostatistical analysis scoring matrix was generated as previously described (Zhao et al., 2001) by assigning numerical values ​​to the stimulation potential of each of the 20 defined AAs at each of the ten positions of the ten-peptide library. Here, these values ​​were calculated as the base 10 logarithm of the median GM-CSF secretion (pg / ml) of three independent experiments in the presence of the mixture, minus the secretion in the absence of the mixture (Figure 3B). According to the model of independent contribution of individual AAs to peptide antigen recognition, the predicted stimulation score for a given peptide is the sum of the matrix values ​​for each AAs contained in the peptide at each position (Zhao et al., 2001). This scoring method was used to rank all natural overlapping 10-mers peptides of protein sequences in the UniProt human protein database according to their irritation score (prediction of their irritation potential). Based on these predictions, the top 50 predicted human natural peptides with the highest scores were synthesized and tested at 5 μg / ml (Figure 4). Unexpectedly, none of the peptides were significantly irritating (Figure 3C). For this TCC, the above method combining positional scanning libraries and biostatistical analysis had lower predictive power than other methods in previous studies. As shown in Figure 3B, the most irritating mixture had the same defined AAs in consecutive positions, suggesting that it is possible to recognize an AA motif in multiple frameworks. In order to be able to use Figure 2 The data were analyzed using the methods outlined in

[14] , and a collection of 22 doubly defined mixtures (i.e., positional scanning mixtures with two defined positions) were designed, synthesized, and tested ( FIG3D ); the results confirmed the presence of a unique recognition motif (LHSXFEV, SEQ ID NO: 99) with different flanking residues ( FIG3E ). To apply this recognition motif in both frameworks to the original GM-CSF-derived matrix and perform biostatistical analysis, the harmonic mean model (HM) was used to integrate the stimulus responses of certain doubly defined mixtures ( FIG3D , framework 1 / 2-HM mixtures) into the original matrix. Using the new “harmonic enhanced” framework 1 and framework 2 matrices, all naturally overlapping 10-mers peptides from the UniProt human protein database were scored and ranked as Figure 2Schematic representation. The 50 predicted natural peptides with the highest scores for both matrices were synthesized and tested for GM-CSF release (Figure 4). Three peptides that were clearly stimulatory were identified from both matrices (Figure 3F). The two most stimulatory peptides were NVLHSAFEVG (SEQ ID NO: 16) predicted by the harmonic enhancement framework 1 and DNVLHSAFEV (SEQ ID NO: 15) predicted by the harmonic enhancement framework 2, belonging to the GDP-L-fucose synthase encoded by the TSTA3 gene and overlapping by 9 AA.

[0195] Specifically, Figure 3 shows: A. TCC21.1 produces GM-CSF in response to a complete ten-peptide position scanning library (200 mixtures) presented by BLS cells expressing only DRB1*15:01. B. The scoring matrix design uses the log10 of the median GM-CSF production of three independent experiments. The bold border shows the mixtures selected for the dual-defined mixtures. C. TCC21.1 produces GM-CSF in response to the 50 peptides with the highest scores predicted using the GM-CSF-based scoring matrix. D. TCC21.1 produces GM-CSF in response to 22 dual-defined mixtures. The mixtures with defined AAs with TCR motifs in frame 1 are shown in gray and those in frame 2 are shown in black. The stimulatory responses of the mixtures shown in bold (frame 1 / 2-HM) were integrated into the original matrix using the harmonic mean model. E. TCR motifs and dual-defined mixture activity values ​​selected based on the harmonic mean model and incorporated into the original matrix, frame 1-HM is gray and frame 2-HM is black. F. TCC21.1 produces GM-CSF in response to 50 peptides with higher scores predicted using the reconciled boosting framework 1 and 2 scoring matrices. BLS cells expressing DRB1*15:01 presented the complete decapeptide library, dually defined mixtures, and individual decapeptides. Mixtures were tested at 200 μg / ml and individual decapeptides were tested at 5 μg / ml. Histograms show the mean ± standard error (SEM) of three independent experiments and dot plots show the mean of three independent experiments. The total cytokine released is expressed as pg / ml released by TCC21.1 in response to stimulation minus pg / ml released without stimulation (negative control).

[0196] Figure 4 shows a summary of the human decapeptides predicted by the biostatistical method that have been synthesized and then tested for stimulatory capacity based on GM-CSF release at 5 mg / ml. The peptides are ranked from 1 to 50 for HM reinforcement frameworks 1 and 2, respectively.

[0197] 3. RNASeq / transcriptome and proteome data demonstrate the expression of GDP-L-fucose synthase in brain tissue

[0198] Figure 5Transcript levels of GDP-L-fucose synthase in autologous brain LI and LIII are shown, expressed as "reads per kilo base of exon model per million mapped reads" (RPKM). Transcript values ​​for other brain-specific genes are also shown as quality controls for the samples and as references for genes expressed at high (MBP, PLP1) and medium (myelin-associated glycoprotein (MAG), myelin-associated oligodendrocyte basic protein (MOBP), and oligodendrocyte myelin glycoprotein (OMG)) levels.

[0199] By proteomic analysis, 17 GDP-L-fucose synthase peptides were identified in white and gray matter brain tissues from other MS patients and non-MS controls. Figure 5 Peptide sequences are listed along with peptide spectrum matches (PSM) in different samples. The percentage of GDP-L-fucose synthase AA sequence covered by the identified peptides is 56%. Positions are referenced to the sequence of UniProtKB / Swiss Prot: Q13630.1. Analysis of other brain-specific proteins as references is also included ( Figure 5 ).

[0200] In conclusion, GDP-L-fucose synthase has been shown to be expressed in the brain (RNA and protein).

[0201] 4. GDP-L-fucose synthase as a major autoantigen in brain-infiltrating TCC

[0202] To identify autoantigens recognized by TCC21.1 in two autologous brain lesions (LI and III (Planas et al., 2015)) in which TCC21.1 was known to be clonally expanded, all natural overlapping 10-mer peptides in protein sequences in the brain proteome database created using RNASeq-based transcriptome data from these two lesions (GSE60943) were then scored and ranked according to their stimulatory scores using the reconciled enhanced framework 1 and framework 2 matrices. Of the 40 predicted natural brain peptides with the highest scores in the framework 1 matrix, 38 peptides had been predicted from the unbiased UniProt human database. For the framework 2 matrix, the top 40 peptides were previously predicted (Figure 4). Two new framework 1 peptides were synthesized and tested. NVLHSAFEVG (GDP-L-fucose synthase 96-105, SEQ ID NO: 16) and DNVLHSAFEV (95-104, SEQ ID NO: 15) were found to stimulate TCC21.1 (Figure 4).

[0203] 5. Characterization of GDP-L-fucose synthase recognition / characterization of response

[0204] As described above, the two GDP-L-fucose synthase peptides recognized by TCC21.1 overlapped by 9 AAs. On BCL cells expressing DRB1*15:01, another 9 10-mer peptides with 9 AAs overlap were synthesized and tested, and another peptide VLHSAFEVGA (97-106, SEQ ID NO: 18) that induced GM-CSF release was identified. The peptide NVLHSAFEVG (96-105, SEQ ID NO: 16) gave the best response with an EC50 of 0.2 μg / ml, presented by DRB1*15:01 and DQB1*06:02 molecules. The common AA of the three stimulatory peptides was VLHSAFEV (97-104) (data not shown).

[0205] Next, the response of TCC21.1 to GDP-L-fucose synthase peptides presented by autologous irradiated PBMCs and BCLs was characterized. GDP-L-fucose synthase peptides presented by two types of APCs were able to induce proliferation. The functional phenotype of the response was also analyzed. TCC21.1 showed a Th2 phenotype, mainly releasing Th2 cytokines and lower levels of IL-22 and IL-10. Unexpectedly, when the peptides were presented by autologous BCLs, they induced higher levels of IFNγ. In addition, TCC21.1 also released GM-CSF and IL-3 in response to GDP-L-fucose synthase peptides. The release of these two cytokines appears to be a specific feature of this TCC compared to other Th1, Th1* or Th1 / 2 TCCs generated from patients with different conditions. Intracellular cytokine staining confirmed the Th2 functional phenotype of TCC21.1 in response to GDP-L-fucose synthase (96-105, SEQ ID NO: 16). After stimulation with GDP-L-fucose synthase (96-105, SEQ ID NO: 16), more than 60% of TCC21.1 cells were IL-4+, while only about 12% were IFNγ+. About 60% of the cells were GM-CSF+, of which 47.7% were also IL-4+. Further characterization of TCC21.1 demonstrated the expression of CD28 and chemokine receptor CRTh2 (data not shown).

[0206] 6. Recognition of GDP-L-fucose synthase by CSF-infiltrating CD4+ T cells from patient 1154SA

[0207] 62 15-mer peptides overlapping 10 AA and covering the entire GDP-L-fucose synthase protein were synthesized (Figure 6) and tested for their ability to induce TCC21.1 proliferation when presented by autologous PBMCs. Seven immunodominant / encephalitogenic myelin peptides (Bielekova et al., 2004), CEF (cytomegalovirus, EBV, influenza virus and tetanus toxoid) peptide pools and control beads were tested in parallel (Figure 6). TCC21.1 recognizes two overlapping GDP-L-fucose synthase peptides, namely 91-105 (SEQ ID NO: 14) and 96-110 (SEQ ID NO: 17), which contain three previously identified stimulatory decapeptides (i.e., 95-104 (SEQ ID NO: 15), 96-105 (SEQ ID NO: 16) and 97-106 (SEQ ID NO: 18)).

[0208] 7. Recognition of GDP-L-fucose synthase by CSF-infiltrating CD4+ T cells from CIS / MS patients

[0209] In order to find out whether specific recognition of GDP-L-fucose synthase occurs in CSF-infiltrating CD4+T cells of patients with different forms of MS (mostly CIS and RRMS), a new protocol was developed to amplify fresh CSF-infiltrating CD4+T cells in large quantities in a single round to minimize the differences in the original T cell pool. CSF-infiltrating CD4+T cells (also derived from T cells in the CNS compartment) from 31 CIS / MS patients with resting PHA amplification were tested in quadruplicate with 62 overlapping GDP-L-fucose synthase peptides presented by autologous irradiated PBMCs, as well as seven myelin peptides, CEF peptide pools and control beads. All stimulation indexes (SI; except control beads) were summarized, and SI values ​​less than 1 were considered to have unit values. Cluster k-means analysis was performed to determine the best critical value to distinguish between SI values ​​with responses and SI values ​​without responses in this patient population. The critical value obtained by K-means clustering was 1.455 to distinguish between positive responses and negative responses. Subsequently, for each patient, all peptides with a median SI (in quadruplicate wells) greater than 1.455 were identified as positive responses.

[0210] Next, a patient score was constructed by calculating the sum of the median SI for each responsive peptide, weighted by the total number of patients who responded to that peptide. In this way, the SI value of each peptide itself as well as the relative immunogenicity of each peptide were taken into account to assess the frequency and intensity of the immune response to a specific antigen. A three-cluster k-means analysis was performed based on the 10 patient scores and clearly divided the patients into three categories: “non-responders”, “moderate responders” and “high responders”. Thus, 19 patients (61.3%) were characterized as non-responders to GDP-L-fucose synthase, 6 (19.35%) were moderate responders, and 6 (19.35%) were high responders (data not shown). No significant differences were found between the three groups for CEF responses or for positive or negative controls. Immunodominant peptides are defined as peptides capable of inducing a positive response in at least 10% of patients. The 14 GDP-L-fucosylated peptides that met this criterion are as follows Figure 7 Functional analysis of the strongest responses to certain immunodominant peptides revealed a Th1 phenotype with predominantly IFN-γ production (data not shown).

[0211] In detail, Figure 7 Shown: Number of CIS / MS patients with CSF-infiltrating CD4+ T cells responding to GDP-L-fucose synthase peptides. Black shows immunodominant peptides that were positive in at least three patients. Black squares are high responders and white squares are intermediate responders.

[0212] In summary, approximately 20-25% of MS patients (CIS, RRMS, SPMS) showed immune responses to different immunodominant GDP-L-fucose synthase peptides; the majority of these GDP-L-fucose synthase-specific T cells had a Th1 phenotype (the most common phenotype in MS patients). Comparison with responses to seven myelin peptides (ETIMS peptides) showed that significantly fewer patients responded to these peptides compared to GDP-L-fucose synthase.

[0213] RASGRP2 specificity

[0214] 8. Identification of TCC14 and Testing of ps-SCL with TCC14 to Identify RASGRP2

[0215] TCC14 was similarly identified from MS patient 1 (homozygous for HLA-DR15), but in this case from a fraction of self-proliferating (proliferating without stimulation) peripheral blood T cells that was enriched for brain-homing T cells. TCC14 was also shown to be clonally propagated in the brain by deep TCR sequencing of cells infiltrating the patient's brain lesions. Figure 1 As shown schematically (right part), TCC14 was generated. The isolation of self-proliferating T cells is shown in more detail in Figure 8In detail, peripheral blood mononuclear cells were plated in duplicate wells after labeling with the dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) and without stimulation. After 7 days of culture, proliferating cells (CFSE dim ) and non-proliferating cells (CFSE hi ) and isolated from proliferating T cells by cell sorting (CFSE dim ). Comparison of MS brain lesions and CFSE dim TCRβV sequences between (self-proliferating) populations. More than 20% CFSE dim TCRβV sequences of the population were also found in MS brain lesions ( Figure 8 B).

[0216] Then as Figure 2As shown, TCC14 was amplified and tested using a positional scanning combinatorial peptide library and biostatistical analysis as described above, using an unbiased approach to identify one or more target antigens of TCC14. TCC14 was amplified well enough to be tested with a full set of 200 positional scanning library mixtures. Because TCC is derived from an MS patient who is homozygous for HLA-DR15, the restriction of TCC14 was tested using BLS cells transfected with an HLA-DR15 haplotype (DR2a, DR2b, or DQw6) expressing an HLA class II allele. After determining its HLA class II restriction (DRB1*15:01), the reactivity against all 200 samples was tested using BLS cells transfected with DRB1*15:01, and positive responses were shown for single or multiple amino acids (aa) in each of the 10 positions. After 72 hours, proliferation based on the thymidine incorporation assay (stimulation index = SI; dashed line SI = 2) was used as a readout of the TCC response (Figure 9, panel I). After testing multiple doses, a scoring matrix was used to summarize the reactivity of TCC14 to all 20 L-aas in each of the 10 positions of the decapeptide library, and the peptides recognized by TCC14 were predicted using a biostatistical analysis process (Figure 9, panel II) (Zhao et al., 2001). The average response from three replicates was used to generate a matrix of optimal amino acid combinations for potential peptide ligands. The brain transcriptome data and corresponding proteins of patient 1 from whom TCC was isolated were used as a search database. 92 sequences were synthesized and tested for recognition by TCC14 (Figure 9, panel III) based on their appearance in the top 50 predicted peptides in at least one of the matrices used (stimulation response of SI>3). As previously shown for other TCCs, there is a good relationship between the predicted high ranking and T cell response, since TCC14 recognizes many high-scoring peptides (Sospedra et al., 2010; Zhao et al., 2001). In order to evaluate the functional affinity of these peptides, dose titration experiments were performed using 33 peptides that gave positive responses. Among them were peptides with SEQ ID NOs: 33 to 35. After 72 hours, the proliferation response of TCC14 was tested against decreasing concentrations of stimulatory peptides using BLSDR2b. (Figure 9, panel IV). The peptide from RASGRP2 (SEQ ID NO: 33) was recognized with high antigen affinity (EC50 = 0.012 μM), but peptides from several other RASGRP isoforms (RASGRP1, -3, 4) and other peptides also gave positive responses (Figure 9, panel IV). Recognition of RASGRP2 peptides by TCC14 resulted in the secretion of Th2 cytokines as well as IFN-γ (data not shown).

[0217] In conclusion, clone TCC14 can recognize multiple RASGRP versions, with RASGRP2 having by far the highest affinity (i.e., at lower antigen concentrations), thus emphasizing its biological relevance.

[0218] 9. RASGRP2 Reactivity in Peripheral Blood Cells

[0219] To test the reactivity of peripheral blood-derived memory T cells to RASGRP2, cryopreserved PBMCs (1×10 8 2 × 10 cells were thawed and then depleted of CD45RA-expressing cells using magnetic cell sorting (Miltenyi). 2 × 10 5 CD45RA-depleted PBMCs (10-15 replicate wells per condition) were treated with vehicle (DMSO), treated with CD2 / CD3 / CD28 beads, or loaded with RASGRP2 peptide pools (final pool concentration was 10 μM) or intact purified RASGRP2 protein (Origene; 0.3 μg / ml). 15mer overlapping peptides covering the entire RASGRP2 protein (SEQ ID NO: 38-98) were divided into 9 peptide pools, each with 7 peptides covering the RASGRP2 sequence from the N-terminus (pool 1) to the C-terminus (pool 9). Thymidine incorporation assays were used to measure the proliferative response to RASGRP2. On day 7, cells were treated with 1 μCi of methyl- 3 H-thymidine (Hartmann Analytic) was loaded and collected on membranes (Tomtec) after 15 hours. Incorporation was measured by β-scintillation counting (Wallac 1450, PerkinElmer). Results are shown as points (mean ± SEM). The stimulation index (SI) was calculated as the ratio of peptide or protein stimulation to vehicle control. SI values ​​> 2 were considered positive ( Fig.10 ).

[0220] all in all, Fig.10 Memory T cells from MS patients with high autoproliferative responses to RASGRP2 were shown. All eight donors responded to single or multiple peptide pools of RASGRP2 peptides (including pool 2, which contained a peptide with SEQ ID NO: 46 that overlapped with the target peptide of TCC 14 (SEQ ID NO: 33)) and / or the whole protein, demonstrating that RASGRP2 is an autoantigen that is widely recognized by MS patients with high autoproliferation.

[0221] 10. RNASeq / transcriptome and proteome data demonstrate expression of RASGRP2 in B cells and in the brain

[0222] The expression of RASGRP1-4 was tested at the RNA and protein levels in peripheral B cells and brain ( Fig.11 RASGRP1-3 were expressed in the transcriptome of both brain and self-proliferating memory B cells of patient 1 ( Fig.11 ).

[0223] In detail, Fig.11 Shown: (A) Active brain lesion III (RPKM) from MS patient 1 and self-proliferation (CFSE) from 6 RRMS (REM) patients dim ) Expression levels of stimulatory peptide-derived transcripts in peripheral blood B cells (RPKM). Expression levels below 0.1 or no transcript expression were set to 0.1. Expression of control transcripts in brain (MOBP) and B cells (CD19) is also shown. (B) Mass spectrometry analysis of RRMS peripheral blood B cells (REM, nihil; n=4) and brain tissue (gray matter, pooled, n=6) of MS patients. Protein coverage (bars) and spectral counts (numbers) of RASGRP1-4 are depicted as measures of protein abundance.

[0224] Brain tissue (white and gray matter) from controls and MS patients was also tested by proteomics using mass spectrometry. Peptides of RASGRP1, 2, and 3 proteins were identified in brain tissue, with RASGRP2 in particular being highly abundant ( Fig.12 ). This position refers to the following sequences: GenBank AAC97349.1 (RASGRP1), GenBank AAI10307.1 (RASGRP2), and GenBank AAY15037.1 (RASGRP3).

[0225] Furthermore, immunohistochemistry (IHC) studies were performed and showed the expression of RASGRP2 protein in the brain gray matter (particularly in cortical neurons) and spleen (data not shown).

[0226] 11. Identification of other immunodominant peptides in GDP-L-fucose synthase and RASGRP2 sequences

[0227] In addition, peptides with potential immunogenicity have been identified based on commonly used search algorithms and assumptions. This method has been adopted from the context of tumor vaccination, where it is standard procedure to find immunogenic peptides in self-proteins (from tumors). In this case, protein sequences are screened for peptides that are expected to bind to disease-related HLA alleles (or HLA alleles of a given tumor patient). Therefore, GDP-L-fucose synthase and RASGRP2 sequences were used, and the recognized NetMHCII (http: / / www.cbs.dtu.dk / services / NetMHCII / ) and IEDB (http: / / www.iedb.org / ) computer peptide binding prediction algorithms were used to predict strong (SB) or weak (WB) binding peptides. Detailed information on how to perform the search can be easily found on the websites of the two search algorithms. In short, the search requires copying the sequence of the target protein into the web tool and selecting the HLA class II (or class I, if interested) allele of interest. The algorithm will then generate peptide sequences and their respective predicted binding to the HLA class II allele. For alleles for which NetMHCII search was not possible, IEDB was used. HLA alleles known to be associated with MS have been used. If one now considers all regions of both proteins that are predicted to be WB (of greatest interest in the context of autoantigens) or SB (NetMHCII search), or have a predicted binding rank of 25% or less (IEDB), these amino acid stretches cover nearly all of the complete GDP-L-fucose synthase and RASGRP2 proteins:

[0228] For the GDP-L-fucose synthase protein, peptide binding was predicted for the region spanning amino acids 1 to 315.

[0229] For the RASGRP2 protein, peptides spanning regions from amino acids 9 to 449 and 457 to 659 were predicted to bind.

[0230] The following alleles were used:

[0231] -HLA-DRB1*15:01(NetMHCII)

[0232] -HLA-DRB5*01:01(NetMHCII)

[0233] -HLA-DRB1*03:01(NetMHCII)

[0234] -HLA-DRB1*13:03(IEDB)

[0235] -HLA-DRB1*08:01(IEDB)

[0236] -HLA-DRB3*02:02(IEDB)

[0237] -HLA-DRB1*04:01(NetMHCII)

[0238] -HLA-DRB1*04:04(NetMHCII)

[0239] -HLA-DQw6(DQA1*01:01;DQB1*06:02)(IEDB)

[0240] The following protein sequences were used:

[0241] GDP-L-fucose synthase: GenBank: AAH93061.1

[0242] RASGRP2: GenBank: AAI10307.1

[0243] In conclusion, the intact protein and the immunodominant peptides are suitable for use in the treatment, diagnosis and / or prevention of MS.

[0244] 12. Making chemically coupled red blood cells

[0245] EDC as a chemical crosslinker

[0246] Peptides synthesized with biotin residues (biotin-PLP1; PLP1 = PLP 139-154) have been used for highly specific detection of peptides using fluorophore-conjugated streptavidin. Briefly, peripheral blood mononuclear cells were loaded with biotin-PLP1 (final concentration 0.05 mg / ml), EDC (final concentration 10 mg / ml) (all in PBS) at 4°C for 1 hour. After two washing steps, cells were stained with fluorophore-conjugated streptavidin and analyzed by flow cytometry (streptavidin-APC).

[0247] like Fig.13 As shown, effective peptide binding was observed only when both (biotin-PLP peptide and EDC) were present simultaneously.

[0248] 13. Further analysis was performed on 105 MS patients (for GDP-L-fucose synthase) and 57 MS patients (for RASGRP2)

[0249] Further analysis was performed on 105 MS patients (for GDP-L-fucose synthase) and 57 MS patients (for RASGRP2) to validate the immunodominance of GDP-L-fucose synthase peptides 51-65, 136-150, 161-175, 246-260 and 296-310 (SEQ ID NOs: 12, 21, 23, 28 and 32) and RASGRP2 peptide 78-92 (SEQ ID NO: 46). The reactivity of CSF-infiltrating CD4+ T cells to the test peptides, which are highly associated with the pathogenicity of MS, was compared with the reactivity to the known immunodominant reference peptides MBP 13-32, MBP 83-99, MBP 111-129, MBP 146-170, MOG 1-20, MOG 35-55 and PLP 139-154.

[0250] The results are shown in Figures 14 and 15. The data show that the test peptides, which are fragments of the proteins GDP-L-fucose synthase and RASGRP2 identified herein, have the same reactivity or even higher reactivity than the known immunodominant reference peptides. Therefore, the test peptides were confirmed to be immunodominant again. It is particularly interesting to study CSF-infiltrating T cells and their response to putative autoantigens because autoreactive T cells that have infiltrated the target organs (i.e., brain, spinal cord or CSF) are considered to be potentially biologically relevant.

[0251] Proliferative responses and IFN-γ secretion were measured as described above in "7." and below in "15. Materials and Methods", unless otherwise specified.

[0252] Specifically, Figure 14 shows: A. Proliferative response, expressed as CSF-infiltrating CD4 + Stimulation index (SI) and IFN-γ secretion (pg / ml) of T cells (single round of PHA expansion) to 5 GDP-L-fucose synthase peptides (51-65, 136-150, 161-175, 246-260 and 296-310), 4 MBP peptides (13-32, 83-99, 111-129 and 146-170), two MOG peptides (1-20 and 35-55), one PLP peptide (139-154) and CEF peptide presented by autologous PBMCs. All peptides were tested in four wells / patient. Each point represents a well, and each peptide was tested in 420 wells (4 wells x 105 patients). Positive wells are wells with SI greater than 2 (dashed line) or IFN-γ greater than 20pg / ml (dashed line). The percentage of positive wells and the ratio between the percentage of IFN-γ and SI positive wells are also shown. B. CSF infiltrating CD4 with or without specificity identified for different peptides +Percentage of positive patients for T cells. Positive patients were defined as patients with more than 2 out of 4 wells positive for SI (left histogram), IFN-γ (middle histogram), and SI or IFN-γ (right histogram).

[0253] Specifically, Figure 15 shows: A. Proliferative response, expressed as CSF-infiltrating CD4 + Stimulation index (SI) and IFN-γ secretion (pg / ml) of T cells (single round of PHA expansion) to one RASGRP2 peptide (78-92), four MBP peptides (13-32, 83-99, 111-129 and 146-170), two MOG peptides (1-20 and 35-55), one PLP peptide (139-154) and CEF peptide presented by autologous PBMCs. All peptides were tested in four wells / patient. Each dot represents a well, and each peptide was tested in 228 wells (4 wells x 57 patients). Positive wells are wells with SI greater than 2 (dashed line) or IFN-γ greater than 20pg / ml (dashed line). The percentage of positive wells and the ratio between the percentage of IFN-γ and SI positive wells are also shown. B. CSF-infiltrating CD4 with or without identified specificity for different peptides + Percentage of positive patients for T cells. Positive patients were defined as patients with more than 2 out of 4 wells positive for SI (left histogram), IFN-γ (middle histogram), and SI or IFN-γ (right histogram).

[0254] 14. In vivo tolerance induction with myelin peptides in a phase Ib trial

[0255] Tolerance induction was tested in 10 patients diagnosed with MS. In particular, in a Phase Ib trial, the safety and tolerability of peptide-coupled, EDC-fixed erythrocytes in vivo and indicators of tolerance induction were tested by intravenous (iv) injection of autologous erythrocytes chemically (via EDC) coupled to a panel of myelin peptides (MBP13-32, MBP 83-99, MBP 111-129, MBP146-170, MOG 1-20, MOG 35-55 and PLP 139-154) (SEQ ID No: 261-267). Briefly, blood was drawn from each patient and erythrocytes were separated. The erythrocytes were then chemically coupled to myelin peptides ex vivo under sterile conditions and injected intravenously (iv) into the patient. Two patients received 1x10 10 cells, 3 patients received 1x10 11 cells, 5 patients received 3x10 11 The day of injection was defined as day 0.

[0256] Blood was also collected 6 weeks before injection (before tolerance treatment) and 12 weeks after injection (after tolerance treatment). On these dates, peripheral blood lymphocytes were obtained and examined by flow cytometry using fluorescently labeled antibodies for the presence of a variety of different cell types, including the following subpopulations: T cells, B cells, monocytes and dendritic cells, natural killer cells, including those expressing markers of induced T regulatory cells (Tr1) or natural T regulatory cells (nTregs). The latter two cell types can be characterized by the following markers:

[0257] -T regulatory 1 (Tr1) cells (CD3+CD4+CD45RA-CD49b+LAG3+)

[0258] -FoxP3+ natural T regulatory (nTreg) cells (CD4+CD25hi FOXP3+)

[0259] In addition, T cell reactivity to all seven peptides was measured separately for peripheral blood T cells and for CSF-infiltrating T cells, as described below in “Testing of large numbers of CSF-derived T cells from CIS and RRMS patients against GDP-L-fucose synthase and myelin peptides” and “T cell stimulation” on the same days, i.e., 6 weeks before and 12 weeks after injection.

[0260] Figure 16 shows that signs of antigen-specific tolerance induction following injection of coupled erythrocytes are detectable, as measured by an increase in Tr1 and FoxP3+nTreg cells and a decrease in peptide-specific T cell reactivity. These data indicate that tolerance to individual immunodominant peptides can be induced by administering immunodominant peptides.

[0261] In detail, Figure 16 shows:

[0262] A. Percentage of Tr1 cells in CD4+ memory cells 6 weeks before injection (= before tolerization treatment) and 12 weeks after injection (= after tolerization treatment). B. Percentage of FoxP3+ nTreg cells in CD4+ memory cells 6 weeks before injection (= before tolerization treatment) and 12 weeks after injection (= after tolerization treatment). C. Percentage of Tr1 cells in CD4+ memory cells 6 weeks before injection and 12 weeks after injection with 3x10 11 T cell reactivity in 5 patients with 10 cells (grey dots: before injection; black dots: after injection). The upper graph shows the percentage of cells that responded to all peptides. The lower graph shows the proliferation of each individual microwell (60 in total).

[0263] 15. Materials and Methods

[0264] Patient Materials

[0265] GDP-L-fucose synthase

[0266] Patient 1154SA: CSF-derived mononuclear cells and PBMCs were obtained from a SPMS patient with type II demyelinating lesions as previously described (Planas et al., 2015). The HLA class I and II types in this patient were: A*32:01, A*33:01, B*14:02, B*51:01, DRB1*15:01, DRB5*01:01, DQB1*06:02, DQA1*01:02.

[0267] Methods: CSF and paired peripheral blood were collected from diagnostic lumbar punctures of 31 untreated MS patients: 8 patients with CIS, 20 with RRMS, and 3 with SPMS. Patients were recruited from the inims clinic and day hospital at the University Medical Center Hamburg-Eppendorf and the inims department of the neurology clinic at the University Hospital Zurich. MS diagnosis was based on the modified McDonald criteria. All CIS patients had CSF-specific oligoclonal bands detected by isoelectric focusing (IEF). Patients who had not taken steroids for at least 4 weeks before enrollment or any immunomodulators or immunosuppressants in the past 3 months were considered untreated and included in the study. Fresh CSF cells from these patients were expanded in vitro (see below). PBMCs were freshly isolated from EDTA-containing blood tubes by Ficoll density gradient centrifugation (PAA, Pasching, Austria) and cryopreserved. The Ethik Kommission Hamburg approved the use of the FDA-approved standard of care. The study procedures were approved by the Ethical Committee of the Canton of Zurich (Ethical Committee of the Hamburg, Project No. 2758) and the Cantonal Ethical Committee of Zurich (Research Project EC-No: 2013-0001). Informed consent was obtained from all patients or their families.

[0268] Brain autopsy tissue from 13 MS patients (7 SPMS, 5 PPMS and 1 primary relapsing (PR) MS) and 7 non-MS controls was obtained from the UK Multiple Sclerosis Tissue Bank (UK Multicentre Research Ethics Committee, MREC / 02 / 2 / 39).

[0269] For further analysis of GDP-L-fucose synthase (13 of "Example"), CSF was collected from 105 patients. Among the 105 patients, the female:male ratio was 1.9, the average age was 35.68 years (range 17-58 years), 4 patients were diagnosed with RIS, 10 patients were diagnosed with CIS, 82 patients were diagnosed with RRMS, 4 patients were diagnosed with SPMS, and 5 patients were diagnosed with PPMS.

[0270] RASGRP type 2

[0271] PBMCs were isolated from patient 1154SA by Ficoll density centrifugation.

[0272] Further analysis of RASGRP2 (13 of "Example"). CSF was collected from 57 patients. These 57 patients belonged to 105 patients tested for GDP-L-fucose synthase. Among the 57 patients, the ratio of female to male was 2.5, the average age was 35.33 years (range 17 to 55 years), 2 patients were diagnosed with RIS, 8 patients were diagnosed with CIS, 43 patients were diagnosed with RRMS, 1 patient was diagnosed with SPMS, and 3 patients were diagnosed with PPMS.

[0273] Self-proliferation assay

[0274] PBMCs were thawed in complete IMDM medium (GE Healthcare) containing 100 U / mL penicillin / streptomycin (Corning), 50 μg / mL gentamicin (Sigma-Aldrich), 2 mmol / L L-glutamine (PAA) and 5% heated decomplemented human serum (HS, PAA), and then washed once with serum-free AIM-V medium (GIBCO, Thermo Fisher Scientific) containing human albumin. Cells were incubated in AIM-V medium containing 50 U / ml DNase (Roche) at 37°C for 15 minutes to avoid cell clump formation. After two washing steps with PBS containing 0.1% HS, cells were plated at 10 × 10 6 The cells were resuspended in PBS / 0.1% HS at a concentration of 10 cells / ml and then labeled with a final concentration of 0.5 μM CFSE (Sigma-Aldrich) for 3 minutes at room temperature. The labeling was terminated by quenching with a 5x excess volume of cold complete RPMI (PAN-Biotech) medium containing 10% HS. After another washing step with AIM-V, CFSE-labeled cells were plated at 2×10 cells / ml at 37° C., 5% CO2, without exogenous stimulation (= self-proliferation). 5PBMC / 200 μl / well were seeded in AIM-V in a 96-well U-bottom microtiter plate (Greiner Bio one) (10-12 replicate wells for each donor and condition). For conventional T cell responses, for the same donor, PHA (0.5 μg / ml) was used as a TCR-independent stimulator, tetanus toxoid (TTx, 5 μg / ml, Novartis Behring) was used as a foreign antigen stimulator, and mixed lymphocyte reaction (MLR) was used as an allogeneic antigen stimulator. After 7 days, CFSE-labeled cells were collected and cells from replicate wells were combined, washed with PBS, Fc-blocked with human IgG (Sigma-Aldrich), and incubated at 4°C with Aqua (Invitrogen, Thermo Fisher Scientific). After washing with cold PBS containing 2 mM EDTA and 2% FCS, cells were directly stained for surface markers using fluorochrome-conjugated antibodies (Key Resources Table). Measurements were performed on an LSR Fortessa flow cytometer (BD Biosciences) and data were analyzed with FlowJo (Tree Star). The assay was further used to test competition from self-proliferation by incubating CFSE-labeled PBMCs in the presence of anti-HLA-DR, anti-CD4, anti-IFN-γ, and anti-GM-CSF antibodies (10 μg / ml) or appropriate isotype controls for 7 days. For the thymidine incorporation assay, 2 × 10 5 PBMCs / well (10-12 replicate wells per donor and condition) were cultured in serum-free AIM-V medium in 96-well U-bottom microtiter plates at 37°C, 5% CO2, and treated with 1 μCi of methyl- 3 H-thymidine (Hartmann Analytic) was loaded and cells were harvested 15 hours later (Tomtec). Incorporation was determined by β-scintillation counting (Wallac 1450, PerkinElmer).

[0275] T cell clones

[0276] To generate TCCs from the self-proliferating compartment, CFSE from MS patient 1 (used for TCR Vβ sequencing, as described above) was used. dim 500 CFSE cells were sorted from a cell bank (20,000 cells) dimCells were added and limiting dilution was performed as described previously (Aly et al., 2011). TCCs were enriched by using an amplification scheme of PHA (Sigma) and human IL-2 (Aly et al., 2011). As described previously (Yousef et al., 2012), sequencing of TCR rearrangements of generated TCCs was analyzed. In order to evaluate the cytokine response of TCCs, each TCC in 7 replicate wells was stimulated using MACSibead particles (Miltenyi) loaded with anti-CD2 / CD3 / CD28 antibodies, wherein each well had 200,000 cells in X-Vivo culture medium (Lonza). As described above, supernatants were collected after 48 hours and cytokine responses were measured. For the expression of chemokine receptors, after thawing and resting the cells overnight, TCCs were stained with Live / dead Aqua and antibodies for CXCR3 and CCR6.

[0277] Transcriptome analysis

[0278] Transcriptomic analysis of brain lesions was performed as previously described (Planas et al., 2015). The data discussed are deposited in the Gene Expression Omnibus at NCBI and can be accessed through GEO Series Accession No. GSE60943.

[0279] Proteomic analysis

[0280] For proteomic analysis, pressure-assisted protein extraction and digestion were performed using a pressure cycler (barocycler) (2320EXT, BioSciences, Inc, South Easton, MA). Before the sample was digested with Lys-C and trypsin, the homogenate was reduced and alkylated. The peptide was desalted on a solid phase extraction column (C18 Finisterre, Wicom Germany), vacuum dried, redissolved and measured (Nanodrop 1000, spectrophotometer (Thermo Scientific, Wilmington, DE, USA)). The resulting peptides were purified by hydrophilic interaction chromatography (HILIC, Agilent LC1200, equipped with a polyamide II column 250x 3.0mm 5 μm) for purification and separation, followed by injection into the nano-liquid chromatography system Easy nLC connected to an Orbitrap Fusion instrument (Thermo Fisher). Data analysis was performed using the human UniProtKB / Swiss Prot protein database (March 22, 2016, 40912 entries) using MASCOT software. Search parameters were 0.05 Da fragment mass tolerance and 10 ppm precursor mass, a minimum number of peptides 2, an FDR (false discovery rate) of 0.1%, and 2 false cleavages of tryptic fragments were allowed. Carbamidomethylation at cysteine ​​was set as a fixed modification, and oxidation of methionine, n-terminal acetylation were set as variable modifications.

[0281] Positional scanning of peptide libraries and individual peptides

[0282] GDP-L-fucose synthase

[0283] A synthetic N-acetylated, C-amide L-amino acid (AA) decapeptide combinatorial library (200 mixtures) and 22 doubly defined mixtures were prepared in a positional scanning format. Individual peptides (Figures 4 and 6) were synthesized by Peptides and Elephants GmbH (Potsdam, Germany).

[0284] RASGRP2

[0285] A L-aa decapeptide position scanning library (N-acetylated and C-amide TPI 2040) was prepared using standard methods. Each of the 200 mixtures of the library (at 40, 120 and 200 μg / ml) was tested for proliferation activity by TCC14 using a thymidine incorporation assay. Since TCCs were derived from MS patients homozygous for HLA-DR15, TCC14 was tested using restricted BLS cells transfected with HLA-DR15 haplotypes expressing HLA class II alleles (DR2a (=DRB5*01:01), DR2b (=DRB1*15:01) or DQw6 (=DQB1*0602)). HLA class II expression of the BLS cell line was verified with specific antibodies against DR2a, DR2b and DQ, and the test cells were negative for mycoplasma. The results are organized into four matrices (data not shown): three matrices each represent the activity at one of the above-mentioned doses, and one matrix uses a concentration that achieves 3 times of proliferation to combine all three doses into a single activity. Utilize the biostatistical analysis process (Zhao et al., 2001) of the transcript histone database from the brain of MS patient 1154SA to generate a list of predicted peptides for each of the four matrices. Due to a large amount of consistency between the prediction lists, a total of 92 different decamer peptides have occurred in the first 50 predicted peptides in the prediction list of at least one matrix. These peptides (Peptides and Elephants GmbH, Potsdam, Germany) are selected to be synthesized and tested.

[0286] Cells and culture conditions

[0287] As previously reported, large numbers of CSF-derived monocytes from patient 1154SA were expanded (Planas et al., 2015). Briefly, 2000 cells per well were seeded in 96-well U-bottom microtiter plates along with 2 x 10 5Allogeneic irradiated PBMC (45 Gy), 1 μg / ml PHA-L (Sigma, St Louis, MO) and IL-2 supernatant (500 U / ml). The culture medium consisted of IMDM (PAA) containing 100 U / ml penicillin / streptomycin (PAA), 50 μg / ml gentamicin (BioWhittaker, Cambrex), 2 mM L-glutamine (Gibco, Invitrogen, Carlsbad, CA) and 5% heated decomplemented human serum (PAA). Additional IL-2 was added every 3-4 days. CSF-infiltrating CD4+ T cells were positively selected using anti-CD4 magnetic beads (CD4 Micro Beads human MACS, Miltenyi Biotec Inc, CA, USA) and restimulated again with PHA-L, IL-2 and allogeneic irradiated PBMC.

[0288] As previously described, TCC21.1 was established from CSF-infiltrating cells and TCC14 was established from PBMC-derived self-proliferating T cells (Planas et al., 2015).

[0289] Testing of bulk T cells derived from CSF of CIS and RRMS patients against GDP-L-fucose synthase and myelin peptides

[0290] Fresh bulk CSF-derived mononuclear cells from 31 CIS / MS patients were mixed with 5 × 10 6 Allogeneic irradiated PBMCs were mixed and CD4+ T cells were positively selected using anti-CD4 magnetic beads. The CD4+ fraction was then seeded at 1500 cells per well in a 96-well U-bottom microtiter plate, along with 1.5 x 10 5 Allogeneic irradiated PBMC, 1 μg / ml PHA-L and IL-2 supernatant. The culture medium consists of RPMI 1640 without Hepes (Pan-Biotech, Aidenbach, Germany) but supplemented with 2mM glutamine (Pan-Biotech), 1% (vol / vol) non-essential amino acids (Gibco), 1% (vol / vol) sodium pyruvate (Gibco), 50μg / ml penicillin-streptomycin (Corning, NY, USA), 0.00001% β-mercaptoethanol (Gibco) and 5% human serum (BloodBank Basel). Additional IL-2 is added every 4 days. The growth wells are transferred to 48-well plates and finally to 75cm3 flasks until the cells are fully resting (20-25 days). The cells are highly expanded in a single round of stimulation.

[0291] For further analysis of GDP-L-fucose synthase peptide and RASGRP2 peptide (13 of "Example"), the same method was performed.

[0292] Autologous BCLs of patient 1154SA were generated by EBV transformation. BLS cells were transfected with single HLA class II molecules DR2a (DRA1*01:01, DRB5*01:01), DR2b (DRA1*01:01, DRB1*15:01), and DQw6 (DQA1*01:02, DQB1*06:02).

[0293] T cell stimulation

[0294] TCC responses to single / dual defined peptide mixtures or individual decapeptides were assessed by inoculating 2 x 10 4 T cells and 5x 10 4 1x 10 irradiated BLS cell lines or autologous BCL or 1x 10 5 irradiated PBMCs (as indicated). 2.5 μg / ml PHA and 10 -7 M PMA (Sigma), 1 μg / ml surface-coated anti-CD3 (OKT3, Ortho Biotech Products, Raritan, NJ), and 0.5 μg / ml soluble anti-CD28 (Biolegend, San Diego, CA), and T cell activation kit (anti-CD3, anti-CD28, anti-CD2 beads) (Miltenyi Biotec) served as positive controls.

[0295] The responses of PHA-expanded CSF-infiltrating CD4+ T cells to GDP-L-fucose synthase, myelin, and CEF peptides (Figure 6) were assessed by inoculating 6 x 10 4 T cells and 2x 10 5 Irradiated autologous PBMCs were tested. For EdU experiments, BLSDRB1*15:01 was used as APC. A T cell activation kit was used as a positive control.

[0296] For further analysis of GDP-L-fucose synthase peptides and RASGRP2 peptides (13 of "Example"), the same method as for the response of PHA-expanded CSF-infiltrating CD4+ T cells to GDP-L-fucose synthase was performed.

[0297] Cytokine measurements

[0298] GDP-L-fucose synthase

[0299] Cytokines in the supernatants of stimulated TCC21.1 and expanded CSF cells were measured 48 hours after stimulation using Human T Helper Cytokine Panel LEGENDplex bead-based immunoassay (Biolegend), GM-CSF ELISA (BD Biosciences, Franklin Lakes, NJ), and IL-3 ELISA (Biolegend) according to the manufacturer's instructions.

[0300] For intracellular cytokine staining, TCC21.1 was analyzed 48 hours after stimulation. After 5 hours in the presence of GolgiStop protein transport inhibitor (BD Biosciences), T cells were labeled with Cytofix / Cytoperm (Invitrogen). After fixation and permeabilization with Cytofix / Cytoperm (BD Biosciences), cells were stained with antibodies against CD4 (APC-Cy7, Biolegend), IFN-γ (FITC, Biolegend), IL-4 (PE, BD Biosciences), GM-CSF (APC, Biolegend), and IL-3 (PE, Biolegend) in PBS containing saponin and BSA and analyzed by flow cytometry.

[0301] For further analysis of GDP-L-fucose synthase peptides and RASGRP2 peptides (13 of "Example"), IFN-γ secretion in the supernatants of stimulated and unstimulated expanded CSF-infiltrating T cells was measured after 48 h of culture using IFN-γ ELISA (Biolegend) in duplicate in all individual wells according to the manufacturer's instructions.

[0302] Proliferative response

[0303] GDP-L-fucose synthase

[0304] Proliferation was measured 72 hours after stimulation by 3H-thymidine (Hartmann Analytic, Braunschweig, Germany) incorporation in a scintillation counter (Wallac 1450, PerkinElmer, Rodgau-Jürgesheim, Germany). The stimulation index (SI) was calculated as follows: SI = median (repeated cpm peptide) / median (repeated cpm without peptide). Proliferation was also measured using the Click-iTTM-EdU flow cytometry assay kit (APC, Molecular Probes, Invitrogen) according to the manufacturer's instructions. Cells were stained with the following antibodies: anti-CD3 (PE-Cy-7, e-Bioscience, San Diego, CA) and anti-TRBV-21 (FITC, Beckman-Coulter, Brea, CA) and analyzed by flow cytometry.

[0305] RASGRP2

[0306] The proliferative response was tested as described in 9.

[0307] For further analysis of GDP-L-fucose synthase and RASGRP2 peptides (13 of "Examples"), proliferative responses were measured as described above for GDP-L-fucose synthase.

[0308] Surface receptor expression

[0309] Resting TCC21.1 were stained with antibodies against CD4 (PE-Texas Red, Thermo Fischer, Waltham, MA), TRBV21 (FITC, Beckman Coulter), CD28 (PE-Cy7, BioLegend), CCR4 (APC, BioLegend), CCR6 (BV785, BioLegend), and CRTh2 (PE, BioLegend) and analyzed by flow cytometry.

[0310] Flow cytometric analysis

[0311] Sample acquisition was performed using an LSR Fortessa flow cytometer (BD Biosciences) with Diva software, and data were analyzed using FlowJo (Tree Star, Ashland, OR).

[0312] RT-PCR and sequencing of TCR rearrangements

[0313] RNA extraction, reverse transcription, and TCR α / β chain (TRA / BV) sequencing of TCC21.1 were evaluated as previously reported (Planas et al., 2015). TCR gene nomenclature conforms to the IMGT nomenclature (ImMunoGeneTics, www.IMGT.org).

[0314] HLA

[0315] HLA class I and class II molecular typing was performed on individuals at Histogenetics LLC, NY, USA. DNA was isolated from whole blood at a final concentration of 15 ng / μl using a standard DNA isolation protocol using Triton lysis buffer and proteinase K treatment. Samples were typed into HLA class I (A* and B*) and HLA class II (DRB1*, DRB3*, DRB4*, DRB5*, DQA1*, and DQB1*) using high-resolution HLA sequence-based typing (SBT). HLA class II binding prediction was performed using the IEDB Analysis Resource Consistency Tool.

[0316] Statistical analysis

[0317] A three-cluster k-means analysis was performed on the patient scores to stratify the patients into three categories. Associations between response levels of peptides, patients and HLA status were performed using Fisher's Exact Test, with a Bonferroni-Holm correction applied when appropriate, with a 5% significance level.

[0318] Implementation

[0319] 1. A GDP-L-fucose synthase protein or a protein of the RASGRP protein family, or a fragment, derivative or splice variant thereof, or a nucleotide sequence encoding any one of the protein or its fragment, derivative or splice variant, for use in the treatment, diagnosis and / or prevention of multiple sclerosis (MS).

[0320] Particularly preferred are GDP-L-fucose synthase proteins or proteins of the RASGRP protein family, in particular RASGRP2, or fragments thereof.

[0321] 2. The protein, fragment, derivative or splice variant according to embodiment 1,

[0322] GDP-L-fucose synthase protein

[0323] a) having the amino acid sequence shown in SEQ ID NO: 1, or

[0324] b) having an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence shown in SEQ ID NO: 1, or

[0325] c) having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the amino acid sequence shown in SEQ ID NO: 1, or

[0326] d) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence shown in SEQ ID NO: 1, and the protein or fragment or splice variant thereof binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof, or

[0327] e) encoded by the TSTA3 gene, in particular encoded by the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11, or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11;

[0328] and / or

[0329] Members of the RASGRP protein family

[0330] f) having an amino acid sequence as shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or

[0331] g) having an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or

[0332] h) has an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or

[0333] i) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, and the protein or its fragment or splice variant binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the respective amino acid sequences shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 or fragments thereof, or

[0334] j) is encoded by the RASGRP gene, in particular by the following gene sequence:

[0335] - nucleotides 38488101 to 38565575 of NC_000015.10,

[0336] - nucleotides 64726911 to 64745456 of NC_000011.10,

[0337] - nucleotides 33436324 to 33564750 of NC_000002.12, or

[0338] - nucleotides 38409051 to 38426305 of NC_000019.10,

[0339] or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of:

[0340] - nucleotides 38488101 to 38565575 of NC_000015.10,

[0341] - nucleotides 64726911 to 64745456 of NC_000011.10,

[0342] - nucleotides 33436324 to 33564750 of NC_000002.12, or

[0343] -nucleotides 38409051 to 38426305 of NC_000019.10.

[0344] Particularly preferred is a GDP-L-fucose synthase protein having the amino acid sequence shown in SEQ ID NO: 1 or a protein having at least 90% identity or a fragment thereof. Also particularly preferred is a RASGRP protein having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 or a protein having at least 90% identity or a fragment thereof.

[0345] 3. The protein, fragment, derivative or splice variant according to embodiment 1 or 2, wherein the fragment comprises 5 to 50, preferably 5 to 20, more preferably 10 to 15 amino acids, even more preferably 15 amino acids.

[0346] Particularly preferred are fragments with a length of 10 to 15 amino acids.

[0347] 4. A protein, fragment, derivative or splice variant according to embodiment 2 or 3, wherein the fragment is

[0348] a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to the respective corresponding amino acid sequence, or

[0349] b) is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the respective corresponding amino acid sequence, or

[0350] c) is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the respective corresponding amino acid sequence and binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the respective amino acid sequence.

[0351] Particularly preferably, the fragments are at least 90% identical to the respective corresponding amino acid sequence.

[0352] 5. The protein, fragment, derivative or splice variant according to any of the preceding embodiments, wherein the fragment comprises, preferably consists of, a sequence selected from the group comprising SEQ ID NO: 10 to 98, preferably SEQ ID NO: 10 to 35.

[0353] The fragment preferably comprises a sequence selected from the group comprising SEQ ID NOs: 10 to 35.

[0354] 6. A protein, fragment, derivative or splice variant according to any one of the preceding embodiments for use in identifying a human subject suitable for tolerization to autoantigens in MS, preferably early MS. Particularly preferred is the use of said protein or a fragment thereof.

[0355] 7. The protein, fragment, derivative or splice variant according to any one of embodiments 1 to 5 for use in diagnosing mode II MS in a human subject. Particularly preferred is the use of said protein or a fragment thereof.

[0356] 8. A carrier comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of embodiments 1 to 5. It is particularly preferred that the carrier comprises at least one protein, fragment or nucleotide sequence.

[0357] 9. The carrier according to embodiment 8, wherein the carrier is coupled to at least one protein, fragment, derivative and / or splice variant, and / or the carrier comprises at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence.

[0358] It is particularly preferred that the carrier is coupled to at least one protein or fragment and / or that the carrier comprises a nucleotide sequence.

[0359] 10. The carrier according to embodiment 8 or 9, wherein the carrier is selected from the group comprising cells (preferably blood cells), proteins, lipids, glycolipids, beads, nanoparticles, virus-like particles (VLPs) and molecules (e.g. sugar molecules) and any combination thereof.

[0360] The carriers are preferably blood cells.

[0361] 11. The carrier according to embodiment 10, wherein the protein, fragment, derivative and / or splice variant is expressed by a cell, preferably a blood cell.

[0362] It is particularly preferred that the protein is expressed by blood cells.

[0363] 12. The carrier according to embodiment 10 or 11, wherein the blood cells are red blood cells or white blood cells.

[0364] 13. The carrier according to any one of embodiments 10 to 12, wherein the carrier is a blood cell and the blood cell is chemically coupled to at least one protein, fragment, derivative and / or splice variant via a coupling agent, preferably 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI / EDC). Preferably, at least one protein or fragment is coupled to the blood cell via EDC.

[0365] 14. A method of producing the chemically coupled blood cells of embodiment 13, the method comprising isolating blood cells from a human subject, adding the at least one protein, fragment, derivative and / or splice variant, and subsequently adding a coupling agent, preferably EDC. Preferably, at least one protein or fragment is added.

[0366] 15. A pharmaceutical composition comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of embodiments 1 to 5 and a pharmaceutically acceptable carrier. The pharmaceutical composition preferably comprises at least one protein or a fragment or nucleotide sequence thereof and a pharmaceutically acceptable carrier.

[0367] 16. A method for inducing antigen-specific tolerance to a self-antigen in a human subject suffering from or at risk of developing MS, the method comprising the steps of applying to the human subject

[0368] a) at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of embodiments 1 to 5, and / or

[0369] b) at least one carrier according to any one of embodiments 8 to 13.

[0370] Preferred is a method for inducing antigen-specific tolerance, wherein at least one protein or a fragment thereof or a carrier coupled to said at least one protein or fragment or a carrier comprising a nucleotide sequence is applied.

[0371] 17. The method according to embodiment 16, wherein at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence is applied by nasal, inhalation, oral, subcutaneous (sc), intracavitary (ic), intramuscular (im), intradermal (id), transdermal (td) or intravenous (iv) administration, preferably by iv, sc, id, td, oral, inhalation, nasal or coupled to a carrier, preferably red blood cells.

[0372] 18. The method according to embodiment 16 or 17, for inducing antigen-specific tolerance to self-antigens in early MS.

[0373] 19. A method for identifying a human subject suitable for tolerization to self-antigens in MS, preferably early MS, the method comprising isolating T cells and / or antibodies from the subject's blood, CSF or other body fluids, and measuring the reactivity of the T cells and / or antibodies against a protein, fragment, derivative and / or splice variant according to any one of embodiments 1 to 5. Particularly preferred is measuring the reactivity of the T cells and / or antibodies against a fragment.

[0374] 20. The fragment according to any one of embodiments 3 to 5, preferably the fragment according to embodiment 5, for use as a medicament. The fragment preferably comprises a sequence selected from the group comprising SEQ ID NOs: 10 to 35.

[0375] 21. Use of a protein, fragment, derivative and / or splice variant according to any one of embodiments 1 to 5 for the in vitro diagnosis of MS. Particularly preferred is the use of said protein or a fragment thereof.

[0376] 22. A method for in vitro diagnosis of MS using the protein, fragment, derivative and / or splice variant according to any one of embodiments 1 to 5. Particularly preferred is the use of said protein or a fragment thereof.

[0377] 23. Use of a protein, fragment, derivative and / or splice variant according to any one of embodiments 1 to 5 in an in vitro pre-test for diagnosing a human subject suffering from MS or a human subject at risk of developing MS. Particularly preferred is the use of said protein or a fragment thereof.

[0378] 24. Use of a protein, fragment, derivative and / or splice variant according to any one of embodiments 1 to 5 for the manufacture of a medicament for the treatment, diagnosis and / or prevention of MS. Particularly preferred is the use of said protein or a fragment thereof.

[0379] 25. A protein, fragment, derivative and / or splice variant according to any one of embodiments 1 to 5, wherein the derivative is an amino acid sequence that shares at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homology or identity with the corresponding part of the reference amino acid sequence over its entire length.

[0380] 26. An in vitro method for identifying a human subject suitable for tolerization to autoantigens in MS, preferably early MS, comprising using T cells and / or antibodies previously obtained from the subject's blood, CSF or other body fluids, and measuring the reactivity of said T cells and / or antibodies against a protein, fragment, derivative and / or splice variant according to any one of embodiments 1 to 5. Particularly preferred is the use of said protein or a fragment thereof.

[0381] 27. At least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of embodiments 1 to 5, and / or at least one carrier according to any one of embodiments 8 to 13, for use in a method of inducing antigen-specific tolerance to autoantigens in a human subject suffering from MS or at risk of developing MS, the method comprising the step of applying to the human subject at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of embodiments 1 to 5, and / or at least one carrier according to any one of embodiments 8 to 13. Particularly preferred is the use of the protein or fragment thereof and / or at least one carrier coupled to the protein or fragment thereof.

[0382] 28. At least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to embodiment 27 and / or at least one carrier according to embodiment 27, wherein the at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence is applied by nasal, inhalation, oral, subcutaneous (sc), intracavitary (ic), intramuscular (im), intradermal (id), transdermal (td) or intravenous (iv) administration, preferably by iv, sc, id, td, oral, inhalation, nasal or coupled to a carrier, preferably erythrocytes. Particularly preferred is the use of the protein or fragment thereof and / or at least one carrier coupled to the protein or fragment thereof.

[0383] 29. At least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of embodiments 1 to 5, and / or at least one carrier according to any one of embodiments 8 to 13, for inducing antigen-specific tolerance to autoantigens in early MS. Particularly preferred is the use of said protein or fragment thereof and / or at least one carrier coupled to said protein or fragment thereof.

Claims

1. A GDP-L-fucose synthase protein or a protein of the RASGRP protein family, or a fragment, derivative or splice variant thereof, or a nucleotide sequence encoding any one of the protein or its fragment, derivative or splice variant, for use in the treatment, diagnosis and / or prevention of multiple sclerosis (MS).

2. The protein, fragment, derivative or splice variant used according to claim 1, GDP-L-fucose synthase protein a) having the amino acid sequence shown in SEQ ID NO: 1, or b) having an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence shown in SEQ ID NO: 1, or c) having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the amino acid sequence shown in SEQ ID NO: 1, or d) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence shown in SEQ ID NO: 1, and the protein or fragment or splice variant thereof binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof, or e) encoded by the TSTA3 gene, in particular encoded by the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11, or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of nucleotides 143612618 to 143618048 of NC_000008.11; and / or Members of the RASGRP protein family f) having an amino acid sequence as shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or g) having an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or h) has an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or i) has an amino acid sequence that is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, and the protein or its fragment or splice variant binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize the respective amino acid sequences shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 or fragments thereof, or j) is encoded by the RASGRP gene, in particular by the following gene sequence: - nucleotides 38488101 to 38565575 of NC_000015.10, - nucleotides 64726911 to 64745456 of NC_000011.10, - nucleotides 33436324 to 33564750 of NC_000002.12, or - nucleotides 38409051 to 38426305 of NC_000019.10, or encoded by a gene that is at least 80%, preferably at least 90%, even more preferably at least 95% identical to the gene sequence of: - nucleotides 38488101 to 38565575 of NC_000015.10, - nucleotides 64726911 to 64745456 of NC_000011.10, - nucleotides 33436324 to 33564750 of NC_000002.12, or -nucleotides 38409051 to 38426305 of NC_000019.

10.

3. The protein, fragment, derivative or splice variant for use according to claim 1 or 2, wherein the fragment comprises 5 to 50, preferably 5 to 20, more preferably 10 to 15 amino acids, even more preferably 15 amino acids.

4. The protein, fragment, derivative or splice variant for use according to claim 2 or 3, wherein the fragment a) is at least 85%, preferably at least 90%, more preferably at least 95% identical to the respective corresponding amino acid sequence, or b) is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the respective corresponding amino acid sequence, or c) is at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% homologous to the respective corresponding amino acid sequence and binds to self-HLA alleles, is recognized by T cells and / or is recognized by antibodies that bind to or recognize said respective amino acid sequence.

5. The protein, fragment, derivative or splice variant for use according to any one of the preceding claims, wherein the fragment comprises, preferably consists of, a sequence selected from the group comprising SEQ ID NO: 10 to 98, preferably SEQ ID NO: 10 to 35.

6. A protein, fragment, derivative or splice variant for use according to any one of the preceding claims for identifying human subjects suitable for tolerization to autoantigens in MS, preferably early stage MS.

7. A protein, fragment, derivative or splice variant for use according to any one of claims 1 to 5 for diagnosing mode II MS in a human subject.

8. A carrier comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of claims 1 to 5, for use in the treatment, diagnosis and / or prevention of MS.

9. The carrier according to claim 8, wherein the carrier is coupled to the at least one protein, fragment, derivative and / or splice variant, and / or the carrier comprises the at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence.

10. The carrier according to claim 8 or 9, wherein the carrier is selected from the group comprising cells, preferably blood cells, proteins, lipids, glycolipids, beads, nanoparticles, virus-like particles (VLPs) and molecules such as sugar molecules, and any combination thereof.

11. The carrier according to claim 10, wherein the protein, fragment, derivative and / or splice variant is expressed by cells, preferably blood cells.

12. The carrier according to claim 10 or 11, wherein the blood cells are red blood cells or white blood cells.

13. The carrier according to any one of claims 10 to 12, wherein the carrier is a blood cell and the blood cell is chemically coupled to at least one protein, fragment, derivative and / or splice variant via a coupling agent, preferably via 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI / EDC).

14. A method of producing chemically coupled blood cells according to claim 13, said method comprising isolating blood cells from a human subject, adding said at least one protein, fragment, derivative and / or splice variant, and subsequently adding a coupling agent, preferably an EDC.

15. A pharmaceutical composition comprising at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

16. A method for inducing antigen-specific tolerance to a self-antigen in a human subject suffering from or at risk of developing MS, the method comprising the steps of applying to the human subject: a) at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence according to any one of claims 1 to 5, and / or b) At least one carrier body according to any one of claims 8 to 13.

17. The method according to claim 16, wherein the at least one protein, fragment, derivative, splice variant, nucleotide sequence and / or gene sequence is applied by nasal, inhalation, oral, subcutaneous (sc), intracavitary (ic), intramuscular (im), intradermal (id), transdermal (td) or intravenous (iv) administration, preferably by iv, sc, id, td, oral, inhalation, nasal or coupled to a carrier, preferably red blood cells.

18. A method according to claim 16 or 17 for inducing antigen-specific tolerance to self-antigens in early MS.

19. A method for identifying a human subject suitable for tolerization to self-antigens in MS, preferably early MS, the method comprising isolating T cells and / or antibodies from the subject's blood, CSF or other body fluids, and measuring the reactivity of the T cells and / or antibodies to the protein, fragment, derivative and / or splice variant according to any one of claims 1 to 5.

20. The fragment according to any one of claims 3 to 5, preferably the fragment according to claim 5, for use as a medicament.

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