Multiple sclerosis associated autoantigens and their use in therapy and diagnosis

Abstract

The present invention provides multiple sclerosis associated autoantigens and their use in therapy and diagnosis, as well as tolerogenic compositions for use in methods of treating multiple sclerosis (MS) in multiple sclerosis (MS) patients exhibiting T-cell autoreactivity against endogenous epitopes, such as T-cell autoreactivity against endogenous epitopes. The present invention relates to a composition comprising an endogenous epitope corresponding to a T cell epitope comprised in the amino acid sequence of SEQ ID NO: 5, comprising a therapeutic T cell epitope comprising a sequence of 8 contiguous amino acid residues differing from the subsequence of SEQ ID NO: 5 by 0-2 residue substitutions, deletions and / or insertions, or the composition comprises a nucleic acid encoding the therapeutic T cell epitope.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of March 29, 2018, the invention name of "Multiple Sclerosis-Associated Autoantigens and Their Use in Therapy and Diagnosis", PCT application number PCT / SE2018 / 050341, and patent application number 201880022977.X. Technical Field

[0002] The present invention relates to the treatment and diagnosis of multiple sclerosis. Background of the Invention

[0004] Multiple sclerosis

[0005] Multiple sclerosis (MS, ICD 10 code G35) is an immune-mediated, chronic disease of the central nervous system (CNS). The current paradigm proposes that it is an autoimmune inflammatory disease leading to demyelination and axonal destruction. Mainly affecting young people between 20 and 40 years of age, with a total of 2.5 million affected people worldwide, MS is one of the leading causes of disability among young people in developed countries and causes substantial morbidity in the population as well as high costs to society due to the care required.

[0006] MS is characterized by the infiltration of autoreactive T cells into the CNS through the blood-brain barrier (BBB), where they may be activated by antigen presenting cells (APCs). Subsequently, these autoreactive T cells cause the typical features of MS, neuroinflammation, demyelination and axonal destruction; creating characteristic histopathological signs - plaques. Focal destruction leads to various pathological clinical manifestations involving motor, sensory, visual and autonomic systems. Inflammation is usually short-lived, and some remyelination occurs between inflammatory episodes, leading to obvious sudden attacks of increased neurological dysfunction (called relapses), followed by periods of partial recovery. However, over time, recovery becomes lacking and persistent symptoms accumulate.

[0007] T cells and autoantigens in MS

[0008] CD4 + T cells and MS

[0009] CD4 + The main physiological role of T cells is to recognize foreign antigens presented by APCs via MHC class II molecules, and then activate and release cytokines to regulate immune responses. + T cell clones have a specific T cell receptor (TCR) expressed on the cell surface and are sensitive to a specific antigen. +T cells, commonly referred to as T helper cells, can be further divided into subgroups based on function and the cytokines they produce. Briefly, the main function of type 1 helper (Th1) T cells is to coordinate immune responses against intracellular pathogens, Th2 cells against parasitic infections and extracellular pathogens, and Th17 against fungal and bacterial infections.

[0010] As part of the adaptive immune response, CD4 + The purpose of T cells and their TCRs is to have specificity against foreign pathogens. However, since receptors are generated by random rearrangements of the encoding genes, T cells with TCRs specific for self structures may occur, causing autoreactivity. In healthy individuals, autoreactive T cells are negatively selected and removed, but defects in central and peripheral tolerance can generate persistent autoreactive T cells. It is believed that such CD4 + T cells play a central role in the pathogenesis of MS. Certain genes encoding MHC class II are closely associated with the disease and a large number of CD4 + The fact that T cells are involved in the antigen presentation by APCs and the subsequent CD4 + T cell activation plays a crucial role in disease. + T cells are also present in MS lesions, and although their exact role in the disease pathophysiology remains unclear, they likely play a role as well. Experimental autoimmune encephalitis (EAE), a widely used mouse model that mimics MS, is induced via immunization of mice with myelin-derived peptides, further suggesting that autoreactivity plays a role in MS. Organ-specific autoimmune diseases such as MS are generally considered to be Th1-mediated, but recent studies suggest that Th17 cells can similarly drive autoimmune responses.

[0011] Both T cells and APCs are present in a fraction of cells commonly referred to as peripheral blood mononuclear cells (PBMCs). PBMCs are a heterogeneous population of cells derived from peripheral blood that contain T cells, B cells, natural killer cells, monocytes, and dendritic cells.

[0012] T cell cytokines

[0013] After activation by APC, CD4 + Different subsets of T cells produce a variety of different cytokines, whose function can be to induce the proliferation and maturation of other cells or to regulate the overall intensity and duration of inflammation.

[0014] Interferon gamma (IFNγ) is a multipotent proinflammatory cytokine that is highly expressed by Th1 cells and serves as the major product of Th1 cells. IFNγ promotes the cytotoxic activity of other cells, activates macrophages, regulates the expression of MHC class I and class II, and contributes to the further Th1 cell differentiation of naive T cells. When APCs activate antigen-specific CD4 + When T cells are activated, a large amount of IFNγ is released to drive T cells to differentiate into Th1. Interleukin 17A (IL-17A) is a CD4 + IL-17A is a major cytokine of the Th17 T cell subset and upregulates the production and secretion of proinflammatory cytokines, chemokines, and metalloproteinases by other cells. IL-17A has been shown to be involved in MS and has been found to be upregulated in mouse models of EAE and in patients with other autoimmune diseases such as RA, psoriasis, and inflammatory bowel disease. Interleukin 22 (IL-22) is present in activated T cells, primarily by memory CD4 + It is expressed by T cells, Th17 cells, and the recently characterized Th22 cells. It has been found to promote BBB disruption and CNS inflammation together with IL-17A and is considered an important cytokine in the pathogenesis of MS.

[0015] Autoantigens in MS

[0016] In order to make CD4 +T cells are activated, and they must recognize specific antigens presented by APCs. In the case of autoreactive T cells, the antigen is a self-protein, a so-called autoantigen. Several different autoantigens in MS have been proposed and studied (Elong Ngono A, Pettre S, Salou M, Bahbouhi B, Soulillou JP, Brouard S, et al. Frequency of circulating autoreactive Tcells committed to myelindeterminants in relapsing-remitting multiple sclerosis patients. Clin Immunol. 2012; 144 (2): 117-26.). The most studied include antigens derived from myelin, astrocytes, and neurons, but there are other suggestions (Riedhammer C, Weissert R. Antigen Presentation, Autoantigens, and Immune Regulation in Multiple Sclerosis and Other Autoimmune Diseases. Front Immunol. 2015; 6: 322). Among the candidate autoantigens studied are myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin associated glycoprotein (MAG), myelin oligodendrocyte basic protein (MOBP), CNPase, S100β, and transaldolase, with MBP being the most thoroughly studied. For these candidates, T cell autoreactivity or autoantibodies have been found in some human studies and animal models. However, the results are inconclusive and the data lack consistency. Although evidence is difficult to find, it is still believed that autoantigens and their CD4 + The activation of T cells plays a key role in the pathogenesis of MS (Hohlfeld R, Dornmair K, Meinl E, Wekerle H. The search for the target antigens of multiple sclerosis, part 1: autoreactive CD4+T lymphocytes as pathogenic effectors and therapeutic targets. Lancet Neurol. 2015).

[0017] T cell epitopes

[0018] T cells specific for an antigen do not recognize the entire amino acid (aa) sequence of the antigen, but rather recognize a much shorter specific T cell epitope contained somewhere in the antigen. When presented in MHC class I molecules, the epitope is typically between 8-11aa long, and when presented in MCH class II molecules, the epitope is typically between 13-17aa long. When APC internalizes the antigen, it is digested into shorter peptide fragments, which are then presented to the T cell via the MHC molecules on the APC surface. The fragments of these digested antigens are potential specific T cell epitopes.

[0019] Antigen-specific immunotherapy

[0020] Antigen-specific immunotherapy is considered a potentially effective future treatment for MS. The goal of this treatment is to induce immune tolerance by depleting autoantigen-specific, disease-driving T cells or inducing a favorable immune response (regulatory). One of the main methods to achieve this goal is to apply autoantigens, such as immunodominant peptide epitopes, in a tolerogenic manner via oral, cutaneous or subcutaneous injection routes, or to induce regulatory responses using antigen-specific T cells or their receptors as vaccines. The common theme of different approaches is the antigenic target used. This treatment strategy is generally successful because it has been determined that T cell tolerance can be induced via various methods. Encouraging results have been reported in mouse models of MS, experimental autoimmune encephalomyelitis (EAE), but so far limited success has been achieved in human trials, resulting in ineffective or small effects. One of the main reasons is that, unlike EAE, the target autoantigens in MS are not yet fully known, and the best targets or sufficient targets may not yet be used; "One of the major obstacles to the discovery and development of antigen-specific therapies is certainly our ignorance of the target antigens for multiple sclerosis" (Hohlfeld R, Dornmair K, Meinl E, Wekerle H. The search for the target antigens of multiple sclerosis, part 1: autoreactive CD4+T lymphocytes as pathogenic effectors and therapeutic targets. Lancet Neurol. 2015).

[0021] Knowledge gap

[0022] In summary, activation of CD4 by APC and its recognition of self-antigens + T cells are believed to play a key role in the pathogenesis of MS. The association between MHC class II and MS, the effectiveness of immunomodulatory therapies targeting T cells (such as natalizumab), and the role of CD4 +The discovery of T cells, as well as the discovery of some autoreactive T cells in early studies, reinforced this hypothesis. However, exactly which autoantigens trigger autoreactive T cells and drive inflammation remains unknown, although several have been studied. Given the promise of antigen-specific immunotherapy, the identification of autoantigens is becoming increasingly important. However, findings to date have been inconclusive, and there is a need in the art to identify additional antigens involved in the pathogenesis of MS.

[0023] It is therefore an object of the present invention to provide improved or alternative means and methods for determining multiple sclerosis-related autoimmunity in a subject, and to provide improved means, methods and compositions for treating MS.

[0024] definition

[0025] Sequence identity is expressed as a percentage, defined as a value determined by comparing two optimally aligned sequences over a comparison window, wherein for optimal alignment of the two sequences, a portion of the sequence in the comparison window may include an addition or deletion (i.e., a gap) compared to a reference sequence (which does not include an addition or deletion). The number of matching positions is generated by determining the number of positions of the identical amino acid residues present in both sequences, the number of matching positions is divided by the total number of positions in the comparison window and the result is multiplied by 100 to obtain the percentage of sequence identity, to calculate the percentage. Unless otherwise indicated, the comparison window is the entire length of the sequence referenced. In this case, the optimal alignment is the alignment generated by the BLASTP algorithm, which is implemented online by the US National Center for Biotechnology Information (US National Center for Biotechnology Information) (see NCBI Manual [Internet], Chapter 16), and its input parameters are as follows: word length = 3, matrix = BLOSUM62, gap cost = 11, gap extension cost = 1.

[0026] The term antigen in the context of the present invention refers to a molecule (usually a polypeptide) containing a specific T-cell epitope.

[0027] The term specific T cell epitope is defined as the part of an antigen that is recognized by a T cell. Typically, a specific T cell epitope is an amino acid sequence of 8-11 aa in length when presented in an MHC class I molecule and 13-17 aa in length when presented in an MCH class II molecule.

[0028] The term MS antigen refers to antigens that are involved in the pathology of multiple sclerosis (MS).

[0029] Endotoxins, such as lipopolysaccharide (LPS), consist of covalently linked lipid and polysaccharide subunits found on the outer cell wall of Gram-negative bacteria such as Escherichia coli.

[0030] CD4 + T cells or T helper cells are cells that coordinate immune responses through cytokine secretion. They can inhibit or enhance other immune cells, such as stimulating antibody class switching of B cells, amplification of cytotoxic T cells, or enhancing phagocytes. They are activated by antigen presentation via MHC class II on APCs, and they express T cell receptors (TCRs) that are specific for segments of about 13-17 amino acids within a specific antigen (so-called T cell epitopes).

[0031] CD8 + T cells or cytotoxic T cells are cells that kill tumor cells, infected cells, or cells that are damaged in other ways. + Unlike T cells, they do not require specialized APCs for activation. Their T cell receptors recognize antigen-derived peptides (approximately 8-11 amino acids long) presented by MHC class I, a protein expressed on all nucleated cells.

[0032] Antigen-specific T cell activation is a process that requires interaction between the TCR and defined peptides presented on MHC (HLA) molecules combined with co-stimulation.

[0033] Antigen presenting cells (APCs) are typically dendritic cells (DCs), B cells, or macrophages, which are cells that phagocytose or internalize extracellular organisms or proteins (i.e., antigens) and, after processing, present antigen-derived peptides on MHC class II to CD4 + T cells. Monocytes are the most abundant antigen-presenting cells in the blood.

[0034] Phagocytic particles are defined as particles that are capable of being phagocytosed by cells of the immune system, particularly monocytes.

[0035] Peripheral blood mononuclear cells (PBMC) are a fraction of human blood prepared by density gradient centrifugation of whole blood. The PBMC fraction consists primarily of lymphocytes (70-90%) and monocytes (10-30%), while erythrocytes, granulocytes and plasma have been depleted.

[0036] Protein epitope signature tag (PrEST) is a short recombinant 10-12 kDa peptide that represents a unique portion of a human protein (Lindskog M, Rockberg J, Uhlen M, Sterky F. Selection of protein epitopes for antibody production. Biotechniques. 2005; 38(5):723-7.).

[0037] The term peptidomimetic is defined in the context of this application as a peptide-like polymer chain designed to structurally mimic a peptide but having different or improved properties in some respects.

[0038] The term treatment in this context refers to treatment that produces a beneficial effect on the subject or patient suffering from the condition to be treated, including any degree of relief, including mild relief, substantial relief, major relief and cure. Preferably, the degree of relief is at least mild relief. Since MS is a disease characterized by episodic relapses, treatment in this context also involves preventing relapses or reducing the likelihood of relapses.

[0039] The term prevention in this context refers to preventive measures that result in any degree of reduction in the likelihood of developing the condition to be prevented or of the recurrence or recurrence of the condition, including a slight, substantial or major reduction in the likelihood of developing or re-developing the condition, as well as total prevention. Preferably, the degree of reduction in likelihood is at least a slight reduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A-Figure 1C . Autoantigen screening of patients with multiple sclerosis against a library of 125 proteins. Comparison of T cell activation of PBMCs from MS patients with that of PBMCs from healthy controls when stimulated with an antigen library containing PrESTs from 1-2 different human proteins. Figure 1A , IFNγ-FluoroSpot activation assay. Figure 1B , IL17-FluoroSpot activation assay. Figure 1C , IL22-FluoroSpot activation assay. Patients' T cells responded significantly more to certain proteins in the library. Open squares and solid circles represent the mean activation in PBMCs of patients and controls, respectively, and staples represent the mean CI95%. P values ​​were determined using two-way ANOVA. Asterisks represent P values.

[0042] Figure 2A-2D IFNγ / IL-22 / IL-17 Fluorospot assay comparing T cell activation in multiple sclerosis (MS) patients and healthy controls when stimulated with suspected autoantigens in MS. Results are based on the same data as in Figure 1, representing each of the new MS markers contained in this application. 16 patients and 9 controls were included. Figure 2A , activation upon stimulation with antigen pool 18 (containing FABP7 (SEQ ID NOs: 1 and 6) and CYB561D2). Figure 2B , activation upon stimulation with antigen pool 23 (containing PROK2 (SEQ ID NO: 2) and NOVA2). Figure 2C, activation upon stimulation with antigen library 26 (containing RTN3 (SEQ ID NO: 3) and SDK2). Figure 2D , Activation upon stimulation with antigen pool 29 (containing SNAP91 (SEQ ID NO: 4) and SNAP25). P values ​​were determined using the Mann-Whitney U test and are written where found. Staple lines represent the mean with CI 95%.

[0043] Figure 3 .Split of the antigen library from the antigen screening. The antigen library from the screening experiment was further split for which significant differences in activation between patients and controls were detected. Each of these libraries contained PrESTs from 2 different human proteins, referred to as #1 and #2 in the figure. The 4 patients who responded with a high degree of activation in the screening were tested again with independent antigens in the IFNγ / IL-22 / IL-17 Fluorospot. All tests were performed in triplicate. P values ​​were determined using the Student's t-test, comparing the number of activated cells in each well (n=12 per antigen). The stapled lines represent the SD. The positive antigens were: 18#2–FABP7.23#2–PROK2.26#1–RTN3.29#2–SNAP91.

[0044] Figure 4 .Reactivity profiles between patients. The pattern of activation observed between patients. Each line connects the data points of one patient. For profiles 1a, 1b and 3, the dotted line represents the control mean + 2SD. For profile 2, the dotted line represents the average IL-22 profiles of three patients. Only 3 patients reacted significantly to antigen 26 (RTN3), and only the activation of cells producing IFNγ made the results from the previous figure about antigen 26 non-optimal for showing these specific patients. However, the figure clearly shows different groups of patients reacting to this antigen, referred to herein as profile 2. Such a pattern was not identified between healthy controls.

[0045] Figure 5A-5F ROC curves for individual antigens, sensitivity and specificity when used as diagnostic markers. Individually, FABP7 (from library 18) showed the most promise as a marker for disease and achieved 75% and 85% sensitivity and specificity, respectively. Figure 5A-5D ROC curves are shown for each antigen used in isolation. Figure 5E , for each specific individual (same applies to patients and controls) the higher of the activated cell numbers of PROK2 or SNAP91 is used. This way of selecting and analyzing only the highest responses produces even better ROC curves than analyzing them individually. Using the average (of PROK2 and SNAP91) results in a similarly improved ROC curve, although not as good as using only the highest. Fig. 5F Based on the full-length recombinant form of Antigen 18, FABP7 isoform 2 (SEQ ID NO: 1).

[0046] Figure 6 . IFNγ / IL-22 / IL-17 Fluorospot assay comparing T cell activation in multiple sclerosis (MS) patients and healthy controls when stimulated with full-length recombinant FABP7 isoform 2. 13 patients (7 of which consisted of new sampling one year after the first sampling of previously included patients, and 6 not previously tested, open squares) and 7 controls (none of which were included in previous testing) were further tested with an in-house produced recombinant form of FABP7 isoform 2 protein (SEQ ID NO: 1). P values ​​were calculated using the Mann-Whitney test. Stapled lines represent mean values ​​and CI 95%.

[0047] Figure 7A-7B IFNγ / IL-22 / IL-17 Fluorospot assay of patient cells stimulated with overlapping peptides from autoantigens. Overlapping peptides (15aa long, 10aa overlap) spanning the entire FABP7 isoform 2 and PROK2 were pooled in pools containing 5-7 peptides each. Cells from 6 MS patients were tested against these in the FluoroSpot assay. Fig. 7A , activation upon stimulation with 6 different overlapping peptide pools from FABP7 (SEQ ID NO: 1). Figure 7B , activation when stimulated with 3 different overlapping peptide pools from PROK2 (SEQ ID NO: 2). Detailed peptide information is provided in Table 4. Stapled lines indicate CI 95%. Negative numbers for activated T cells in the graph are due to the average response being lower than the negative control.

[0048] Figure 8A-8D IFNγ / IL-22 / IL-17 Fluorospot assay comparing T cell activation in multiple sclerosis (MS) patients and healthy controls when stimulated with full-length recombinant antigens (FABP7, PROK2, RTN3, SNAP91). 52 MS patients and 24 healthy controls were tested in the FluoroSpot assay for T cell activation against recombinant full-length forms of self-antigens. Fig. 8A : FABP7 isoform 2 (SEQ ID NO: 1). Figure 8B :PROK2 (SEQ ID NO: 2). Figure 8C :RTN3 SEQ ID NO:3. Fig.8D: SNAP91 SEQ ID NO: 4. P values ​​calculated using Mann-Whitney test. Stapled lines represent mean values ​​and CI 95%.

[0049] Figure 9- Fig.9E . ROC curves of full-length antigens, sensitivity and specificity when used as diagnostic markers. Individually, the four antigens performed similarly as diagnostic markers for the disease, all achieving a sensitivity of -50% and a specificity of 95%. Fig.9A :FABP7 isoform 2. Fig. 9B :PROK2. Fig. 9C :RTN3. Fig.9D :SNAP91. Fig.9E : Using a combination of all four antigens as a diagnostic test achieves higher sensitivity and specificity. Specifically, using the ratio of the average IL-17 response to the four antigens divided by the square root of the IFNγ response achieves a sensitivity of 70% with a specificity of >95%.

[0050] Figures 10A-10D .Examples of the degree of binding affinity of FABP7 and PROK2 peptides to HLA DRB5 01:01. Overlapping peptides spanning the entirety of FABP7 (SEQ ID NOs: 1 and 6) and PROK2 (SEQ ID NO: 2) were tested for their affinity to multiple sclerosis-associated HLA DRB5 01:01 in a competitive binding assay. The degree of affinity was classified into four categories, and representative binding curves are presented: Fig. 10A :-No binding, Fig. 10B : + weak binding, Fig. 10C :++Moderately combined, Fig. 10D :+++Strong binding. Black dots indicate binding of the reference H1N1 influenza peptide, and triangles indicate the tested autoantigen peptides. See Table 4 for complete results. SUMMARY OF THE INVENTION

[0052] The present invention is based on the findings of a screen performed by the inventors on a T cell reactivity platform previously disclosed in PCT / EP2016 / 081141.

[0053] Samples of T cells from multiple sclerosis (MS) patients and controls were screened against a library of 125 candidate antigens in 45 pools (Example 1). Positive antigen pools 18, 23, 26, and 29 were split into single proteins and retested, allowing FABP7 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3), and SNAP91 (SEQ ID NO: 4) to be identified as novel autoantigens in MS (Example 2). Individual patients were also found to display several different types of reactivity profiles to novel autoantigens (Example 3).

[0054] The diagnostic utility of the novel autoantigens was demonstrated by receiver-operator-characteristic (ROC) analysis (Example 4). Among the individual antigens, sensitivity and specificity (always a trade-off) were most promising for FABP7, which showed 75% sensitivity and 85% specificity. combination , sensitivity and specificity were most promising, with a sensitivity of 70% and a specificity of >95%.

[0055] The autoantigens were further validated by testing the full-length recombinant form of each antigen (Example 7), and the identification principle of specific T cell epitopes was demonstrated using overlapping peptides (15aa, 10aa overlap) covering the entire sequence of FABP7 and PROK2 (Example 6). In addition, the HLA binding properties of different peptide epitopes were demonstrated (Example 6).

[0056] Given that the prior art has demonstrated that MS can be successfully treated with tolerogenic compositions comprising disease-associated T cell epitopes, the inventors recognize that the discovery of autoantigens further provides a new therapeutic approach for MS (see Example 8).

[0057] As discussed in the background section, methods for inducing T cell tolerance are known, but treatment of MS is hampered by the lack of suitable antigens that can induce T cell tolerance. It is well recognized in the art that the biological mechanism of the inventive MS treatment is based on the induction of antigen-specific T cell tolerance.

[0058] While the initial academic studies were based on the concept of the four novel autoantigens as independent entities (due to their natural biological background), it was later recognized that it was possible and advantageous to consider the pooled sequences of all four novel autoantigens as a single source of specific T cell epitopes (SEQ ID NO: 5) for use in the present invention, given the short nature of the T cell epitopes used in both diagnosis and therapy and the fact that combining all four autoantigens improved diagnostic results (ROC analysis). The sequence of SEQ ID NO: 5 is a combination of sequences of unique portions of FABP7 isoform 2 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3), SNAP91 (SEQ ID NO: 4), and FABP7 isoform 1 (SEQ ID NO: 6).

[0059] The present invention specifically relates to the following items. The subject matter disclosed in the following items should be deemed to be disclosed in the same manner as the subject matter disclosed in the patent claims.

[0060] 1. A tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5,

[0061] The composition includes a therapeutic T cell epitope comprising a sequence of n consecutive amino acid residues, wherein the sequence of n consecutive amino acid residues:

[0062] a. a subsequence identical to SEQ ID NO: 5; or

[0063] b. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0064] wherein n is at least 8, and m is 0, 1 or 2.

[0065] 2. A tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5,

[0066] The composition includes a nucleic acid encoding a therapeutic T cell epitope comprising a sequence of n consecutive amino acid residues, wherein the sequence of n consecutive amino acid residues:

[0067] a. a subsequence identical to SEQ ID NO: 5; or

[0068] b. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0069] wherein n is at least 8, and m is 0, 1 or 2.

[0070] 3. A tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5,

[0071] The composition comprises an antigen presenting cell exposed ex vivo to a therapeutic T cell epitope comprising a sequence of n consecutive amino acid residues, wherein the sequence of n consecutive amino acid residues:

[0072] a. a subsequence identical to SEQ ID NO: 5; or

[0073] b. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0074] wherein n is at least 8, and m is 0, 1 or 2.

[0075] 4. A composition for use according to any of the preceding items, wherein the method of treatment comprises determining the patient's T cell autoreactivity against a T cell epitope contained in the amino acid sequence of SEQ ID NO: 5.

[0076] 5. The composition for use according to item 4, wherein the determination is performed by a method according to any one of items 43-110.

[0077] 6. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised within residues 1 to 166 of SEQ ID NO:5.

[0078] 7. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised within residues 167-295 of SEQ ID NO:5.

[0079] 8. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised within residues 296-1327 of SEQ ID NO:5.

[0080] 9. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised within residues 1328-2234 of SEQ ID NO:5.

[0081] 10. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised within residues 2235-2250 of SEQ ID NO:5.

[0082] 11. A composition for use according to any one of items 1-5, wherein the subsequence is comprised of residues 1-2234 of SEQ ID NO:5.

[0083] 12. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised in the sequence of a peptide of any one of Table 4.

[0084] 13. A composition for use according to any of the preceding items, wherein n is at least 11.

[0085] 14. A composition for use according to any of the preceding items, wherein n is at least 13.

[0086] 15. A composition for use according to any of the preceding items, wherein n is at least 15.

[0087] 16. A composition for use according to any of the preceding items, wherein n is at least 17.

[0088] 17. A composition for use according to any of the preceding items, wherein n is at least 19.

[0089] 18. A composition for use according to any of the preceding items, wherein n is at least 50, at least 75, more preferably at least 100.

[0090] 19. A composition for use according to any of the preceding items, wherein m is 2.

[0091] 20. The composition for use according to any one of items 1 to 18, wherein m is 1.

[0092] 21. A composition for use according to any one of items 1 to 18, wherein m is 0.

[0093] 22. A composition for use according to any of the preceding items, wherein the T cell epitope differs from the subsequence by no more than m residue substitutions and comprises no substitutions or deletions compared to the subsequence.

[0094] 23. A composition for use according to item 1 or 3 or any sub-item thereof, wherein the therapeutic T-cell epitope is a peptide or a peptidomimetic.

[0095] 24. A composition for use according to item 23, wherein the therapeutic T cell epitope is a peptide or peptide mimetic having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to a subsequence of SEQ ID NO:5.

[0096] 25. A composition for use according to item 1 or any of its dependent items, wherein the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitopes, each of which meets the criteria described in item 1.

[0097] 26. A composition for use according to item 2 or any of its dependent items, wherein the composition comprises nucleic acids encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitopes, each of which meets the criteria described in item 2.

[0098] 27. A composition for use according to item 3 or any of its dependent items, wherein the antigen presenting cells have been exposed to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitopes, each of which meets the criteria described in item 3.

[0099] 28. A composition for use according to any one of items 25-27, wherein the different therapeutic T-cell epitopes are selected from one, two, three, four, five or six of the sequence intervals described in items 6-11.

[0100] 29. A composition for use according to item 1 or any of its dependencies, wherein the composition comprises a therapeutic T cell epitope coupled to a solid support, such as a biocompatible polymer, particle or cell.

[0101] 30. The composition for use according to item 29, wherein the coupling is via a covalent bond.

[0102] 31. A composition for use according to any one of the preceding items, formulated for inducing T cell tolerance to said therapeutic T cell epitope in a patient.

[0103] 32. A composition for use according to item 1 or any of its dependencies, comprising a therapeutic T cell epitope that is capable of specifically binding to the same T cell receptor as the endogenous epitope.

[0104] 33. A composition for use according to item 2 or any of its dependent items, comprising a nucleic acid encoding a therapeutic T cell epitope that is capable of specifically binding to the same T cell receptor as the endogenous epitope.

[0105] 34. A composition for use according to item 3 or any of its dependencies, comprising an antigen presenting cell exposed to a nucleic acid of a therapeutic T cell epitope, wherein the therapeutic T cell epitope is capable of specifically binding to the same T cell receptor as the endogenous epitope.

[0106] 35. A composition for use according to any one of the preceding items, the method comprising selecting the therapeutic T cell epitope such that it corresponds to an epitope against which the patient exhibits T cell autoreactivity.

[0107] 36. A composition for use according to item 35, wherein the therapeutic T cell epitope is selected based on its ability to specifically bind to the same TCR as the endogenous epitope.

[0108] 37. A composition for use according to any of the preceding items, wherein the method of treatment comprises administering the composition to a subject in a tolerogenic manner, thereby inducing T cell tolerance to said T cell epitope in the patient.

[0109] 38. A composition for use according to any of the preceding items, wherein the method of treatment comprises administering the composition orally, mucosally, intradermally, transdermally or subcutaneously.

[0110] 39. A composition for use according to item 1 or 2 or any of its dependent items, wherein the method of treatment comprises administering the composition to antigen presenting cells in vitro and then administering the antigen presenting cells to a patient.

[0111] 40. A composition for use according to item 2 or any of its dependencies, wherein the nucleic acid is included in a vector, operably coupled to a promoter allowing expression in a cell, preferably an antigen presenting cell.

[0112] 41. The composition for use according to item 40, wherein the vector is a gene therapy vector or a viral vector.

[0113] 42. A composition for use according to item 3 or any sub-item thereof, wherein the antigen presenting cells are dendritic cells, monocytes, macrophages, B cells (preferably derived from PBMC) or microglia.

[0114] 43. A method for determining the extent of multiple sclerosis (MS)-related autoimmunity in a test subject, comprising:

[0115] a. Providing a test sample containing live T cells derived from a test subject;

[0116] b. quantifying in vitro the antigen-specific activation of T cells in response to a test sample comprising a test antigen comprising a T cell epitope, wherein the T cell epitope comprises an amino acid sequence of n consecutive residues, wherein the amino acid sequence of n consecutive residues:

[0117] i. a subsequence identical to SEQ ID NO: 5; or

[0118] ii. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0119] wherein n is at least 8, and m is 0, 1 or 2; and

[0120] c. Comparing the quantified antigen-specific activation with a relevant reference to determine the extent of MS-related autoimmunity in the test subject.

[0121] 44. The method according to item 43, wherein the subsequence is comprised of residues 1-166 of SEQ ID NO:5.

[0122] 45. The method according to item 43, wherein the subsequence is comprised of residues 167-295 of SEQ ID NO:5.

[0123] 46. ​​The method according to item 43, wherein the subsequence is comprised of residues 296-1327 of SEQ ID NO:5.

[0124] 47. The method according to item 43, wherein the subsequence is contained in residues 1328-2234 of SEQ ID NO:5.

[0125] 48. The method according to item 43, wherein the subsequence is contained in residues 2235-2250 of SEQ ID NO:5.

[0126] 49. The method according to item 43, wherein the subsequence is comprised of residues 1-2234 of SEQ ID NO:5.

[0127] 50. The method according to item 43, wherein the subsequence is contained in the sequence of any one of the peptides in Table 4.

[0128] 51. A method according to any of the preceding method items, wherein antigen-specific activation is quantified for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different T cell epitopes, each of which satisfies the criteria described in item 43.

[0129] 52. The method according to item 51, wherein the different T cell epitopes are selected from one, two, three, four, five or six of the sequence intervals described in items 44-49.

[0130] 53. A method according to any of the preceding method items, wherein antigen-specific activation is quantified for at least two, three or four test antigens, each of which comprises a specific T cell epitope corresponding to a different MS antigen selected from the group consisting of FABP7 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3) and SNAP91 (SEQ ID NO: 4).

[0131] 54. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 1-82 of FABP7 (SEQ ID NO: 1).

[0132] 55. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 83-166 of FABP7 (SEQ ID NO: 1).

[0133] 56. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 105-166 of FABP7 (SEQ ID NO: 1).

[0134] 57. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 34-74 of PROK2 (SEQ ID NO: 2).

[0135] 58. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 106-128 of PROK2 (SEQ ID NO: 2).

[0136] 59. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 81-217 of RTN3 (SEQ ID NO: 3).

[0137] 60. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 345-483 of RTN3 (SEQ ID NO: 3).

[0138] 61. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 378-480 of SNAP91 (SEQ ID NO: 4).

[0139] 62. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 481-572 of SNAP91 (SEQ ID NO: 4).

[0140] 63. The method according to any of the preceding method items, wherein the subsequence is comprised in the sequence of residues 584-691 of SNAP91 (SEQ ID NO: 4).

[0141] 64. The method according to any of the preceding method items, wherein n is at least 11.

[0142] 65. The method according to any of the preceding method items, wherein n is at least 13.

[0143] 66. The method according to any of the preceding method items, wherein n is at least 15.

[0144] 67. The method according to any of the preceding method items, wherein n is at least 17.

[0145] 68. The method according to any of the preceding method items, wherein n is at least 19.

[0146] 69. The method according to any of the preceding method items, wherein n is at least 50, at least 75, more preferably at least 100.

[0147] 70. The method according to any of the preceding method items, wherein m is 2.

[0148] 71. The method according to any one of items 43-69, wherein m is 1.

[0149] 72. The method according to any one of items 43-69, wherein m is 0.

[0150] 73. A method according to any of the preceding method items, wherein the T cell epitope differs from a subsequence of a selected MS antigen by no more than m residue substitutions and comprises no substitutions or deletions compared to the subsequence.

[0151] 74. The method according to any of the preceding method items, wherein the T cell epitope is a peptide or a peptide mimetic.

[0152] 75. A method according to any of the preceding method items, wherein the antigen comprising a specific T cell epitope is a peptide or peptide mimetic having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with any one of SEQ ID NO:1-4, SEQ ID NO:5 or SEQ ID NO:6.

[0153] 76. The method according to any of the preceding method items, wherein the subject is a patient diagnosed with MS.

[0154] 77. The method according to any one of items 43-75, wherein the subject is an individual suspected of having MS.

[0155] 78. The method according to any of the preceding method items, wherein the subject is a human.

[0156] 79. The method according to any of the preceding method items, wherein the test sample is derived from a blood sample.

[0157] 80. The method according to any of the preceding method items, wherein the test sample is a PBMC sample.

[0158] 81. The method according to any of the preceding method items, wherein the test sample further comprises antigen presenting cells.

[0159] 82. The method according to any one of the preceding method items, wherein the method further comprises:

[0160] a. Providing live antigen presenting cells;

[0161] b. contacting the test antigen with an antigen presenting cell;

[0162] c. contacting the test sample in vitro with antigen-presenting cells contacted with a test antigen under conditions that allow antigen-specific activation of T cells in response to the antigen presented by the antigen-presenting cells; and

[0163] d. Quantification of antigen-specific T cell activation in test samples.

[0164] 83. The method according to any of the preceding method items, wherein the method comprises providing the test antigen tightly associated with the phagocytosable particles.

[0165] 84. A method according to item 83, wherein the method further comprises the steps of (a') tightly associating the test antigen with the phagocytable particles and / or (a") subjecting the test antigen associated with the particles to a denaturing wash resulting in endotoxin levels low enough not to interfere with subsequent steps.

[0166] 85. The method according to any of items 83-84, wherein the maximum dimension of the particles is less than 5.6 μm, preferably less than 4 μm, more preferably less than 3 μm, even more preferably in the interval 0.5-2 μm or most preferably about 1 μm.

[0167] 86. The method according to item 85, wherein the particles are substantially spherical.

[0168] 87. A method according to any one of items 84-86, wherein the denaturing wash involves subjecting the particles with associated test antigen to high pH, ​​such as at least pH 13, more preferably at least pH 14, most preferably at least pH 14.3.

[0169] 88. A method according to any one of items 84-87, wherein the denaturing wash involves subjecting the particles with associated test antigen to low pH.

[0170] 89. A method according to any one of items 84-88, wherein the denaturing wash involves subjecting the particles with associated test antigen to elevated temperatures, such as at least 90°C, more preferably at least 92°C, most preferably at least 95°C.

[0171] 90. A method according to any one of items 84-89, wherein the denaturing wash involves subjecting the particles with associated test antigen to a denaturing agent such as urea or guanidine hydrochloride at a sufficient concentration (such as at least 5M, 6M, 7M or 8M).

[0172] 91. A method according to any one of items 84-90, wherein the denaturing wash results in an amount of endotoxin in the test antigen such that in the method, the final concentration of endotoxin is less than 100 pg / ml, preferably less than 50 pg / ml, more preferably less than 25 pg / ml and most preferably less than 10 pg / ml.

[0173] 92. The method according to any one of items 83 to 91, wherein the particles have paramagnetic properties.

[0174] 93. The method according to any one of items 83-92, wherein the test antigen is covalently linked to the particles or is linked to the particles via a metal chelate.

[0175] 94. The method according to any of the preceding method items, wherein quantification of antigen-specific T-cell activation in the test sample is performed using ELISpot or FluoroSpot technology or by measuring T-cell proliferation.

[0176] 95. The method according to any of the preceding method items, wherein quantifying antigen-specific T cell activation in a test sample comprises determining the T cell response by measuring the secretion of IFN-γ.

[0177] 96. The method according to any of the preceding method items, wherein quantifying antigen-specific T cell activation in a test sample comprises determining the T cell response by measuring the secretion of IL-17.

[0178] 97. The method according to any of the preceding method items, wherein quantifying antigen-specific T cell activation in a test sample comprises determining the T cell response by measuring the secretion of IL-22.

[0179] 98. The method according to any of the preceding method items, wherein quantitative testing of T cell activation in a cell sample involves determining the fraction of activated T cells in the sample.

[0180] 99. The method according to any of the preceding method items, wherein quantification of antigen-specific T cell activation comprises the following steps:

[0181] a. providing phagocytosable particles having a test antigen tightly associated therewith, wherein the particles having the associated test antigen have been subjected to a denaturing wash resulting in endotoxin levels low enough not to interfere with subsequent steps;

[0182] b. Providing live antigen presenting cells;

[0183] c. contacting the washed particles with antigen-presenting cells under conditions that allow phagocytosis of the particles by the antigen-presenting cells;

[0184] d. Providing a test sample to be assayed containing live T cells;

[0185] e. contacting the test sample in vitro with the antigen presenting cells in contact with the particles under conditions that allow antigen-specific activation of T cells in response to antigens presented by the antigen presenting cells; and

[0186] f. Quantifying antigen-specific T cell activation in a test sample.

[0187] 100. The method according to any of the preceding method items, wherein the reference is an equivalent quantitative antigen-specific activation in a reference sample from a reference subject without pathological MS-related autoimmunity.

[0188] 101. The method according to any one of items 43-99, wherein the reference is the mean value of equivalent quantitative antigen-specific activation in a group of reference samples from a group of reference subjects without pathological MS-related autoimmunity.

[0189] 102. The method according to item 101, wherein the group comprises at least 10 reference subjects.

[0190] 103. A method according to any one of items 43-99, wherein the reference is an equivalent quantification of antigen-specific activation in samples from the same subject taken at different time points.

[0191] 104. The method according to any of the preceding method items, wherein an increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is at least 2-fold, preferably 3-fold, more preferably 5-fold, most preferably 10-fold higher compared to the reference.

[0192] 105. A method according to any of the preceding method items, wherein an increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is higher than the mean value of a group of equally quantified reference samples from a group of reference subjects without pathological MS-related autoimmunity by 2 times the standard deviation of the reference sample group.

[0193] 106. The method according to any of the preceding method items, wherein an increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is statistically significantly higher than the reference with a p-value calculated with Student's T-test of less than 0.05.

[0194] 107. The method according to any of the preceding method items, wherein an increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is statistically significantly higher than the reference with a p-value calculated using the Mann-Whitney U test of less than 0.05.

[0195] 108. The method according to any of the preceding method items, wherein:

[0196] a. Methods for diagnosing MS in a subject;

[0197] b. the reference represents an equivalent quantitative antigen-specific activation in a reference subject without pathological multiple sclerosis-related autoimmunity; and

[0198] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of multiple sclerosis in the test subject.

[0199] 109. The method according to any of the preceding method items, wherein:

[0200] a. Methods for following the course of MS in subjects;

[0201] b. the reference represents an equivalent quantification of antigen-specific activation in samples taken from the same subject at different time points; and

[0202] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of a higher multiple sclerosis disease activity in the test subject, and vice versa.

[0203] 110. The method according to any of the preceding method items, wherein:

[0204] a. A method for prognosing the course of MS in a subject;

[0205] b. the reference represents an equivalent quantification of antigen-specific activation in samples taken from the same subject at different time points; and

[0206] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of increased multiple sclerosis disease activity in the test subject, and vice versa.

[0207] 111. The method according to any of the preceding method items, wherein:

[0208] a. Methods used to assess a subject's response to therapeutic treatment;

[0209] b. the reference represents an equivalent quantitative antigen-specific activation in a sample taken from the same subject before administration of the therapeutic treatment to be evaluated;

[0210] c. the test sample is from the same subject after initiation of therapeutic treatment; and

[0211] d. A lower degree of quantitative antigen-specific activation in the test sample compared to the reference indicates therapeutic efficacy against multiple sclerosis disease activity in the test subject, and an unchanged or higher degree of quantitative antigen-specific activation indicates a lack of therapeutic efficacy against multiple sclerosis disease activity in the test subject.

[0212] 112. Use of a peptide or peptide mimetic in the diagnosis or treatment of MS, wherein the peptide or peptide mimetic comprises a specific T cell epitope corresponding to an MS antigen, and the T cell epitope comprises an amino acid sequence of n consecutive residues that differs from the subsequence of SEQ ID NO:5 by no more than m residue substitutions, deletions and / or insertions, wherein m is 0, 1 or 2.

[0213] 113. The use according to item 112, wherein the subsequence is selected from residues 1-166 of SEQ ID NO:5, residues 167-295 of SEQ ID NO:5, residues 296-1327 of SEQ ID NO:5, residues 1328-2234 of SEQ ID NO:5, residues 2235-2250 of SEQ ID NO:5 and / or residues 1-2234 of SEQ ID NO:5.

[0214] 114. The use according to item 112 or 113, wherein n is at least 11.

[0215] 115. The use according to item 112 or 113, wherein n is at least 13.

[0216] 116. The use according to item 112 or 113, wherein n is at least 15.

[0217] 117. The use according to item 112 or 113, wherein n is at least 17.

[0218] 118. The use according to item 112 or 113, wherein n is at least 19.

[0219] 119. The use according to any one of items 112 to 118, wherein m is 1.

[0220] 120. The use according to any one of items 112 to 118, wherein m is 0. DETAILED DESCRIPTION OF THE INVENTION

[0222] Tolerogenic compositions for treating multiple sclerosis

[0223] In a first aspect, the present invention provides a tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a MS patient who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5,

[0224] The composition includes a sequence comprising n consecutive amino acid residues Therapeutic T cell epitopes , the sequence of n consecutive amino acid residues:

[0225] a. a subsequence identical to SEQ ID NO: 5; or

[0226] b. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0227] wherein n is at least 8, and m is 0, 1 or 2.

[0228] In a second aspect, provided is a tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5, the composition comprising Encoding therapeutic T cell epitopes Nucleic Acid , the therapeutic T cell epitope is as defined in the first aspect.

[0229] In a third aspect, provided is a tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5, the composition comprising In vitro exposure to therapeutic T cells Antigen presenting cells , the therapeutic T cell epitope is as defined in the first aspect.

[0230] It will be understood that it is implied that the method of treatment comprises administering the composition to a patient.

[0231] The method of treatment may include a step of determining the patient's T cell autoreactivity against a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5 prior to administering the composition, preferably by the method disclosed below as the fourth aspect of the present invention.

[0232] It should be noted that the therapeutic T cell epitope can be included as part of virtually any larger polypeptide (e.g., by genetic engineering or chemical peptide synthesis), given that the patient's antigen presenting cells (APCs) will digest the larger polypeptide and present the fragments to the T cells. In other words, due to digestion by the APCs, there is little or no difference in the sequence context in which the therapeutic T cell epitope is found.

[0233] The compositions of the preferred first aspect comprise more than one therapeutic T cell epitope, for example 2,3,4,5,6,7,8,9,10,12,14,16,18,20,30,40,50,100 or more, each of which satisfies the above criteria. As discussed above and in the background section, antigen presenting cells will digest any protein for antigen presentation into small fragments, so it is possible or even preferred to include one or more therapeutic T cell epitopes in the larger polypeptide or peptide mimetics that will be used in the same treatment paradigm. In order to avoid any uncertainty, it is obvious that therapeutic T cell epitopes can also be independent chemical entities.

[0234] This also applies to the composition of the second aspect, which has the following modifications, the composition comprises one or more nucleic acids encoding more than one therapeutic T cell epitope, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100 or more therapeutic T cell epitopes, each epitope meeting the above criteria. The nucleic acid can encode more than one therapeutic T cell epitope, because as described above, the cell digests any expressed protein for antigen display into small fragments. Alternatively or in addition, for the above reasons, the therapeutic T cell epitope can be included as part of a longer sequence, and the longer sequence comprises a sequence that is not a specific T cell epitope.

[0235] The antigen presenting cells of the third composition may be exposed to more than one therapeutic T cell epitope, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100 or more, each of which meets the above criteria. As previously mentioned, even in this case, the T cell epitope may be included in one or more larger polypeptides.

[0236] It should be understood that "exposing" in the context of the third aspect may refer not only to contacting an antigen presenting cell with a peptide or peptide mimetic comprising a therapeutic T cell epitope, but also to transfecting an antigen presenting cell with a nucleic acid encoding a therapeutic T cell epitope, resulting in the cell expressing the therapeutic T cell epitope, thereby exposing the cell.

[0237] Subsequence

[0238] The subsequence of the first, second or third aspect may be comprised within i) residues 1-166 of SEQ ID NO: 5, ii) residues 167-295 of SEQ ID NO: 5, iii) residues 296-1327 of SEQ ID NO: 5, iv) residues 1328-2234 of SEQ ID NO: 5 or v) residues 2235-2250 of SEQ ID NO: 5. Preferably, the subsequence is comprised within vi) residues 1-2234 of SEQ ID NO: 5.

[0239] The composition of the first aspect may comprise more than one different therapeutic T cell epitope as defined above. Preferably, the more than one different therapeutic T cell epitope is selected from two, three, four, five or six different intervals presented as i)-vi) above.

[0240] The same principles apply to the nucleic acid of the second aspect, mutatis mutandis. Preferably, the nucleic acid encodes one or more different therapeutic T cell epitopes as defined above. Preferably, the one or more differently encoded T cell epitopes are selected from two, three, four, five or six different intervals presented above as i)-vi).

[0241] Likewise, the cells of the third aspect may have been exposed to more than one different therapeutic T cell epitope as defined above. Preferably, the more than one different therapeutic T cell epitope is selected from two, three, four, five or six different intervals presented as i)-vi) above.

[0242] The subsequence may be comprised in the sequence of any one of the peptides in Table 4. Preferably, the subsequence is comprised in the sequence of any one of the peptides in Table 4, wherein HLA binding is indicated as "++" or "+++", most preferably "+++".

[0243] Preferably, n is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. n can be at least 50, at least 75, even more preferably at least 50. n can be any interval formed by the above numbers, for example 8-22, 9-21, 8-17, 8-15, 8-12 or 8-100. When m=0, most preferably n is 8-19 or 8-17. Preferably, m is 2, more preferably m is 1, and most preferably m is 0.

[0244] Preferably, the therapeutic T cell epitope is identical to the subsequence (m=0). However, it is very likely that in most cases the sequence is slightly changed and still a fully or substantially equivalent therapeutic T cell epitope is functionally present compared to a therapeutic T cell epitope having sequence identity with a subsequence of SEQ ID NO:5. For example, substitution of an amino acid with another amino acid having similar characteristics in terms of polarity, charge or hydrophobicity may be tolerable. Small insertions or deletions within a therapeutic T cell epitope may also be tolerable, provided that they produce fully or substantially equivalent therapeutic T cell epitopes functionally present compared to a therapeutic T cell epitope having sequence identity with a subsequence of SEQ ID NO:5. Preferably, m is 2, more preferably m is 1, and most preferably m is 0.

[0245] Preferably, the therapeutic T cell epitope may differ from a subsequence by no more than m residue substitutions and contains no substitutions or deletions compared to the subsequence.

[0246] The therapeutic T cell epitope of the first or third aspect may be a peptide or peptide mimetic. The therapeutic T cell epitope of the first or third aspect may be a peptide or peptide mimetic having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to a subsequence of SEQ ID NO:5.

[0247] The nucleic acid of the second aspect may be DNA, PNA or any other nucleic acid capable of encoding a protein for expression in mammalian cells.

[0248] Composition preparation

[0249] The composition is formulated for inducing T cell tolerance to said therapeutic T cell epitope in a patient, which may be a human or a non-human mammal, preferably a human.

[0250] The composition of the first aspect may comprise a therapeutic T cell epitope coupled to a solid support, such as a biocompatible polymer (eg PLGA), a liposome, a solid particle or a cell.

[0251] The coupling is preferably via a covalent bond, but may also be other couplings, including metal chelate binding, hydrophobic interactions or ionic interactions. EP2205273 discloses a suitable coupling scheme for coupling antigenic peptides to cells. Gholamzad and colleagues disclose a suitable coupling scheme for coupling antigenic peptides to PLGA-microparticles (Iranian Journal of Allergy, Asthma and Immunology 2017.16(3):271-281). Pearson and colleagues disclose a suitable scheme for coupling antigenic peptides to polymer carriers (Mol Ther. 2017 Jul 5; 25(7):1655-1664.doi:10.1016 / j.ymthe.2017.04.015).

[0252] The composition according to the first aspect may comprise a therapeutic T cell epitope capable of specifically binding to the same T cell receptor as said endogenous epitope.In the context of the present invention, specific binding means binding that is high enough to result in T cell activation in a biological environment in vitro or in vivo.

[0253] The composition according to the second aspect may comprise a nucleic acid encoding a therapeutic T-cell epitope capable of specifically binding to the same T-cell receptor as the endogenous epitope.

[0254] The antigen presenting cells of the third aspect may have been exposed to a therapeutic T cell epitope capable of specifically binding to the same T cell receptor as the endogenous epitope.

[0255] Selecting therapeutic T cell epitopes

[0256] The treatment method of the first, second or third aspect may comprise selecting the therapeutic T cell epitope to correspond to an endogenous T cell epitope against which the patient exhibits T cell autoreactivity.

[0257] Therapeutic T cell epitopes can be selected based on their ability to specifically bind to the same TCR as the endogenous epitope.

[0258] The therapeutic T cell epitopes of the first, second or third composition can be individually tailored or selected to match the autoreactivity of the individual patient or subject to be treated. However, in many cases, it may be more practical to administer a composition comprising several therapeutic T cell epitopes corresponding to the most common endogenous T cell epitopes, as customizing a composition is time consuming, expensive and may pose regulatory challenges in many jurisdictions.

[0259] Application composition

[0260] The treatment methods of the first to third aspects preferably comprise administering the composition to the subject in a tolerogenic manner, thereby inducing T cell tolerance to the patient's therapeutic T cell epitopes.

[0261] Treatment methods may include oral, mucosal, intradermal, transdermal or subcutaneous administration of the composition. Administration may be by injection.

[0262] The treatment method of the first or second aspect may comprise administering the composition ex vivo to antigen presenting cells and then administering the antigen presenting cells to the subject.

[0263] The therapeutic dose is preferably titrated from a low dose to a higher dose over a period of weeks / months. Treatment is preferably started with a low first dose, and then the dose is increased (e.g., doubled) at each subsequent administration. After a titration period of weeks / months, a maintenance level 10-100 times higher than the starting dose may be achieved and maintained for a period of time.

[0264] The literature describes several protocols for inducing T cell tolerance in MS and other conditions, which can be adapted for use in the present invention simply by replacing the T cell antigen to which tolerance is induced with the therapeutic T cell epitopes described herein.

[0265] Cathaway and colleagues (Cathaway J, Martin K, Barrell K, Sharrack B, Stolt P, Wraith DC. Effects of ATX-MS-1467 immunotherapy over 16 weeks in relapsing multiple sclerosis. Neurology 2018) conducted an open-label study to evaluate the safety, tolerability, and efficacy of antigen-specific immunotherapy using different treatment regimens to induce tolerance to the antigen in patients with relapsing multiple sclerosis.

[0266] In a double-blind, placebo-controlled cohort study, Walczak and colleagues (Walczak A, Siger M, Szczepanik M, Selmaj K. Transdermal application of myelin peptides in multiple sclerosis treatment. JAMA Neurol. 2013) have demonstrated effective antigen-specific therapy in multiple sclerosis using transdermal application of myelin peptides in human patients.

[0267] Lutterotti and colleagues have published a tolerogenic protocol in MS patients using a single infusion of autologous peripheral blood mononuclear cells chemically coupled to seven myelin peptides (Sci Transl Med. 2013 Jun 5; 5(188): 188ra75). The same team has also published a similar protocol in a patent publication in which the peptides were coupled to red blood cells (EP2205273).

[0268] Gholamzad and colleagues (Iranian Journal of Allergy, Asthma and Immunology 2017. 16(3): 271-281) disclosed a protocol involving intravenous injection of PLGA microparticles coated with myelin oligodendrocyte glycoprotein (MOG), which has a tolerogenic effect, in experimental autoimmune encephalomyelitis, a disease model of MS.

[0269] Pujol-Autonell and colleagues (Nanomedicine (Lond). 2017 Jun; 12(11): 1231-1242. Doi: 10.2217 / nnm-2016-0410) disclosed a scheme for immunotherapy based on phosphatidylserine-liposomes, which had a therapeutic effect on a multiple sclerosis disease model, in which MOG-peptide served as antigen.

[0270] Pearson and colleagues (Mol Ther. 2017 Jul 5; 25(7): 1655-1664. Doi: 10.1016 / j.ymthe.2017.04.015) reported an experimental protocol using antigenic MOG peptides conjugated to poly(lactide-co-glycolide) in relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a mouse model of multiple sclerosis. The polymer-conjugated peptides effectively inhibited the disease.

[0271] By modifying the antigenic peptide according to the first aspect, any of the schemes and regimens described above may be used with the composition of the first aspect.

[0272] Gene therapy

[0273] In general, the nucleic acid of the second aspect is preferably included in a vector, operably coupled to a promoter that allows expression in the patient's cells, preferably antigen presenting cells. The vector can be a gene transfer vector, or a viral vector known in the art, such as a retroviral vector or an adeno-associated viral vector or an adenoviral vector. Naked DNA gene transfer vectors can be administered by any means known in the art, including electroporation, gene guns, sonoporation, magnetofection or hydrodynamic delivery. Chemical methods that can be used to enhance vector delivery include liposomes, lipoplexes, polymer vesicles, polymer complexes, dendrimers, inorganic or organic nanoparticles or cell penetrating peptides.

[0274] Suitable promoters are known in the art depending on the tissue in which expression of the sequence encoding the therapeutic T cell epitope is desired.

[0275] Keeler and colleagues (Mol Ther. 2018 Jan 3; 26 (1): 173-183) have demonstrated that antigen-specific regulatory T cells (Tregs) induced by gene therapy can inhibit neuroinflammation and reverse disease in MS, using a liver-targeted gene transfer vector that expresses full-length myelin oligodendrocyte glycoprotein (MOG) in hepatocytes. The materials and methods used can be applied to a method of treatment according to the second aspect, wherein the modification of the expressed nucleic acid sequence is replaced by a therapeutic T cell epitope defined herein for the second aspect.

[0276] Therapy using antigen presenting cells

[0277] The antigen presenting cell of the third aspect can be a dendritic cell, a monocyte, a macrophage or a B cell, which can preferably be derived from a peripheral blood mononuclear cell. Alternatively, the antigen presenting cell can be a microglia, which can be CNS derived. Preferably, the cells are autologous to the patient, but they can also come from different individuals with respect to the donor matching of the MHC receptor. In addition, it is also envisioned to use genetically engineered APC cell lines from different individuals or even different species, wherein the MHC receptor is engineered to match the patient.

[0278] Cells are exposed to the composition (therapeutic T cell epitopes) of the third aspect in vitro, so that the cells take up the epitopes and present the epitopes to the patient's immune system after being administered to the patient. Since the cells process and digest the polypeptides before displaying any polypeptides on the surface, the epitopes can be given to the cells as part of a larger protein or several different proteins. It is also applicable to transfect cells with nucleic acid constructs encoding therapeutic T cell epitopes so that the epitopes are expressed in the cells.

[0279] In a review article, Phillips et al. (Front Immunol. 2017;8:1279) discuss the use of tolerogenic dendritic cells and ongoing clinical studies for MS and other autoimmune diseases.

[0280] Jones and Hawiger (Front Immunol. 2017 May 9;8:532. doi:10.3389 / fimmu.2017.00532) discuss experiments showing that antigen-specific Treg cells (pTreg cells) formed in an extrathymic form in the peripheral immune system can ameliorate or even completely prevent neuroinflammation during tolerogenic responses to relevant neuronal antigens, and the relevance of these findings to MS treatment.

[0281] Iberg et al. (Trends Immunol. 2017 Nov; 38(11): 793-804. doi: 10.1016 / j.it.2017.07.007) discuss how dendritic cell function conferred by specific delivery of T cell antigens can be exploited for tolerance induction in a clinical setting.

[0282] Huang et al. reported that autoantigen-pulsed dendritic cells induced tolerance to experimental allergic encephalomyelitis (EAE) in Lewis rats (Clin Exp Immunol (2000) 122(3):437–44. doi:10.1046 / j.1365-2249.2000.01398.x).

[0283] Menges et al. reported that repeated injections of dendritic cells matured with tumor necrosis factor alpha induced antigen-specific protection from autoimmunity in mice (J Exp Med (2002) 195(1):15–21. Doi:10.1084 / jem.20011341).

[0284] The means and methods discussed in the above-mentioned original studies and reviews (including the original studies cited therein) can be adapted for use with the compositions of therapeutic T cell epitopes of the present invention.

[0285] Methods for determining the extent of autoimmunity associated with multiple sclerosis (MS)

[0286] In a fourth aspect, the present invention provides a method for determining the extent of multiple sclerosis (MS)-related autoimmunity in a test subject, comprising:

[0287] a. Providing a test sample containing live T cells derived from a test subject;

[0288] b. quantifying in vitro the antigen-specific activation of T cells of a test sample in response to a test antigen, wherein the test antigen comprises a specific T cell epitope corresponding to an MS antigen, wherein the T cell epitope comprises an amino acid sequence of n consecutive residues, wherein the amino acid sequence of n consecutive residues:

[0289] i. a subsequence identical to SEQ ID NO: 5; or

[0290] ii. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0291] wherein n is at least 8, and m is 0, 1 or 2; and

[0292] c. Comparing the quantified antigen-specific activation with a relevant reference to determine the extent of MS-related autoimmunity in the test subject.

[0293] The method of the fourth aspect for determining the extent of multiple sclerosis (MS)-related autoimmunity in a test subject may comprise:

[0294] a. Providing a test sample containing live T cells derived from a test subject;

[0295] b. quantifying in vitro the antigen-specific activation of T cells of a test sample in response to a test antigen, the test antigen comprising a specific T cell epitope corresponding to an MS antigen; and

[0296] c. comparing the quantified antigen-specific activation to a relevant reference to determine the extent of MS-related autoimmunity in the test subject;

[0297] wherein the MS antigen is selected from the group consisting of FABP7 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3) and SNAP91 (SEQ ID NO: 4);

[0298] The T cell epitope comprises an amino acid sequence of n consecutive residues, wherein the amino acid sequence of n consecutive residues is:

[0299] i. is identical to a subsequence of a selected MS antigen; or

[0300] ii. differing from the selected subsequence of the MS antigen by no more than m residue substitutions, deletions and / or insertions; and

[0301] wherein n is at least 8, and m is 0, 1 or 2.

[0302] It should be noted that T cell epitopes can be included in virtually any larger polypeptide test antigen (e.g., by genetic engineering), given that the test antigen will be digested by antigen presenting cells (APCs). Since the test antigen will be digested, there is no difference in the context in which the specific T cell epitope is found in the test antigen. Thus, any test antigen can be used, provided that the test antigen contains as part of its sequence a specific T cell epitope corresponding to one of the following novel MS antigens: FABP7, PROK2, RTN3, and SNAP91 (all contained in SEQ ID NO: 5).

[0303] As discussed in the background section, the shortest epitope is usually 8aa long, but can be longer depending on individual circumstances. Therefore, the present invention requires that the T cell epitope has sequence identity or similarity to SEQ ID NO: 5 (i.e., one of the novel MS antigens), at least sharing similarity for a continuous stretch of n amino acids.

[0304] The value of n can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Preferably, n is at least 11, more preferably n is at least 13, still more preferably n is at least 15, still more preferably n is at least 17, and even more preferably n is at least 19. Alternatively, n can be at least 50, preferably at least 75, or most preferably at least 100. n can be any interval formed by the above numbers, such as 8-22, 9-21, 8-17, 8-15, 8-12 or 8-100. Most preferably, when m=0, n is 8-19 or 8-17.

[0305] Preferably, the T cell epitope is identical to the subsequence (m=0). However, in most cases, the sequence can be slightly altered and still have a specific T cell epitope that is functionally complete or substantially equivalent to the MS antigen. For example, substitution of an amino acid for another amino acid with similar characteristics in terms of polarity, charge or hydrophobicity may be tolerated. Small insertions or deletions within a specific T cell epitope may also be tolerated, provided that they produce a specific T cell epitope that is functionally complete or substantially equivalent to the selected MS antigen.

[0306] Preferably, the differences amount to substitutions only, i.e. no deletions or insertions. More preferably, the differences amount to substitutions only involving the substitution of an amino acid with another amino acid having similar characteristics in terms of polarity, charge and / or hydrophobicity. Preferably, m is 2, more preferably m is 1, most preferably m is 0.

[0307] Preferably, antigen-specific activation is quantified against at least two, three or four test antigens, each of which comprises a specific T-cell epitope corresponding to a different MS antigen selected from the group consisting of FABP7 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3) and SNAP91 (SEQ ID NO: 4). As shown in Example 2, not all MS patients exhibit T-cell-related autoimmunity to all novel MS antigens. Therefore, in particular in a screening setting, it is advantageous to test for MS-related autoimmunity in a test subject against more than one specific T-cell epitope, which are simultaneously derived from different MS antigens. It is also contemplated that the determination of autoimmunity against a novel MS marker may be advantageously combined with the determination of other known MS markers.

[0308] The test antigen comprising a specific T cell epitope may be a peptide or peptide mimetic having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to any one of SEQ ID NO: 1-4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0309] Subsequences in diagnostic methods

[0310] The subsequence of the fourth aspect may be comprised within i) residues 1-166 of SEQ ID NO: 5, ii) residues 167-295 of SEQ ID NO: 5, iii) residues 296-1327 of SEQ ID NO: 5, iv) residues 1328-2234 of SEQ ID NO: 5 or v) residues 2235-2250 of SEQ ID NO: 5. Preferably, the subsequence is comprised within vi) residues 1-2234 of SEQ ID NO: 5.

[0311] The method of the fourth aspect may comprise quantifying antigen-specific activation in response to one or more different T cell epitopes as defined above. Preferably, the one or more different T cell epitopes are selected from two, three, four, five or six different intervals presented as i)-vi) above.

[0312] Preferably, the method of the fourth aspect may comprise quantifying antigen-specific activation in response to more than one therapeutic T cell epitope, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100 or more therapeutic T cell epitopes, each of which meets the above criteria.

[0313] The subsequence may be comprised in the sequence of any one of the peptides in Table 4. Preferably, the subsequence is comprised in the sequence of any one of the peptides in Table 4, wherein HLA binding is indicated as "++" or "+++", most preferably "+++".

[0314] The subsequence may preferably be derived from a specific portion of the MS antigen described herein, which corresponds to the specific antigenic protein used in the Examples.

[0315] The subsequence may be comprised within the sequence of residues 1-82, residues 83-166, or residues 105-166 of FABP7 (SEQ ID NO: 1).

[0316] The subsequence may be comprised within the sequence of residues 34-74 or residues 106-128 of PROK2 (SEQ ID NO: 2).

[0317] The subsequence may be comprised within the sequence of residues 81-217 or residues 345-483 of RTN3 (SEQ ID NO:3).

[0318] The subsequence may be included in the sequence of residues 378-480, residues 481-572, or residues 584-691 of SNAP91 (SEQ ID NO: 4).

[0319] Test subjects

[0320] The subject may be a patient diagnosed with MS, or an individual suspected of having MS. Preferably, the subject is a human.

[0321] Test samples

[0322] The test sample is preferably derived from a blood sample, and more preferably a PBMC sample.

[0323] Most preferably, the test sample further comprises antigen presenting cells (APCs) which can be used to present the test antigen to T cells of the sample. Utilizing APCs from the same individual in the quantification (details below) eliminates any uncertainty caused by individual genetic variation of the MCH receptors on the APCs.

[0324] Details on preferred methods for determining specific T cell activation

[0325] A T cell reactivity platform that can be used to determine MS-related autoimmunity has been previously disclosed by the inventors in PCT / EP2016 / 081141.

[0326] The method of the present invention may further comprise:

[0327] a. Providing live antigen presenting cells;

[0328] b. contacting the test antigen with an antigen presenting cell;

[0329] c. contacting the test sample in vitro with antigen-presenting cells contacted with a test antigen under conditions that allow antigen-specific activation of T cells in response to the antigen presented by the antigen-presenting cells; and

[0330] d. Quantification of antigen-specific T cell activation in test samples.

[0331] Preferably, the antigen presenting cells are derived from the test subject.

[0332] The method may include providing a test antigen tightly associated with a phagocytosable particle. The particle and the test antigen are phagocytosed by an antigen presenting cell. The test antigen is enzymatically digested by the APC, and the digested antigen epitope is presented to the T cell.

[0333] The particular advantage of making the test antigen tightly associated with the phagocytosable particles is that any contaminating endotoxins can be removed by denaturing washing. Unfortunately, a common problem with assays that determine T cell activation is that even low levels of endotoxins in contact with T cells can lead to activation, masking the usually very low levels of antigen-specific activation. Only a small fraction of the tested T cell population reacts to a given antigen in an antigen-specific manner (1 / 10000 order of magnitude from the blood of a subject who has recently encountered the antigen), while cells in large fractions react to endotoxins, thereby producing a high level of background. In view of the ubiquitous endotoxin contamination, this may be a major problem in practice.

[0334] Thus, the method may further comprise the steps of (a') tightly associating the test antigen with the phagocytable particles and / or (a") subjecting the particle-associated test antigen to a denaturing wash resulting in endotoxin levels low enough not to interfere with subsequent steps.

[0335] The particles preferably have a largest dimension of less than 5.6 μm, preferably less than 4 μm, more preferably less than 3 μm, even more preferably in the interval 0.5-2 μm or most preferably about 1 μm. The particles are preferably substantially spherical.

[0336] Denaturing washes may involve subjecting the particles with associated test antigen to high pH, ​​such as at least pH 13, more preferably at least pH 14, and most preferably at least pH 14.3. Denaturing washes may involve subjecting the particles with associated test antigen to low pH. Denaturing washes may involve subjecting the particles with associated test antigen to elevated temperatures, such as at least 90° C., more preferably at least 92° C., and most preferably at least 95° C. Denaturing washes may involve subjecting the particles with associated test antigen to a denaturing agent, such as urea or guanidine hydrochloride at a sufficient concentration, such as at least 5M, 6M, 7M, or 8M.

[0337] Preferably, the denaturing wash results in an amount of endotoxin in the test antigen such that the final concentration of endotoxin in the method is less than 100 pg / ml, preferably less than 50 pg / ml, more preferably less than 25 pg / ml and most preferably less than 10 pg / ml.

[0338] It is advantageous if the particles have paramagnetic properties, allowing easy handling by magnetic retention.

[0339] Preferably, the test antigen is covalently linked to the particle or is linked to the particle via a metal chelate.

[0340] Antigen presenting cells (APC) and T cell samples

[0341] In the context of the present invention, APCs are professional antigen presenting cells, such as monocytes / macrophages or dendritic cells. APCs may be primary cells or immortalized cells.

[0342] The APCs must be compatible with the T cells of the T cell sample so that they can present antigens to the T cells in an antigen-specific environment (MHC restriction) to which the T cells can react. The APCs and T cell sample are preferably obtained from the same species and are donor matched with respect to MHC receptors. However, it is also contemplated to use genetically engineered APCs from different species.

[0343] If the antigen presenting cell and T cell samples originate from the same individual, any possibility of mismatch between APCs and T cells is avoided.

[0344] Antigen presenting cells and T cell samples can be derived from the same blood sample, which is advantageous from a practical point of view. Antigen presenting cells and T cell samples can be derived from PBMC samples from the same individual. Obtaining PBMC from peripheral blood samples is a conventional approach, which provides a convenient source for both APCs and T cells from the same individual.

[0345] PBMC samples can be used fresh or subjected to freezing. From a logistical point of view, the possibility of using frozen cells has great practical advantages.

[0346] The T cell sample may originate from a tumor, preferably a lymphatic vessel in a tumor.

[0347] T cell samples can also be derived from ascites fluid.

[0348] The T cell sample may contain whole PBMCs including both CD4+ and CD8+ T cells, purified T cell populations, or PBMCs lacking one or more specific T cell populations.

[0349] Quantification of antigen-specific T cell activation

[0350] Quantification of antigen-specific T cell activation can include the following steps:

[0351] a. providing phagocytosable particles having a test antigen tightly associated therewith, wherein the particles having the associated test antigen have been subjected to a denaturing wash resulting in endotoxin levels low enough not to interfere with subsequent steps;

[0352] b. Providing live antigen presenting cells;

[0353] c. contacting the washed particles with antigen-presenting cells under conditions that allow phagocytosis of the particles by the antigen-presenting cells;

[0354] d. Providing a test sample to be assayed containing live T cells;

[0355] e. contacting the test sample in vitro with the antigen presenting cells in contact with the particles under conditions that allow antigen-specific activation of T cells in response to antigens presented by the antigen presenting cells; and

[0356] f. Quantifying antigen-specific T cell activation in a test sample.

[0357] Antigen-specific T cell activation in a test sample can be quantitatively tested using ELISpot or FluoroSpot technology or by measuring T cell proliferation. It is important to note that although this example uses expression of a specific cytokine as a readout, there are many other methods that can be used to determine antigen-specific T cell activation. For example, using T cell proliferation as a readout (the utility of which was demonstrated earlier in PCT / EP2016 / 081141) can be used to eliminate the need to measure any specific cytokine.

[0358] Antigen-specific T cell activation in quantitative test samples may preferably include determining T cell responses by measuring the secretion of IFN-γ, IL-17 and / or IL-22. Among them, IL-17 and IL-22 are particularly preferred because they give the most robust results (see Table 3).

[0359] Preferably, quantitative testing of T cell activation in a cell sample involves determining the number of activated T cells in the sample. Fraction Quantitative testing of T cell activation in a cell sample may involve determining the ratio of activated T cells in a sample detected using two different measurements. The numbers may be normalized by numerical operations, such as taking the logarithm or square root. A particularly preferred quantification involves determining a measurement obtained by dividing the number of IL-17 positive cells by the square root of the number of IFN-γ positive cells.

[0360] Related references

[0361] Preferably, the relevant reference to which the quantified antigen-specific activation is compared is an equivalent quantified antigen-specific activation in a reference sample from a reference subject without pathological MS-related autoimmunity.

[0362] The reference may be the mean of equally quantitative antigen-specific activation in a group of reference samples from a group of reference subjects without pathological MS-related autoimmunity. The group may comprise at least 10 reference subjects.

[0363] The reference may be an equal quantification of antigen-specific activation in a sample from the same subject taken at a different time point.

[0364] Diagnostic Applications

[0365] The diagnostic utility of the method is as shown in Examples 3-7. As demonstrated by the receiver operating characteristic (ROC) analysis, there is always a trade-off between sensitivity and selectivity. If the threshold for determining the presence of pathology is set lower, sensitivity increases (i.e., the number of false negatives decreases), but at the expense of reducing selectivity (i.e., the number of false positives increases). If the threshold is raised instead, sensitivity is reduced and selectivity is improved. Depending on the setting, the tolerance for false negatives and false positives will be different. For example, in a full population screening where millions of people are tested, the number of false positives must be very low, otherwise the number of patients who need to be followed up will be overwhelming. On the other hand, if a diagnostic test is used as part of an expert evaluation of a single patient, where several other factors will be weighed before reaching a diagnosis, a larger proportion of false positives can be considered tolerable. Therefore, the threshold for making a conclusion needs to be set in most cases considering the environment for analysis, and it is neither appropriate nor necessary to determine a threshold that is generally applicable.

[0366] If the quantified antigen-specific activation in the test sample is at least 2-fold, preferably 3-fold, more preferably 5-fold, most preferably 10-fold higher compared to the reference, it can be concluded that the extent of MS-related autoimmunity is increased.

[0367] An increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is higher than the mean of a group of equally quantified reference samples from a group of reference subjects without pathological MS-related autoimmunity by 2 times the standard deviation of the reference sample group.

[0368] If the quantified antigen-specific activation in the test sample is statistically significantly higher than the reference with a p-value calculated with the Student's T-test of less than 0.05, an increased degree of MS-related autoimmunity is concluded.

[0369] If the quantified antigen-specific activation in the test sample is statistically significantly higher than the reference with a p-value calculated with the Mann-Whitney U test of less than 0.05, an increased degree of MS-related autoimmunity is concluded.

[0370] Application in specific environments

[0371] There is provided a method according to a fourth aspect, wherein:

[0372] a. Methods for diagnosing MS in a subject;

[0373] b. the reference represents an equivalent quantitative antigen-specific activation in a reference subject without pathological multiple sclerosis-related autoimmunity; and

[0374] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of multiple sclerosis in the test subject.

[0375] The method is particularly suitable for detecting the presence of MS in a patient.

[0376] There is provided a method according to a fourth aspect, wherein:

[0377] a. Methods for following the course of MS in subjects;

[0378] b. the reference represents an equal amount of antigen-specific activation in a sample taken from the same subject at a different time point (preferably earlier); and

[0379] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of a higher multiple sclerosis disease activity in the test subject, and vice versa.

[0380] The method is particularly suitable for following the course of MS in a subject known to have MS.

[0381] There is provided a method according to a fourth aspect, wherein:

[0382] a. A method for prognosing the course of MS in a subject;

[0383] b. the reference represents an equivalent quantitative antigen-specific activation in a sample taken at an earlier time point from the same subject; and

[0384] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of increased multiple sclerosis disease activity in the test subject, and vice versa.

[0385] The method is particularly suitable for prognosing the course of MS in a subject known to suffer from MS.Given that MS is characterised by relapses with intermittent recovery, it is valuable to detect an impending relapse in advance to allow for the preemptive administration of treatment.

[0386] There is provided a method according to a fourth aspect, wherein:

[0387] a. Methods used to assess a subject's response to therapeutic treatment;

[0388] b. the reference represents an equivalent quantitative antigen-specific activation in a sample taken from the same subject before administration of the therapeutic treatment to be evaluated;

[0389] c. the test sample is from the same subject after initiation of therapeutic treatment; and

[0390] d. A lower degree of quantitative antigen-specific activation in the test sample compared to the reference indicates therapeutic efficacy against multiple sclerosis disease activity in the test subject, and an unchanged or higher degree of quantitative antigen-specific activation indicates a lack of therapeutic efficacy against multiple sclerosis disease activity in the test subject.

[0391] The method is particularly suitable for assessing a subject's response to therapeutic treatment. In particular, the method is valuable in conducting clinical trials, selecting appropriate drugs for individual patients and performing dose finding for individual patients.

[0392] Uses of peptides or peptide mimetics

[0393] In a fifth aspect, there is provided a use of a peptide or peptide mimetic in the diagnosis or treatment of MS, wherein the peptide or peptide mimetic comprises a specific T cell epitope contained in SEQ ID NO: 5, wherein the T cell epitope comprises an amino acid sequence of n consecutive residues that differs from a subsequence of a selected MS antigen by no more than 0, 1 or 2 residue substitutions, deletions and / or insertions. The use may be in vitro, in particular for diagnostic use.

[0394] The subsequence can be selected from residues 1-166 of SEQ ID NO:5, residues 167-295 of SEQ ID NO:5, residues 296-1327 of SEQ ID NO:5, residues 1328-2234 of SEQ ID NO:5, residues 2235-2250 of SEQ ID NO:5, or residues 1-2234 of SEQ ID NO:5.

[0395] Preferably, the T cell epitope may correspond to an MS antigen selected from the group consisting of FABP7 isoform 2 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3), SNAP91 (SEQ ID NO: 4) and FABP7 isoform 1 (SEQ ID NO: 6).

[0396] The value of n may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Preferably, n is at least 11, more preferably n is at least 13, still more preferably n is at least 15, still more preferably n is at least 17, even more preferably n is at least 19. Preferably m is 1, more preferably m is 0.

[0397] The T cell epitope of the fifth aspect may be as defined as the therapeutic T cell epitope of the first aspect.

[0398] General aspects related to the present disclosure

[0399] The term "comprising" should be interpreted as including but not limited to. The arrangement of the present disclosure into sections with headings and subheadings is only for improving readability and should not be interpreted as limiting in any way. In particular, the division does not in any way exclude or limit the combination of features under different headings and subheadings with each other.

[0400] All references are hereby incorporated by reference. Example

[0401] The following examples should not be construed as limiting. For further information on experimental details, the skilled reader is referred to the separate section entitled "Materials and Methods".

[0402] Example 1: Identification of autoantigens in multiple sclerosis by FluoroSpot autoantigen screening

[0403] The inventors used IFNγ / IL-22 / IL-17A FluoroSpot as an assay for T cell activation, measuring T cell activation in response to a library of 125 PrESTs divided into 45 pools and coupled to beads. The screen identified possible autoantigens by detecting those antigen pools that stimulated higher T cell responses in PBMCs from MS patients compared to healthy controls. Figure 1A As shown in Figure 2C, in a screen of 16 patients and 9 healthy controls, statistically significant differences in the number of activated T cells between the patient mean and the control mean were seen when analyzed using a two-way ANOVA. For activated T cells secreting IL-22, differences were seen for the following five antigen pools: antigen #6 (P < 0.0001), antigen #18 (P < 0.0001), antigen #23 (P < 0.0001), antigen #29 (P < 0.0001), and antigen #33 (P < 0.05) ( Figure 1A For IL-17-secreting T cells, differences between patients and controls were seen for the same five antigens: antigen #6 (P < 0.05), antigen #18 (P < 0.0001), antigen #23 (P < 0.01), antigen #29 (P < 0.0001), and antigen #33 (P < 0.05) ( Figure 1B For IFNγ, differences were only seen for antigen #18 (p<0.0001) ( Figure 1C ).

[0404] Figure 2A -D shows the same data presented as activation for individual patients and controls against the above antigen pools.

[0405] Example 2: Determination of immunogenic antigens contained in the antigen library.

[0406] Antigen pools 18, 23, 26 and 29 described in Example 1 were split into the individual proteins contained therein. PBMCs from the four patients who showed high degrees of T cell activation against these specific antigen pools in Example 1 were tested again for activation against the individual proteins. The methods and protocols used were the same as in Example 1. In each pool, one specific protein triggered a significant majority of T cell activation. The proteins tested were: Antigen pool 18, #1CYB561D2 and #2FABP7 (SEQ ID NO: 1). Antigen pool 23, containing #1NOVA2 and #2PROK2 (SEQ ID NO: 2). Antigen pool 26, containing #1RTN3 (SEQ ID NO: 3) and #2SDK2. Antigen pool 29, containing #1SNAP25 and #2SNAP91 (SEQ ID NO: 4). The results are presented in Figure 3 middle.

[0407] Example 3: Grouping patients based on responsiveness profiles.

[0408] When T cell activation against the 4 candidate autoantigens was analyzed at the level of the individual subjects, four different reactivity profiles were observed in the patients. When stimulated with all candidate antigens except #26, 4 patients responded with significant activation of cells producing all cytokines, referred to as "Profile 1a". 5 patients responded against #18 and 1 or 2 more antigens with significant activation of cells producing all cytokines, referred to as "Profile 1b". 3 patients responded against #26 with only activated IFNγ-producing cells, referred to as "Profile 2". 4 patients were non-responders, referred to as "Profile 3". The profiles are presented in Figure 4 middle.

[0409] There were too few patients in Profile 2 to produce a clear statistical difference between mean patient and control activation (see Figures 1 and 2C), but had a strikingly unique profile not found in any healthy controls.

[0410] Example 4: Demonstration of diagnostic utility

[0411] T cell activation against these antigens can be used as a diagnostic tool and biomarker for multiple sclerosis. Based on the data from Example 1, receiver operating characteristic (ROC) curves were created to assess sensitivity and specificity if T cell activation is used as a diagnostic tool. These graphs are presented in Figure 5A -F and the data in Tables 1 and 3. Activation of IL22- and IL17-producing T cells was particularly promising when stimulated with antigen #18 (containing FABP7 PrEST) or full-length FABP7 isoform 2 (SEQ ID NO: 1), reaching approximately 80% sensitivity and specificity, respectively. Testing patient and control PBMCs against a mixture of all of these antigens will likely further improve these numbers. As can be seen in Table 2, the combination of antigens 23 and 29 gave a more sensitive test than each antigen alone.

[0412] The full-length protein can also be used in a similar manner, see Fig.9A -D. Using all four full-length antigens, using the average response of each individual to the four antigens further improves sensitivity and specificity ( Fig.9E Combining different readouts, such as analyzing the ratio between IL-17 and IFNγ, could further increase the sensitivity of the test ( Fig.9E , Table 2).

[0413] Table 1. Sensitivity and specificity when using a specific number of activated IL17-producing cells as cut-off. For this specific antigen and read-out, when the cut-off for a "positive test" was set at >3 IL-17 points, the sensitivity and specificity reached 75% and 100%.

[0414] Number of IL-17 activated cells for 18PrEST Sensitivity (%) Specificity (%) 0.25 93.75 33.33 0.75 93.75 55.56 1.25 81.25 55.56 1.75 81.25 66.67 2.25 75 77.78 2.75 75 88.89 3.25 75 100 4.25 62.25 100 5.5 50 100

[0415] Table 2. Sensitivity and specificity when using the ratio between the mean number of IL-17-producing cells reactive to antigen and the mean number of IFNγ-producing cells. ROC curves based on the ratio of the number of IL-17-producing and IFNγ-producing cells rather than the absolute number produce an even stronger diagnostic test. The ratio is calculated as the mean number of IL-17-producing cells reactive to antigen divided by the square root of the mean number of IFNγ-producing cells.

[0416]

[0417]

[0418] Table 3. Figure 5A - Descriptive parameters of the ROC curves presented in D.

[0419]

[0420] Example 5: Validation of FABP7 as an MS antigen using the full-length recombinant form

[0421] A recombinant form of human FABP7 isoform 2 (SEQ ID NO: 1) was produced in E. coli. The protein was produced in house according to a standard E. coli recombinant protein protocol. It was purified via his tag purification and coupled to paramagnetic beads and washed according to the previously described protocol. T cell activation of 13 patients (7 of which consisted of new samples from previously included patients and 6 of which were not previously tested) and 7 controls (not included in the previous testing) was tested in the previously described FluoroSpot assay.

[0422] The results are presented in Figure 6 The results from FABP7 PrEST in Examples 1 and 3, and the full-length recombinant form used in this Example were very similar to each other.

[0423] Example 6: Identification of specific T cell epitopes

[0424] Overlapping peptides covering the entire span of FABP7 (15aa long with 10aa overlap), the largely overlapping isoforms 2 and 1 (SEQ ID NO: 1 and SEQ ID NO: 6, respectively), and PROK2 (SEQ ID NO: 2) were pooled into 6 fractions for FABP7 and 3 fractions for PROK2 and used to stimulate PBMCs in the FluoroSpot assay. PBMCs from 6 patients were tested against these pools. Fig. 7A and 7B As shown, for each antigen, patients responded more highly to a single pool (pool 5 for FABP7, pool 1 for PROK2). Compared to previous examples using longer proteins, this example shows that short peptides with amino acid sequences containing specific T cell epitopes can also be used to identify autoreactive T cells in MS patients.

[0425] Further splitting the positive peptide pool and iteratively repeating the experiment will allow even more detailed identification of specific T cell epitopes within each MS antigen. Peptides even shorter than 15 aa can then be designed and tested to fully elucidate specific T cell epitopes.

[0426] In addition, a complementary way to discover possible T cell epitopes was by performing HLA binding experiments. The same overlapping peptides were tested for binding affinity to the multiple sclerosis-associated HLA DRB5 01:01 molecule in a competitive binding assay. The binding affinity was then compared to the binding affinity of a known strong binding peptide derived from the H1N1 influenza virus. For both FABP7 and PROK2, the strongest binding peptide was found in the same peptide pool that the patient also reacted to, indicating that it is a T cell epitope candidate. The results are presented in Table 4. Representative examples of different binding affinities are shown in Fig. 10A -D.

[0427] Table 4. Affinity of overlapping peptides from FABP7 and PROK 2 to HLA DRB5 01:01.

[0428] Affinity was tested in a competitive binding assay. Legend: - no binding, + weak binding, ++ moderate binding, +++ strong binding.

[0429]

[0430]

[0431]

[0432] Example 7: Validation of autoantigen screening using recombinant full-length antigens

[0433] Recombinant full-length forms of the antigens FABP7 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3), SNAP91 (SEQ ID NO: 4) were produced in-house in E. coli according to standard recombinant protein production protocols and purified via his-tag purification. 52 patients and 24 controls were tested for reactivity against the antigens in the IFNγ / IL17 / IL22 FluoroSpot assay as previously described. Similar to the previously tested PrESTs, the reactivity of PBMCs from patients with multiple sclerosis against all four antigens was significantly higher for all cytokines analyzed, except for IFNγ for SNAP91 (see Fig. 8A -D).

[0434] Example 8: Antigen-specific immunotherapy

[0435] There are well-described previous studies using subcutaneous, intradermal or transdermal administration of previously known multiple sclerosis autoantigenic peptide-epitopes in antigen-specific immunotherapy. For example, Walczak et al. (Walczak A, Siger M, Szczepanik M, Selmaj K. Transdermal application of myelin peptides in multiple sclerosis treatment. JAMA Neurol. 2013) used a mixture of known epitopes from the known autoantigens myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) which were applied transdermally over a 1-year period with significant but modest reductions in disease activity. Chataway et al. (Cathaway J, Martin K, Barrell K, Sharrack B, Stolt P, Wraith DC. Effects of ATX-MS-1467 immunotherapy over 16 weeks in relapsing multiple sclerosis. Neurology 2018) used a mixture of four peptide epitopes from myelin basic protein (MBP) subcutaneously injected at increasing doses over 4 weeks, and then the highest dose every two weeks over 16 weeks. Similarly, a significant positive but modest effect was also seen. This suggests that the method of antigen-specific immunotherapy via induction of T cell tolerance to self-antigens is a feasible approach.

[0436] Based on the examples previously described in this document and known methods of epitope mapping, we will first identify specific peptide epitopes for FABP7, PROK2, RTN3, and SNAP 91. Then, as previously described, patients will be screened for T cell reactivity against these autoantigens. Based on the screening, a patient-based mixture of peptide epitopes will be selected and then used for treatment according to established protocols for antigen-specific immunotherapy. Treatment with peptide epitopes from more autoantigens than previously used can be expected to be more effective and tolerable because the personalized approach (and therefore excluding patient-irrelevant peptide epitopes) remains the same.

[0437] Materials and methods

[0438] Covalent coupling of proteins to paramagnetic beads containing free carboxylic acid groups.

[0439] Using a 1 μm diameter MyOne TM Carboxylic acid (ThermoFischer Scientific), and the coupling procedure was performed according to the manufacturer's protocol (two-step method using NHS (N-Hydroxysuccinimide) and EDC (ethylcarbodiimide)).

[0440] The beads were washed twice with MES buffer (25 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 6). The carboxylic acid groups were then activated by adding 50 mg / ml NHS (N-hydroxysuccinimide) and 50 mg / ml EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) in MES buffer to the beads and incubating at room temperature for 30 minutes. The beads were collected with a magnet, the supernatant was removed, and the beads were washed twice with MES buffer. The protein was diluted to a concentration of 1 mg / ml in MES buffer, a total of 100 ug, and added to the beads and incubated at room temperature for 1 hour. The beads were collected with a magnet, the supernatant was removed and stored for protein concentration measurement. Unreacted activated carboxylic acid groups were quenched with 50 mM Tris pH 7,4 for 15 minutes. The beads were then washed with PBS pH 7.4 and then stored at -80°C.

[0441] To measure the amount of protein coupled to the beads, the protein concentration of the protein before coupling and the protein concentration in the supernatant after coupling were measured using a BCA protein assay kit (Pierce BCA Protein Assay Kit, ThermoFisher Scientific). The BCA assay was used according to the manufacturer's protocol.

[0442] Endotoxins were removed by denaturing washes.

[0443] The beads were coupled to recombinant proteins produced in E. coli. The protein-coupled beads were washed under several different denaturing conditions to ensure the removal of endotoxins. To remove endotoxins, the beads were washed with the following 3 different wash buffers: 2M NaOH pH 14.3, 0.5M L-arginine, and 0.1% Triton X100, all in sterile water at room temperature. The beads were suspended in the buffer and shaken for 4 minutes, and the supernatant was collected and removed with a magnet. Repeat 5 times. The beads were then washed 5 times with sterile PBS to remove any remaining wash buffer. The remaining endotoxins were measured using a monocyte reactivity assay (IL1B / IL6FluoroSpot assay, MABTECH, Sweden).

[0444] patient

[0445] A total of 24 MS patients treated with natalizumab at the Neurological Clinic Karolinska University Hospital, Solna and Huddige donated 80 ml of venous blood at their routine treatment visits. Six of these patients were sampled again 6-12 months after the original sampling for further experiments (Example 5).

[0446] Table 5. Inclusion and exclusion criteria for MS patients

[0447]

[0448] Age- and sex-matched healthy controls were recruited among the staff of the institution and clinic. The controls underwent the same blood sampling protocol as the patients.

[0449] PBMCs were isolated from venous blood samples (obtained in BD Vacutainer EDTA tubes) by Ficoll-Paque (GE Healthcare, Uppsala, Sweden) gradient centrifugation according to standard protocols. Cells were frozen in freezing medium (45% FCS, 45% RPMI, 10% DMSO) and stored at -150°C.

[0450] A second, larger cohort was collected to test the full-length antigen. In this cohort, 52 patients and 24 healthy controls were included. The inclusion / exclusion criteria (Table 5) as well as the sampling and PBMCs isolation were identical.

[0451] Candidate Antigen Library

[0452] The antigens used were from the Royal Institute of Technology (KTH, Sweden) and the Swedish Human Protein Atlas project (Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015; 347(6220): 1260419). They consist of protein epitope signature tags (PrESTs) (short recombinant 10-12 kDa peptides representing unique parts of human proteins). PrESTs have been produced in E. coli and purified via the tag. The PrESTs for this project were selected by the following criteria: 1) Proteins assumed to be involved in MS based on published data. 2) Major structural proteins of the myelin sheet. 3) Proteins of interest, selected by communication with experts in the field. 4) Highly expressed CNS-specific proteins that have not been previously associated with the disease. A total of 125 PrESTs from 70 different proteins were used in this experiment.

[0453] Production of full-length antigens

[0454] The antigens selected from the PrEST screening described in Examples 1 and 2 (FABP7, PROK2, RTN3 and SNAP91) were selected for further testing of the proteins in full-length form. The full-length antigens were produced by transforming the plasmid encoding the antigen and the histamine tag into E. coli. After growth, the bacteria were lysed and the protein was purified from the supernatant of the bacterial lysate via 6x histidine tag purification on an immobilized metal affinity chromatography column. The antigens were then coupled to beads and endotoxins were washed as described above.

[0455] FluoroSpot T Cell Activation Assay

[0456] FluoroSpot assays were performed under sterile conditions in a cell culture hood. Cells were thawed in a 37°C water bath and washed with cRPMI (RPMI 1640 medium, Sigma Aldrich, containing 10% fetal bovine serum, 1% 200 mM L-glutamine, 1% 10,000 U / ml penicillin-10 mg / ml streptomycin). Cells were manually counted using a light microscope (Nikon TMS-F, Nikon, Japan) and subsequently diluted to 2,5x10 in cRPMI. 6FluoroSpot plate (human IFNγ / IL-22 / IL-17AFluoroSpot kit, pre-coated, Mabtech, Sweden) was washed with PBS and then blocked with cRPMI for 30 minutes at room temperature. The blocked cRPMI was then discarded and 100 μl of fresh cRPMI was added to each well of the FluoroSpot plate. Antigen (3x10^6 beads) was added to each well in duplicate according to the specific layout. Anti-CD3 was used as a positive control according to the manufacturer's protocol. Both uncoupled beads and unstimulated culture medium were used as negative controls. PBMC (250,000 cells) in 100 μl cRPMI were added to each well (125,000 cells for anti-CD3). The plate was placed in an incubator (37°C humidified, 5% CO2) for 44 hours. The development of the spots was prepared according to the manufacturer's protocol.

[0457] Overlapping peptides

[0458] Continuous overlapping peptides (15 amino acids in length, 10 of which overlap) spanning the entire FABP7 isoform 1 and 2 and PROK2 were purchased in lyophilized form from a commercial supplier and subsequently suspended in 100% dimethyl sulfoxide (DMSO) to a concentration of 50-100 mg / ml. They were then further diluted to a concentration of 5 mg / ml in sterile PBS. They were pooled into 6 fractions of FABP7 and 3 fractions of PROK2, each containing 5-7 peptides. Each library contained was adjacent to each other on the full-length protein. Cells from 6 MS patients were tested for these in the FluoroSpot assay according to the previously described protocol. The final concentration of each peptide in the cell culture well was 5 μg / ml.

[0459] Antigen-specific immunotherapy

[0460] A clinical trial will be conducted to evaluate the safety, tolerability and efficacy of antigen-specific immunotherapy targeting the identified autoantigens FABP7, PROK2, RTN3 and SNAP91. First, as previously explained in Example 6, the immunodominant epitopes from each antigen are identified. Secondly, the study participants (multiple sclerosis patients) are screened for T cell activity against the autoantigens using the method described in Example 7. Patients who are reactive against the autoantigens will be eligible for inclusion in the trial. The treatment may consist of a mixture of one or several immunodominant peptide epitopes from each autoantigen or a mixture of one or several immunodominant peptide epitopes from autoantigens to which the patient reacts in the pre-inclusion screening.

[0461] The treatment regimen will be based on the previously published successful antigen-specific immunotherapy regimen (Cathaway J, Martin K, Barrell K, Sharrack B, Stolt P, Wraith DC. Effects of ATX-MS-1467 immunotherapy over 16 weeks in relapsing multiple sclerosis. Neurology 2018) (Walczak A, Siger M, Szczepanik M, Selmaj K. Transdermal application of myelin peptides in multiple sclerosis treatment. JAMA Neurol. 2013). In an alternative, the treatment will consist of weekly / biweekly subcutaneous or intradermal injections of peptide-epitope mixtures, starting with a low dose, followed by a dose escalation (up-dosing) phase until the desired higher dose is reached. Then a higher dose injection is performed weekly / biweekly for a limited period of time. Alternatively, the peptide-epitope will be applied in a similar regimen, but in a skin, sublingual or oral manner. Safety and tolerability will be assessed continuously, while efficacy will be assessed after completion of treatment. Endpoints will consist of efficacy parameters such as a combination of MRI-based assessment of the number and volume of lesions and clinical variables including the Expanded Disability Status Score (EDSS), and time to first relapse or frequency of relapse.

[0462] The present invention specifically provides the following embodiments:

[0463] 1. A tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5,

[0464] The composition comprises a therapeutic T cell epitope comprising a sequence of n consecutive amino acid residues, wherein the sequence of n consecutive amino acid residues:

[0465] a. a subsequence identical to SEQ ID NO: 5; or

[0466] b. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0467] wherein n is at least 8, and m is 0, 1 or 2.

[0468] 2. A tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5,

[0469] The composition comprises a nucleic acid encoding a therapeutic T cell epitope comprising a sequence of n consecutive amino acid residues, wherein the sequence of n consecutive amino acid residues:

[0470] a. a subsequence identical to SEQ ID NO: 5; or

[0471] b. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0472] wherein n is at least 8, and m is 0, 1 or 2.

[0473] 3. A tolerogenic composition for use in a method of treating multiple sclerosis (MS) in a patient with MS who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope contained in the amino acid sequence of SEQ ID NO: 5,

[0474] The composition comprises an antigen presenting cell exposed ex vivo to a therapeutic T cell epitope comprising a sequence of n consecutive amino acid residues, wherein the sequence of n consecutive amino acid residues:

[0475] a. a subsequence identical to SEQ ID NO: 5; or

[0476] b. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0477] wherein n is at least 8, and m is 0, 1 or 2.

[0478] 4. A composition for use according to any of the preceding items, wherein the method of treatment comprises determining the patient's T-cell autoreactivity against a T-cell epitope contained in the amino acid sequence of SEQ ID NO:5.

[0479] 5. A composition for use according to item 4, wherein said determination is performed using a method according to any one of items 40-84.

[0480] 6. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised of residues 1 to 166 of SEQ ID NO: 5.

[0481] 7. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised of residues 167-295 of SEQ ID NO:5.

[0482] 8. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised within residues 296-1327 of SEQ ID NO:5.

[0483] 9. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised of residues 1328-2234 of SEQ ID NO:5.

[0484] 10. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised within residues 2235-2250 of SEQ ID NO:5.

[0485] 11. A composition for use according to any one of items 1 to 5, wherein the subsequence is comprised of residues 1-2234 of SEQ ID NO:5.

[0486] 12. A composition for use according to any one of items 1 to 5, wherein the subsequence is contained in the sequence of any one of the peptides in Table 4.

[0487] 13. A composition for use according to any one of the preceding clauses, wherein n is at least 11.

[0488] 14. A composition for use according to any one of the preceding clauses, wherein n is at least 13.

[0489] 15. A composition for use according to any one of the preceding clauses, wherein n is at least 15.

[0490] 16. A composition for use according to any one of the preceding clauses, wherein n is at least 17.

[0491] 17. A composition for use according to any one of the preceding clauses, wherein n is at least 19.

[0492] 18. A composition for use according to any of the preceding clauses, wherein n is at least 50, at least 75, more preferably at least 100.

[0493] 19. A composition for use according to any one of the preceding clauses, wherein m is 2.

[0494] 20. A composition for use according to any one of items 1 to 18, wherein m is 1.

[0495] 21. A composition for use according to any one of items 1 to 18, wherein m is 0.

[0496] 22. A composition for use according to any of the preceding items, wherein the T cell epitope differs from a subsequence by no more than m residue substitutions and comprises no substitutions or deletions compared to the subsequence.

[0497] 23. A composition for use according to item 1 or 3 or any dependent item thereof, wherein the therapeutic T-cell epitope is a peptide or a peptidomimetic.

[0498] 24. A composition for use according to item 23, wherein the therapeutic T cell epitope is a peptide or peptide mimetic having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with a subsequence of SEQ ID NO:5.

[0499] 25. A composition for use according to claim 1 or any of its dependencies, wherein the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitopes, each of which meets the criteria listed in claim 1.

[0500] 26. A composition for use according to claim 2 or any of its dependent claims, wherein the composition comprises nucleic acids encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitopes, each of which meets the criteria listed in claim 2.

[0501] 27. A composition for use according to claim 3 or any of its dependencies, wherein the antigen presenting cells have been exposed to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitopes, each of which meets the criteria listed in claim 3.

[0502] 28. A composition for use according to any one of items 25-27, wherein the different therapeutic T-cell epitopes are selected from one, two, three, four, five or six of the sequence intervals described in items 6-11.

[0503] 29. A composition for use according to item 1 or any of its dependencies, wherein the composition comprises a therapeutic T cell epitope coupled to a solid support, such as a biocompatible polymer, particle or cell.

[0504] 30. A composition for use according to item 1 or any of its dependencies, comprising a therapeutic T cell epitope capable of specifically binding to the same T cell receptor as the endogenous epitope.

[0505] 31. A composition for use according to item 2 or any of its dependencies, comprising a nucleic acid encoding a therapeutic T cell epitope that is capable of specifically binding to the same T cell receptor as the endogenous T cell epitope.

[0506] 32. A composition for use according to item 3 or any of its dependencies, comprising antigen presenting cells exposed to a therapeutic T cell epitope, wherein the therapeutic T cell epitope is capable of specifically binding to the same T cell receptor as the endogenous T cell epitope.

[0507] 33. A composition for use according to any preceding clause, the method comprising selecting the therapeutic T cell epitope such that it corresponds to an epitope against which the patient exhibits T cell autoreactivity.

[0508] 34. A composition for use according to claim 33, wherein the therapeutic T cell epitope is selected based on its ability to specifically bind to the same T cell receptor (TCR) as the endogenous epitope.

[0509] 35. A composition for use according to any one of the preceding clauses, wherein the method of treatment comprises administering the composition to the subject in a tolerogenic manner, thereby inducing T-cell tolerance to the T-cell epitope in the patient.

[0510] 36. A composition for use according to any one of the preceding clauses, wherein the method of treatment comprises administering the composition orally, mucosally, intradermally, transdermally or subcutaneously.

[0511] 37. A composition for use according to item 1 or 2 or any of its dependencies, wherein the method of treatment comprises administering the composition to antigen presenting cells in vitro and then administering the antigen presenting cells to the patient.

[0512] 38. A composition for use according to item 2 or any of its dependencies, wherein the nucleic acid is included in a vector and is operably coupled to a promoter allowing expression in an antigen presenting cell.

[0513] 39. A composition for use according to item 3 or any of its dependencies, wherein the antigen presenting cells are dendritic cells, monocytes, macrophages, B cells (preferably derived from PBMC) or microglia.

[0514] 40. A method for determining the extent of multiple sclerosis (MS)-related autoimmunity in a test subject, comprising:

[0515] a. Providing a test sample containing live T cells derived from the test subject;

[0516] b. quantifying in vitro the antigen-specific activation of T cells of the test sample in response to a test antigen comprising a T cell epitope, wherein the T cell epitope comprises an amino acid sequence of n consecutive residues, the amino acid sequence of n consecutive residues:

[0517] i. a subsequence identical to SEQ ID NO: 5; or

[0518] ii. a subsequence that differs from SEQ ID NO: 5 by no more than m residue substitutions, deletions and / or insertions;

[0519] wherein n is at least 8, and m is 0, 1 or 2; and

[0520] c. comparing the quantified antigen-specific activation to a relevant reference to determine the extent of MS-related autoimmunity in the test subject.

[0521] 41. A method according to item 40, wherein the subsequence is contained in residues 1-166 of SEQ ID NO:5.

[0522] 42. A method according to item 40, wherein the subsequence is contained within residues 167-295 of SEQ ID NO:5.

[0523] 43. A method according to item 40, wherein the subsequence is contained in residues 296-1327 of SEQ ID NO:5.

[0524] 44. A method according to item 40, wherein the subsequence is contained in residues 1328-2234 of SEQ ID NO:5.

[0525] 45. A method according to item 40, wherein the subsequence is contained within residues 2235-2250 of SEQ ID NO:5.

[0526] 46. ​​A method according to item 40, wherein the subsequence is contained in residues 1-2234 of SEQ ID NO:5.

[0527] 47. A method according to item 40, wherein the subsequence is contained in the sequence of any one of the peptides in Table 4.

[0528] 48. A method according to any of the preceding method items, wherein antigen-specific activation is quantified for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different T cell epitopes, each of which satisfies the criteria described in item 40.

[0529] 49. A method according to item 48, wherein the different T cell epitopes are selected from one, two, three, four, five or six of the sequence intervals described in items 41-47.

[0530] 50. A method according to any one of the preceding method items, wherein antigen-specific activation is quantified for at least two, three or four test antigens, each of which comprises a specific T cell epitope corresponding to a different MS antigen selected from the group consisting of FABP7 (SEQ ID NO: 1), PROK2 (SEQ ID NO: 2), RTN3 (SEQ ID NO: 3) and SNAP91 (SEQ ID NO: 4).

[0531] 51. The method according to any of the preceding method items, wherein n is at least 11.

[0532] 52. The method according to any of the preceding method items, wherein n is at least 13.

[0533] 53. The method according to any of the preceding method items, wherein n is at least 15.

[0534] 54. The method according to any of the preceding method items, wherein n is at least 17.

[0535] 55. The method according to any of the preceding method items, wherein n is at least 19.

[0536] 56. The method according to any of the preceding method items, wherein n is at least 50, at least 75, more preferably at least 100.

[0537] 57. The method according to any of the preceding method items, wherein m is 2.

[0538] 58. The method according to any one of items 40-56, wherein m is 1.

[0539] 59. The method according to any one of items 40-56, wherein m is 0.

[0540] 60. A method according to any one of the preceding method items, wherein the T cell epitope differs from a subsequence of the selected MS antigen by no more than m residue substitutions and comprises no substitutions or deletions compared to the subsequence.

[0541] 61. A method according to any of the preceding method items, wherein the antigen comprising a specific T cell epitope is a peptide or peptide mimetic having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with any one of SEQ ID NO:1-4, SEQ ID NO:5 or SEQ ID NO:6.

[0542] 62. The method according to any one of the preceding method items, wherein the subject is a patient diagnosed with MS.

[0543] 63. A method according to any one of items 40-61, wherein the subject is an individual suspected of having MS.

[0544] 64. The method according to any of the preceding method items, wherein the test sample is derived from a blood sample.

[0545] 65. The method according to any of the preceding method items, wherein the test sample is a PBMC sample.

[0546] 66. The method according to any of the preceding method items, wherein the test sample further comprises antigen presenting cells.

[0547] 67. The method according to any one of the preceding method items, wherein the method further comprises:

[0548] a. Providing live antigen presenting cells;

[0549] b. contacting the test antigen with the antigen presenting cell;

[0550] c. contacting the test sample in vitro with the antigen presenting cells contacted with the test antigen under conditions that allow antigen-specific activation of T cells in response to the antigen presented by the antigen presenting cells; and

[0551] d. quantifying antigen-specific T cell activation in the test sample.

[0552] 68. The method according to any of the preceding method items, wherein the method comprises providing the test antigen tightly associated with phagocytable particles.

[0553] 69. The method according to any one of the preceding method items, wherein quantifying the antigen-specific T cell activation in the test sample comprises determining the T cell response by measuring the secretion of IFN-γ.

[0554] 70. The method according to any one of the preceding method items, wherein quantifying said antigen-specific T cell activation in said test sample comprises determining the T cell response by measuring the secretion of IL-17.

[0555] 71. The method according to any one of the preceding method items, wherein quantifying the antigen-specific T cell activation in the test sample comprises determining the T cell response by measuring the secretion of IL-22.

[0556] 72. The method according to any one of the preceding method items, wherein quantification of antigen-specific T cell activation comprises the following steps:

[0557] a. providing phagocytosable particles having the test antigen tightly associated therewith, wherein the particles having the associated test antigen have been subjected to a denaturing wash resulting in endotoxin levels low enough not to interfere with subsequent steps;

[0558] b. Providing live antigen presenting cells;

[0559] c. contacting the washed particles with the antigen-presenting cells under conditions that allow phagocytosis of the particles by the antigen-presenting cells;

[0560] d. providing the test sample to be determined comprising live T cells;

[0561] e. contacting the test sample in vitro with the antigen presenting cells in contact with the particles under conditions that allow antigen-specific activation of T cells in response to antigens presented by the antigen presenting cells; and

[0562] f. quantifying antigen-specific T cell activation in the test sample.

[0563] 73. The method according to any of the preceding method items, wherein the reference is an equivalent quantitative antigen-specific activation in a reference sample from a reference subject without pathological MS-related autoimmunity.

[0564] 74. A method according to any one of items 40-72, wherein the reference is the average of equally quantitative antigen-specific activation in a group of reference samples from a group of reference subjects without pathological MS-related autoimmunity.

[0565] 75. A method according to item 74, wherein the group comprises at least 10 reference subjects.

[0566] 76. A method according to any one of items 40-72, wherein the reference is an equal quantification of antigen-specific activation in samples from the same subject taken at different time points.

[0567] 77. A method according to any of the preceding method items, wherein if the quantified antigen-specific activation in the test sample is at least 2 times, preferably 3 times, more preferably 5 times, most preferably 10 times higher than the reference, an increased degree of MS-related autoimmunity is concluded.

[0568] 78. A method according to any of the preceding method items, wherein if the quantified antigen-specific activation in the test sample is 2 times higher than the mean value of a group of equally quantified reference samples from a group of reference subjects without pathological MS-related autoimmunity by the standard deviation of the reference sample group, a conclusion of an increased degree of MS-related autoimmunity is drawn.

[0569] 79. The method according to any of the preceding method items, wherein an increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is statistically significantly higher than the reference with a p-value calculated using a Student's T-test of less than 0.05.

[0570] 80. A method according to any of the preceding method items, wherein if the quantified antigen-specific activation in the test sample is statistically significantly higher than the reference with a p-value calculated using a Mann-Whitney U test of less than 0.05, an increased degree of MS-related autoimmunity is concluded.

[0571] 81. The method according to any one of the preceding method items, wherein:

[0572] a. The method is used to diagnose MS in the subject;

[0573] b. the reference represents an equivalent quantitative antigen-specific activation in a reference subject without pathological multiple sclerosis-related autoimmunity; and

[0574] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of multiple sclerosis in the test subject.

[0575] 82. The method according to any one of the preceding method items, wherein:

[0576] a. The method is used to track the course of MS in the subject;

[0577] b. the reference represents an equal amount of antigen-specific activation in samples taken from the same subject at different time points; and

[0578] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of a higher multiple sclerosis disease activity in the test subject, and vice versa.

[0579] 83. The method according to any one of the preceding method items, wherein:

[0580] a. The method is used to prognose the course of MS in the subject;

[0581] b. the reference represents an equal amount of antigen-specific activation in samples taken from the same subject at different time points; and

[0582] c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of increased multiple sclerosis disease activity in the test subject, and vice versa.

[0583] 84. The method according to any one of the preceding method items, wherein:

[0584] a. The method is used to assess the subject's response to therapeutic treatment;

[0585] b. said reference represents an equal quantitative antigen-specific activation in a sample taken from the same subject prior to administration of said therapeutic treatment to be evaluated;

[0586] c. said test sample is from the same subject after initiation of said therapeutic treatment; and

[0587] d. A lower degree of quantitative antigen-specific activation in the test sample compared to the reference indicates therapeutic efficacy against multiple sclerosis disease activity in the test subject, and an unchanged or higher degree of quantitative antigen-specific activation indicates a lack of therapeutic efficacy against multiple sclerosis disease activity in the test subject.

[0588] 85. Use of a peptide or peptide mimetic in the diagnosis or treatment of MS, wherein the peptide or peptide mimetic comprises a specific T cell epitope corresponding to an MS antigen, and the T cell epitope comprises an amino acid sequence of n consecutive residues that differs from the subsequence of SEQ ID NO:5 by no more than m residue substitutions, deletions and / or insertions, wherein m is 0, 1 or 2.

[0589] 86. The use according to item 85, wherein the subsequence is selected from residues 1-166 of SEQ ID NO:5, residues 167-295 of SEQ ID NO:5, residues 296-1327 of SEQ ID NO:5, residues 1328-2234 of SEQ ID NO:5, residues 2235-2250 of SEQ ID NO:5 or residues 1-2234 of SEQ ID NO:5.

[0590] 87. The use according to item 85 or 86, wherein n is at least 11.

[0591] 88. The use according to item 85 or 86, wherein n is at least 15.

[0592] 89. The use according to item 85 or 86, wherein n is at least 19.

[0593] 90. The use according to any one of items 85-89, wherein m is 1.

[0594] 91. The use according to any one of items 85-89, wherein m is 0.

Claims

1. A tolerogenic composition for use in a method of inducing immune tolerance in a multiple sclerosis (MS) patient who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope in the amino acid sequence of residues 121-135 of SEQ ID NO: 1, The composition comprises a therapeutic T cell epitope peptide of SEQ ID NO: 1 or a fragment thereof, wherein the fragment comprises a sequence of n consecutive amino acid residues, the sequence of n consecutive amino acid residues being identical to a subsequence of residues 121-135 of SEQ ID NO: 1, wherein n is at least 8, The therapeutic T cell epitope peptide is capable of specifically activating T cells of the MS patient.

2. A tolerogenic composition for use in a method of inducing immune tolerance in a multiple sclerosis (MS) patient who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope in the amino acid sequence of residues 121-135 of SEQ ID NO: 1, The composition comprises a nucleic acid encoding a therapeutic T cell epitope peptide of SEQ ID NO: 1 or a fragment thereof, wherein the fragment comprises an amino acid sequence of n consecutive amino acid residues, the amino acid sequence of n consecutive amino acid residues being identical to a subsequence of residues 121-135 of SEQ ID NO: 21, wherein n is at least 8, The therapeutic T cell epitope peptide is capable of specifically activating T cells of the MS patient.

3. A tolerogenic composition for use in a method of inducing immune tolerance in a multiple sclerosis (MS) patient who exhibits T cell autoreactivity against an endogenous epitope corresponding to a specific T cell epitope in the amino acid sequence of residues 121-135 of SEQ ID NO: 1, The composition comprises an antigen presenting cell exposed ex vivo to a therapeutic T cell epitope peptide of SEQ ID NO: 1 or a fragment thereof, wherein the fragment comprises a sequence of n consecutive amino acid residues, the sequence of n consecutive amino acid residues being identical to a subsequence of residues 121-135 of SEQ ID NO: 1, wherein n is at least 8, The therapeutic T cell epitope peptide is capable of specifically activating T cells of the MS patient.

4. A composition for use according to any preceding claim, wherein the method of inducing immune tolerance comprises determining the patient's T cell autoreactivity to a T cell epitope within the amino acid sequence of residues 121-135 of SEQ ID NO:

1.

5. A composition for use according to any one of the preceding claims, wherein n is at least 11, at least 13, at least 15, at least 17 or at least 19.

6. A composition for use according to claim 1 or 3 or any claim dependent therefrom, wherein the therapeutic T cell epitope peptide is a peptide or a peptidomimetic.

7. A composition for use according to claim 1 or any claim dependent thereon, wherein the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitope peptides, each of which meets the criteria listed in claim 1.

8. A composition for use according to claim 2 or any claim dependent thereon, wherein the composition comprises nucleic acids encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitope peptides, each of which meets the criteria listed in claim 2.

9. A composition for use according to claim 3 or any claim dependent thereon, wherein the antigen presenting cells have been exposed to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic T cell epitope peptides, each of which meets the criteria listed in claim 3.

10. A composition for use according to claim 1 or any claim dependent therefrom, wherein the composition comprises the therapeutic T cell epitope peptide coupled to a solid support.

11. A composition for use according to claim 1 or any claim dependent thereon, comprising a therapeutic T cell epitope peptide capable of specifically binding to the same T cell receptor as the endogenous epitope.

12. A composition for use according to claim 2 or any claim dependent thereon, comprising a nucleic acid encoding a therapeutic T cell epitope peptide capable of specifically binding to the same T cell receptor as the endogenous epitope.

13. A composition for use according to claim 3 or any claim dependent thereon, comprising antigen presenting cells exposed to a therapeutic T cell epitope peptide capable of specifically binding to the same T cell receptor as the endogenous epitope.

14. A composition for use according to any preceding claim, said method comprising selecting said therapeutic T cell epitope peptide such that it corresponds to an epitope against which said patient exhibits T cell autoreactivity.

15. A composition for use according to claim 14, wherein the therapeutic T cell epitope peptide is selected based on its ability to specifically bind to the same T cell receptor (TCR) as the endogenous epitope.

16. A composition for use according to any preceding claim, said method of treatment comprising administering said composition to said subject in a tolerogenic manner, thereby inducing T-cell tolerance to said T-cell epitope in said patient.

17. A composition for use according to any preceding claim, wherein the method of treatment comprises administering the composition orally, mucosally, intradermally, transdermally or subcutaneously.

18. A composition for use according to claim 1 or 2 or any claim dependent therefrom, said method of treatment comprising administering said composition to antigen presenting cells in vitro and then administering said antigen presenting cells to said patient.

19. A composition for use according to claim 2 or any claim appendant thereto, wherein the nucleic acid is comprised in a vector operably coupled to a promoter allowing expression in an antigen presenting cell.

20. A composition for use according to claim 3 or any claim dependent thereon, wherein the antigen presenting cell is a dendritic cell, a monocyte, a macrophage, a B cell or a microglia.

21. Use of a test antigen comprising a T cell epitope peptide in the preparation of a composition for determining the extent of multiple sclerosis (MS) related autoimmunity in a test subject, wherein the determination comprises: a. Providing a test sample containing live T cells derived from the test subject; b. quantifying in vitro the antigen-specific activation of T cells of the test sample in response to the test antigen comprising a T cell epitope peptide; and c. comparing the quantified antigen-specific activation with a relevant reference to determine the extent of MS-related autoimmunity in the test subject, wherein the T cell epitope peptide is SEQ ID NO: 1 or a fragment thereof, wherein the fragment comprises an amino acid sequence of n consecutive residues, wherein the amino acid sequence of n consecutive residues is identical to a subsequence of residues 121-135 of SEQ ID NO: 1, wherein n is at least 8, The T cell epitope peptide is capable of specifically activating T cells of the MS patient.

22. Use according to claim 21, wherein antigen-specific activation is quantified for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different T cell epitope peptides, each of which meets the criteria set out in claim 21.

23. The use according to any one of claims 21-22, wherein antigen-specific activation is quantified for at least two, three or four test antigens, each of which comprises a specific T cell epitope peptide corresponding to a different MS antigen selected from the group consisting of FABP7, i.e., SEQ ID NO: 1; PROK2, i.e., SEQ ID NO: 2; RTN3, i.e., SEQ ID NO: 3; and SNAP91, i.e., SEQ ID NO:

4.

24. Use according to any one of claims 21 to 23, wherein n is at least 11, at least 13, at least 15, at least 17 or at least 19.

25. Use according to any one of claims 21-24, wherein the subject is a patient diagnosed with MS.

26. Use according to any one of claims 21 to 24, wherein the subject is an individual suspected of having MS.

27. Use according to any one of claims 21 to 26, wherein the test sample is derived from a blood sample.

28. Use according to any one of claims 21 to 27, wherein the test sample is a PBMC sample.

29. The use according to any one of claims 21 to 28, wherein the test sample further comprises antigen presenting cells.

30. The method according to any one of claims 21 to 29, wherein said determining further comprises: a. Providing live antigen presenting cells; b. contacting the test antigen with the antigen presenting cell; c. contacting the test sample in vitro with the antigen-presenting cells contacted with the test antigen under conditions that allow antigen-specific activation of T cells in response to the antigen presented by the antigen-presenting cells; and d. quantifying antigen-specific T cell activation in the test sample.

31. Use according to any one of claims 21 to 30, wherein said determining comprises providing said test antigen tightly associated with phagocytable particles.

32. The use according to any one of claims 21 to 31, wherein quantifying the antigen-specific T cell activation in the test sample comprises determining the T cell response by measuring the secretion of IFN-γ, IL-17 and / or IL-22.

33. The method according to any one of claims 21 to 32, wherein the quantification of antigen-specific T cell activation comprises the following steps: a. providing phagocytosable particles having said test antigen tightly associated therewith, wherein said particles having said associated test antigen have been subjected to a denaturing wash resulting in endotoxin levels low enough not to interfere with subsequent steps; b. Providing live antigen presenting cells; c. contacting the washed particles with the antigen-presenting cells under conditions that allow phagocytosis of the particles by the antigen-presenting cells; d. providing the test sample to be determined comprising live T cells; e. contacting the test sample in vitro with the antigen-presenting cells in contact with the particles under conditions that allow antigen-specific activation of T cells in response to antigens presented by the antigen-presenting cells; and f. quantifying antigen-specific T cell activation in the test sample.

34. The use according to any one of claims 21 to 33, wherein the reference is: a. Equivalent quantitative antigen-specific activation in reference samples from reference subjects without pathological MS-related autoimmunity; b. the mean of equally quantitative antigen-specific activation in a group of reference samples from a group of reference subjects without pathological MS-related autoimmunity; or c. Equivalent quantitative antigen-specific activation in samples from the same subject taken at different time points.

35. Use according to claim 34, wherein the group comprises at least 10 reference subjects.

36. Use according to any of claims 21 to 35, wherein an increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is at least 2-fold, preferably at least 3-fold, compared to the reference.

37. Use according to any one of claims 21 to 36, wherein an increased degree of MS-related autoimmunity is concluded if the quantified antigen-specific activation in the test sample is in accordance with: a. a standard deviation of the reference sample group that is 2 times higher than the mean of a group of equally quantified reference samples from a group of reference subjects without pathological MS-related autoimmunity; b. statistically significantly higher than the reference at a p value of less than 0.05 calculated using the Student's T test; or c. Statistically significantly higher than the reference with a p-value less than 0.05 calculated using the Mann-Whitney U test.

38. The use according to any one of claims 21 to 37, wherein: a. The composition is used to diagnose MS in the subject; b. the reference represents an equivalent quantitative antigen-specific activation in a reference subject without pathological multiple sclerosis-related autoimmunity; and c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of multiple sclerosis in the test subject.

39. The use according to any one of claims 21 to 38, wherein: a. The composition is used to track the course of MS in the subject; b. the reference represents an equal quantitative antigen-specific activation in samples taken from the same subject at different time points; and c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of a higher multiple sclerosis disease activity in the test subject, and vice versa.

40. The use according to any one of claims 21 to 39, wherein: a. The composition is used to prognose the course of MS in the subject; b. the reference represents an equal quantitative antigen-specific activation in samples taken from the same subject at different time points; and c. A higher degree of quantitative antigen-specific activation in the test sample compared to the reference is indicative of increased multiple sclerosis disease activity in the test subject, and vice versa.

41. The use according to any one of claims 21 to 40, wherein: a. the composition is used to evaluate the subject's response to therapeutic treatment; b. said reference represents an equal quantitative antigen-specific activation in a sample taken from the same subject prior to administration of said therapeutic treatment to be evaluated; c. the test sample is from the same subject after the start of the therapeutic treatment; and d. A lower degree of quantitative antigen-specific activation in the test sample compared to the reference indicates therapeutic efficacy against multiple sclerosis disease activity in the test subject, and an unchanged or higher degree of quantitative antigen-specific activation indicates a lack of therapeutic efficacy against multiple sclerosis disease activity in the test subject.

42. Use of a peptide or peptide mimetic in the preparation of a composition for diagnosing MS or inducing immune tolerance in MS patients, wherein the peptide or peptide mimetic comprises a specific T cell epitope peptide corresponding to an MS antigen, the T cell epitope peptide is SEQ ID NO: 1 or a fragment thereof, the fragment comprising an amino acid sequence of n consecutive residues identical to a subsequence of residues 121-135 of SEQ ID NO: 1, wherein n is at least 8, The T cell epitope peptide is capable of specifically activating T cells of the MS patient.

43. Use according to claim 42, wherein n is at least 11, at least 15 or at least 19.

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