Composition

Abstract

The present invention relates to compositions comprising peptides derived from myelin basic protein (MBP) peptides for use in treating or preventing uveitis in a subject. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to a composition comprising a peptide or several peptides derived from myelin basic protein (MBP). The composition or peptide may be useful for the prevention and / or suppression of autoimmunity, for example the prevention and / or suppression of effector T helper cells, which is useful for the treatment and / or prevention of uveitis. The present invention also relates to the corresponding method of treatment and the use of the peptide in the manufacture of a medicament for the treatment and / or prevention of uveitis. [Background technology]

[0002] Uveitis refers to a group of diseases involving inflammation of the uvea. The uvea is the region of the eye located between the sclera and the retina, and includes the iris, ciliary body, and choroid. The uvea provides most of the blood supply to the retina. Associated diseases are not limited to those that directly affect the uvea, but adjacent structures such as the retina, optic nerve, lens, vitreous, and sclera can be affected by the development of uveitis.

[0003] All forms of uveitis are characterized by inflammatory cell infiltration, which is typically visualized using a microscope. In 2010, it was estimated that 285 million people had visual impairments, of which 39 million were blind, and 10% of these were due to uveitis (Global data on visual impairments, The World Health Report, WHO (2010) http: / / www.who.int / blindness / GLOBALDATAFINALforweb.pdf).

[0004] Current treatments for uveitis include the use of glucocorticoid steroids and other broad spectrum immunosuppressants such as methotrexate. Summary of the Invention [Problem to be solved by the invention]

[0005] However, there is a need in the art for alternative treatments for uveitis. The present invention addresses this need. [Means for solving the problem]

[0006] The present inventors have identified a number of peptides derived from myelin basic protein (MBP) that may be useful in the prevention and / or treatment of uveitis.

[0007] The present examples demonstrate that MBP peptides according to the invention can significantly reduce the clinical signs of uveitis in an in vivo model of uveitis.

[0008] Thus, the present invention provides a composition comprising a myelin basic protein peptide selected from the following peptides for use in the treatment and / or prevention of uveitis in a subject: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (sequence number 4).

[0009] In one aspect, a composition for use in treating or preventing uveitis in a subject may comprise MBP 30-44 (SEQ ID NO:1), 83-99 (SEQ ID NO:2), 131-145 (SEQ ID NO:3) and 140-154 (SEQ ID NO:4), i.e., the composition may comprise a "cocktail" of the four peptides.

[0010] A composition for use in the treatment or prevention of uveitis in a subject may consist essentially of MBP 30-44 (SEQ ID NO:1), 83-99 (SEQ ID NO:2), 131-145 (SEQ ID NO:3) and 140-154 (SEQ ID NO:4).

[0011] The present invention also provides a method for treating or preventing uveitis in a subject, the method comprising the step of administering to the subject a composition as described herein.

[0012] The peptides have been found to bind to several MHC class II molecules, including HLA-DR and HLA-DQ molecules, and the combination of these apitopes provides greater coverage of the various major histocompatibility complex (MHC) haplotypes found in uveitis patients than therapy using single peptides alone.

[0013] Therapeutic or prophylactic methods according to the invention may include, for example, administration of the composition to an HLA-DQ6 or HLA-DR2 positive subject or to a subject expressing any other HLA type, e.g., DRB1, DRB3, DRB5, DQA1 or DQB1.

[0014] The invention encompasses the use of a composition as described herein in the manufacture of a medicament for treating or preventing uveitis in a subject.

[0015] The invention also encompasses the use of a composition as described herein in the treatment or prevention of uveitis in a subject.

[0016] The present invention also provides a kit comprising a myelin basic protein peptide selected from the following peptides for use in treating or preventing uveitis in a subject: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (SEQ ID NO: 4). In one embodiment, the composition comprises: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (SEQ ID NO: 4) wherein the MBP peptides may be for simultaneous, separate or sequential administration. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 depicts clinical scores for EAU (posterior uveitis). Data are presented as mean±SEM. [Diagram 2] Representative TEFI images. (A) score 0. (B) score 1. (C) score 2, congested blood vessels. (D) score 3. (E) score 4. (F) score 5. (G) score 6. [Diagram 3] FIG. 13. Clinical scores of EAU (anterior uveitis). Data are expressed as mean±SEM. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 4] Representative TEFI images are shown, with a cumulative score of 1-10 being assigned depending on vasodilation, increased angiogenesis, and invasion. [Diagram 5] FIG. 1 depicts clinical scores for EAU (posterior uveitis), huMBP and IRBP immunized groups. Data are presented as mean±SEM. [Figure 6] FIG. 1 shows the clinical score, area under the curve (AUC) of EAU (posterior uveitis). Data are expressed as mean±SEM. Statistical significance: *** p<0.001. [Figure 7A] FIG. 1 depicts anterior uveitis clinical scores (A) in the huMBP+IRBP model. Data are presented as mean±SEM. Statistical significance: for cyclosporine A vs. vehicle: * p<0.05 and **** p<0.0001. for ATX peptide vs. vehicle: # p<0.05 and #### p<0.0001. Bold symbols on the x-axis represent ATX peptide treatment after day 0. Grey shaded area represents the time course of cyclosporine A treatment. [Figure 7B]FIG. 13 depicts area under the curve analysis (AUC) of anterior uveitis clinical scores in the huMBP+IRBP model. Data are presented as mean±SEM. Statistical significance: for cyclosporine A vs. vehicle: * p<0.05 and **** p<0.0001. for ATX peptide vs. vehicle: # p<0.05 and #### p<0.0001. [Figure 8-1] FIG. 13 depicts posterior uveitis clinical scores in the huMBP model (A) and the huMBP+IRBP model (C). Data are presented as mean±SEM. Statistical significance: for cyclosporine A vs. vehicle: * p<0.05, ** p<0.01, **** p<0.0001. for ATX peptide vs. vehicle: # p<0.05, ## p<0.01 and #### p<0.0001. Bold symbols on the x-axis represent ATX peptide treatment after day 0. Grey shaded area represents the time course of cyclosporine A treatment. [Figure 8-2] Figure 13 depicts area under the curve (AUC) analysis of posterior uveitis clinical scores in the huMBP model (B) and the huMBP+IRBP model (D). Data are presented as mean ± SEM. Statistical significance: for cyclosporine A vs. vehicle: * p<0.05, ** p<0.01, **** p<0.0001. For ATX peptide vs. vehicle: # p<0.05, ## p<0.01 and #### p<0.0001. [Figure 9] Figure 1 shows the incidence (%) of posterior uveitis in the huMBP model (A) and the huMBP+IRBP model (B). Statistical significance: for cyclosporine A vs. vehicle: **** p<0.0001. For ATX peptide vs. vehicle: # p<0.05. Bold symbols on the x-axis represent ATX peptide treatment after day 0. Grey shaded area represents the time course of cyclosporine A treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Myelin basic protein Myelin basic protein (MBP) is a protein that can be isolated from human brain white matter. The mature protein has 170 amino acids, and its sequence is widely available in the literature (see, for example, Chou et al (1986) J. Neurochem. 46:47-53, Figure 1; Kamholz et al (1986), PNAS 83:4962-4966, Figure 2; U.S. Patent No. 5,817,629, SEQ ID NO: 1; Roth et al (1987), J. Neurosci. Res. 17:321-328, Figure 4; Medeveczky et al (2006), FEBS Letters 580:545-552, Figure 3). Alternative splice variants / isoforms of MBP may be generated due to alternative splicing of MBP (see, for example, Kamholz, supra).

[0019] The composition according to the invention comprises an MBP peptide selected from the following peptides: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (sequence number 4).

[0020] The composition may include MBP 30-44 (SEQ ID NO:1), 83-99 (SEQ ID NO:2), 131-145 (SEQ ID NO:3) and 140-154 (SEQ ID NO:4).

[0021] The composition may consist essentially of MBP 30-44 (SEQ ID NO:1), 83-99 (SEQ ID NO:2), 131-145 (SEQ ID NO:3) and 140-154 (SEQ ID NO:4).

[0022] The peptides present in the composition can consist of MBP 30-44 (SEQ ID NO: 1), 83-99 (SEQ ID NO: 2), 131-145 (SEQ ID NO: 3) and 140-154 (SEQ ID NO: 4). That is, no other MBP peptides are present. The MBP peptides can therefore consist of SEQ ID NOs: 1 to 4.

[0023] "Consisting of" means including and limited to whatever the phrase "consisting of" follows. That is, "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements can be present. "Consisting essentially of" means including any of the elements listed in the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the listed elements. That is, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0024] peptide Peptides which may be used in compositions and kits for use according to the present invention are as follows: MBP 30-44 (SEQ ID NO: 1): H-Pro-Arg-His-Arg-Asp-Thr-Gly-Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-NH2 MBP 83-99 (SEQ ID NO: 2): H-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro-NH2 MBP 131-145 (SEQ ID NO: 3): H-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Phe-Lys-Gly-Val-Asp-NH2 MBP 140-154 (SEQ ID NO: 4): H-Gly-Phe-Lys-Gly-Val-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ile-Phe-NH2

[0025] The term "peptide" is used in its ordinary sense to mean a series of residues, typically L-amino acids, connected one to the other by peptide bonds, typically between the α-amino and carboxyl groups of adjacent amino acids. The term includes modified peptides and synthetic peptide analogs.

[0026] The peptides of the present invention can be produced using chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin). For example, peptides can be synthesized by solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). Automated synthesis can be achieved, for example, using an ABI 43 1 A peptide synthesizer (Perkin Elmer) following the instructions provided by the manufacturer.

[0027] Alternatively, peptides can be produced by recombinant means or by cleavage from a longer polypeptide. For example, peptides can be obtained by cleavage from myelin basic protein, followed by modification of one or both termini. The composition of the peptide can be confirmed by amino acid analysis or sequencing (e.g., Edman degradation).

[0028] For practical purposes, there are various other properties that a peptide may exhibit. For example, it is important that a peptide is sufficiently stable in vivo to be therapeutically useful. The half-life of a peptide in vivo may be at least 10 minutes, 30 minutes, 4 hours, or 24 hours.

[0029] The peptides may also exhibit good bioavailability in vivo: they may maintain a conformation in vivo that allows them to bind to MHC molecules on the cell surface without significant hindrance.

[0030] In one embodiment, the composition can comprise an MBP peptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to any one of SEQ ID NOs: 1-4. In a preferred embodiment, the peptide has at least 80%, 90%, 95%, 97% or 99% sequence identity to any one of SEQ ID NOs: 1-4.

[0031] Sequence identity can be evaluated by any conventional method. However, to determine the degree of sequence identity between sequences, computer programs that perform multiple alignments between sequences, such as Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22: 4673-4680), are useful. Programs that compare and align pairs of sequences, such as ALIGN (Myers et al., (1988) CABIOS, 4: 1-17), FASTA (Pearson et al., (1988) PNAS, 85:2444-2448; Pearson (1990), Methods Enzymol., 183: 63-98) and Gapped BLAST (Altschul et al., (1997) Nucleic Acids Res., 25: 3389-3402), are also useful for this purpose. In addition, the European Bioinformatics Institute's Dali server provides structure-based alignments of protein sequences (Holm (1993) J. Mol. Biol., 233: 123-38; Holm (1995) Trends Biochem. Sci., 20: 478-480; Holm (1998) Nucleic Acid Res., 26: 316-9).

[0032] Multiple sequence alignments and percent identity can be determined using standard BLAST parameters (using sequences from all available organisms, matrix Blosum 62, gap costs: existence 11, extension 1).

[0033] Alternatively, the following programs and parameters can be used: Program: Align Plus 4, version 4.10 (Sci Ed Central Clone Manager Professional Suite). DNA comparison: Global comparison, Standard Linear Scoring matrix, mismatch penalty = 2, open gap penalty = 4, extension gap penalty = 1. Amino acid comparison: Global comparison, BLOSUM 62 Scoring matrix.

[0034] That is, variants of the sequences described or provided are included within the scope of the invention so long as the variant retains the functional activity of the parent, i.e., the variant is functionally equivalent, in other words, the variant has or retains the activity of the parent peptide as defined herein. Such variants may, for example, include amino acid substitutions, additions or deletions (including truncations at one or both ends) of the parent sequence at one or more positions, e.g., 1-14 amino acids.

[0035] Substitution can be conservative substitution.As used herein, "conservative substitution" refers to changing the identity of an amino acid to replace it at a given position with an amino acid of approximately the same size, charge and / or polarity.Examples of natural conservative substitutions of amino acids include the following eight substitution groups (denoted by the conventional one-letter code): (1) M, I, L, V; (2) F, Y, W; (3) K, R, (4) A, G; (5) S, T; (6) Q, N; (7) E, D; and (8) C, S.

[0036] Also included are functionally equivalent derivatives in which one or more amino acids have been chemically derivatized (eg, replaced with a chemical group).

[0037] Peptides for use according to the invention may comprise portions or fragments of SEQ ID NOs: 1-4, provided that the peptide retains the requisite activity. Portions or fragments of SEQ ID NOs: 1-4 may, for example, be 6 to 16 residues in length, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 residues in length.

[0038] Peptides for use according to the invention may be included among longer peptides, i.e. peptides comprising any of SEQ ID NOs: 1-4. Peptides for use according to the invention may thus comprise 8-30 amino acids, for example 8-25 amino acids, 8-20 amino acids, 8-15 amino acids or 8-12 amino acids. In one aspect, peptides of the invention may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.

[0039] A peptide composition for use according to the invention may comprise an amino acid sequence according to the invention as described herein. In one embodiment, the peptide composition comprises only an amino acid sequence according to the invention as described herein, i.e., does not comprise any additional peptides other than those according to the invention.

[0040] Peptides can be formulated in the composition as neutral or salt form.Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of peptides), such as those formed with inorganic acids, such as hydrochloride or phosphate, or organic acids, such as acetic acid, tartaric acid or malic acid.Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and procaine.

[0041] Apitope In adaptive immune responses, T lymphocytes can recognize internal epitopes of protein antigens. APCs ingest protein antigens and break them down into short peptide fragments. Peptides can bind to major histocompatibility complexes (MHC class II) inside the cell and be carried to the cell surface. When presented on the cell surface together with MHC molecules, peptides can be recognized by T cells (via the T cell receptor (TCR)), in which case the peptides are T cell epitopes.

[0042] Thus, an epitope is an antigen-derived peptide capable of binding to the peptide-binding groove of an MHC molecule and being recognised by a T cell.

[0043] A minimal epitope is the shortest fragment derivable from an epitope that is capable of binding to the peptide-binding groove of an MHC class I or II molecule and being recognized by a T cell. For a given immunogenic region, it is typically possible to generate a "nested set" of overlapping peptides that act as epitopes, all of which contain the minimal epitope but differ in their flanking regions.

[0044] Similarly, it is possible to identify the minimal epitope for a particular MHC molecule:T cell combination by measuring responses to truncated peptides. For example, if a response is obtained to a peptide comprising residues 1-15 in an overlapping library, a set truncated at both ends (i.e., 1-14, 1-13, 1-12, etc. and 2-15, 3-15, 4-15, etc.) can be used to identify the minimal epitope.

[0045] The present inventors have previously identified a correlation between the ability of a peptide to bind to MHC molecules and be presented to T cells without further processing and the ability of the peptide to induce antigen-specific immune tolerance in vivo (WO02 / 16410). If a peptide is too long to bind to the peptide-binding groove of MHC molecules without further processing (e.g., trimming), or binds in an inappropriate conformation, it is not tolerogenic in vivo. On the other hand, if a peptide is of the appropriate size and conformation to directly bind to the MHC peptide-binding groove and be presented to T cells, it can be predicted that this peptide is useful for tolerance induction.

[0046] It is therefore possible to test the tolerogenic potential of a peptide by determining whether it is able to bind to MHC molecules and be presented to T cells without further antigen processing in vitro.

[0047] MBP apitopes (antigen processing-independent epitopes) are capable of binding to MHC molecules and stimulating responses from MBP-specific T cells without further antigen processing. Such apitopes can be predicted to induce tolerance to MBP according to the rule-based method described in WO02 / 16410.

[0048] Peptides that bind to MHC class I molecules are typically 7-13, more usually 8-10 amino acids long. Binding of the peptide is stabilized at its two ends by contacts between atoms in the peptide backbone and in invariant sites in the peptide-binding groove of all MHC class I molecules. There are invariant sites at either end of the groove that bind the amino and carboxy termini of the peptide. Variation in peptide length is accommodated by kinks in the peptide backbone, often at proline or glycine residues that allow flexibility.

[0049] Peptides that bind to MHC class II molecules are typically 8-20 amino acids long, more usually 10-17 amino acids long, and can be longer (e.g., up to 40 amino acids). These peptides adopt an extended conformation along the MHC II peptide-binding groove, which is open at both ends (unlike the MHC class I peptide-binding groove). The peptide is held in place primarily by main-chain atomic contacts with conserved residues that line the peptide-binding groove.

[0050] In a preferred embodiment, peptides derived from MBP are capable of binding to MHC class II molecules without further processing.

[0051] Portion The peptides of the present invention may comprise all or a portion of the MBP-derived peptides shown as SEQ ID NOs:1-4.

[0052] The term "portion" refers to a peptide derived from SEQ ID NO: 1-4 and containing at least a minimal epitope, i.e., the peptide is capable of binding to the peptide-binding groove of an MHC class I or II molecule and being recognized by T cells to induce tolerance.

[0053] Uveitis Clinically, uveitis is generally classified as one of the following based on the part of the eye that is primarily affected: anterior uveitis, intermediate uveitis, posterior uveitis or panuveitis uveitis.

[0054] Anterior uveitis includes iridocyclitis and iritis. Iritis is inflammation of the anterior chamber and iris, while iridocyclitis involves inflammation within the ciliary body.

[0055] Intermediate uveitis (pars planitis) generally refers to vitreous inflammation, which is an inflammation of cells in the vitreous cavity, with the deposition of inflammatory material on the pars plana. Inflamed vitreous, or hemorrhage or protein deposits caused by inflammation, are characterized as intermediate uveitis. In these experiments, symptoms of intermediate uveitis are incorporated into the scoring using TEFI images. As shown in Example 3, a score of 4 (x2) with a maximum total of 8 is caused by whiteout. In FIG. 4, the TEFI image with the highest score reflects intermediate uveitis. In one embodiment of the present invention, the uveitis is intermediate uveitis.

[0056] Posterior uveitis (chorioretinitis) is an inflammation of the retinal and choroidal areas.

[0057] Panuveitis uveitis is a general term that refers to inflammation affecting all layers of the uvea.

[0058] In one aspect, the uveitis is idiopathic uveitis.

[0059] In a preferred embodiment of the invention, the uveitis is posterior uveitis. In another aspect of the invention, the uveitis is anterior uveitis. In another aspect of the invention, the uveitis is intermediate uveitis.

[0060] Uveitis can also be classified as either infectious or non-infectious, as well as uveitis associated with autoimmune disease (i.e., primarily non-infectious), which is more common in developed countries. Common animal models used to study uveitis are also autoimmune driven, showing a clear correlation between the two. It is predicted that 25-30% of cases of uveitis are associated with systemic autoimmune or autoinflammatory disease.

[0061] The uveitis according to the invention described herein is preferably a non-infectious uveitis, even more preferably an autoimmune uveitis.

[0062] Tolerance T cell epitopes play a central role in the adaptive immune response to any antigen, whether self or foreign. The central role played by T cell epitopes in hypersensitivity diseases (including allergies, autoimmune diseases and transplant rejection) has been demonstrated by the use of experimental models. It is possible to induce inflammation or allergic diseases by injection of self antigens or synthetic peptides (based on the structure of T cell epitopes) combined with adjuvants.

[0063] In contrast, it has been shown that it is possible to induce immunogenic tolerance to a particular antigen by administering soluble forms of peptide epitopes.The administration of soluble peptide antigens has been demonstrated as an effective means of inhibiting disease in experimental autoimmune encephalomyelitis (EAE-model for multiple sclerosis (MS)) (Metzler and Wraith (1993) Int. Immunol. 5:1159-1165; Liu and Wraith (1995) Int. Immunol. 7:1255-1263; Anderton and Wraith (1998) Eur. J. Immunol. 28:1251-1261) and experimental models of arthritis, diabetes, and uveoretinitis (reviewed in Anderton and Wraith (1998) above).It has also been demonstrated as a means of treating ongoing disease in EAE (Anderton and Wraith (1998) above).

[0064] Tolerance is the failure to respond to an antigen. Tolerance to self-antigens is a key feature of the immune system, and its loss can result in autoimmune diseases. The adaptive immune system must maintain the ability to respond to a huge variety of infectious agents while avoiding autoimmune attack of self-antigens contained within its own tissues. This is controlled to a large extent by the susceptibility of immature T lymphocytes to apoptotic cell death in the thymus (central tolerance). However, not all self-antigens are detected in the thymus, so the death of self-reactive thymocytes remains incomplete. There are therefore also mechanisms by which tolerance can be acquired by mature self-reactive T lymphocytes in peripheral tissues (peripheral tolerance). A review of the mechanisms of central and peripheral tolerance is given in Anderton et al (1999)(Immunological Reviews 169: 123-137).

[0065] Available data suggest that uveitis may result from reactive T cells generated by retinal proteins, driving inflammation and mimicking the effects of chronic infection. The composition according to the present invention can induce tolerance to self-antigens such as MBP, thereby restoring tolerance to MBP protein and reducing pathogenic immune responses when administered to a subject.

[0066] composition Compositions comprising one or more peptides according to the invention may be for prophylactic or therapeutic use in patients with uveitis.

[0067] When administered for prophylactic use, the composition can reduce or prevent the occurrence of an immune response against MBP. The level of immune response is less than would be obtained if the patient had not been treated with the composition. The term "reduce" indicates that a partial reduction in immune response is observed, for example, a 50%, 70%, 80% or 90% reduction in the response that would have been observed if the patient had not been treated with the composition (or in the response observed in an untreated patient over the same period of time). The term "prevent" indicates that no appreciable immune response against MBP is observed.

[0068] When administered for therapeutic use, the compositions may suppress an already ongoing immune response to MBP. The term "suppress" refers to a reduction in the level of an ongoing immune response compared to the level before peptide treatment or the level that would have been observed at the same time point if no treatment had been given.

[0069] Treatment with a composition according to the invention may result in a reduction in the levels of any or all of the following: (i)MBP autoantibody (ii) MBP-specific inflammatory CD4+ T cells (iii) B cells secreting MBP autoantibodies.

[0070] Detection of all these elements can be performed by techniques known in the art, such as ELISA, flow cytometry, etc.

[0071] Treatment with the composition of the present invention may also or alternatively cause anergy in MBP-specific CD4+ T cells. Anergy can be detected, for example, by subsequent MBP administration in vitro. Treatment with the composition of the present invention may result in the generation of antigen-specific regulatory T cells, characterized, for example, by transcription factors c-Maf and NFIL3, and negative co-stimulatory molecules LAG-3, TIGIT, PD-1 and TIM-3 (see Burton et al. Nature Communications (2014) Article number 4741).

[0072] formulation The compositions according to the present invention as described herein can be prepared as injections, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. The preparations can also be emulsified, or the peptides can be encapsulated in liposomes. The peptides can alternatively be encapsulated in carriers (e.g., nanoparticles) or bound to the surface of carriers. The active ingredient can be mixed with additives that are pharma- ceutically acceptable and compatible with the active ingredient. Suitable additives are, for example, water, saline (e.g., phosphate buffered saline), dextrose, glycerol, ethanol, and the like, and combinations thereof.

[0073] In addition, if desired, the composition can contain small amounts of auxiliary substances such as wetting or emulsifying agents and / or pH buffering agents.Buffer salts include phosphate, citrate, acetate.Hydrogen acid and / or sodium hydroxide can be used to adjust pH.Disaccharides such as sucrose or trehalose can be used for stabilization.

[0074] After formulation, the composition can be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4° C., or it can be freeze-dried.

[0075] Advantageously, the composition is prepared as lyophilized (freeze-dried) powder.Lyophilization allows long-term storage in a stabilized form.Lyophilization procedures are well known in the art, see, for example, http: / / www.devicelink.com / ivdt / archive / 97 / 01 / 006.html.Bulking agents such as mannitol, dextran or glycine are generally used before lyophilization.

[0076] The compositions may be administered in any convenient manner, such as by oral, intravenous (if water soluble), intramuscular, subcutaneous, sublingual, intranasal, intradermal or suppository routes, or by implantation (e.g., using a sustained release molecule or device).

[0077] The composition may advantageously be administered via intranasal, subcutaneous or intradermal routes. In a preferred embodiment, administration is intradermal.

[0078] The methods and pharmaceutical compositions for use according to the present invention can be used to treat human subjects. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject, which will vary with the age, weight and response of the particular patient.

[0079] The compositions as described herein are typically administered in an "effective amount"; that is, in an amount effective to produce, inter alia, any one or more of a therapeutic or prophylactic effect. Those skilled in the art will be able to determine, by routine experimentation, the effective, non-toxic amount to be included in a pharmaceutical composition or to be administered for a desired outcome. In general, the compositions disclosed herein can be administered in a manner that is compatible with the route of administration and the physical characteristics (including health status) of the recipient, and that produces the desired effect (i.e., therapeutically effective and / or prophylactic). For example, the appropriate dosage of the composition may depend on various factors, including, but not limited to, the subject's physical characteristics (e.g., age, weight, sex), and other factors that can be recognized by those skilled in the art. For example, other illustrative examples of general considerations that can be taken into account when determining appropriate dosages of a composition are discussed by Gennaro (2000, "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; and Gilman et al., (Eds), (1990), "Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press).

[0080] It is expected that the amount will fall in a relatively broad range that can be determined through methods known to those of skill in the art, taking into account some of the considerations outlined above.

[0081] The peptides and compositions of the present invention can be used to treat human subjects. The subject according to the present invention preferably has posterior uveitis. The subject can have T cells specific for MBP.

[0082] In a preferred embodiment, a "dose escalation" protocol may be followed in which multiple doses are given to the patient at increasing concentrations. Such an approach has been used, for example, for phospholipase A2 peptides in immunotherapy applications against bee venom allergy (Muller et al (1998) J. Allergy Clin Immunol. 101:747-754 and Akdis et al (1998) J. Clin. Invest. 102:98-106).

[0083] kit Peptides derived from MBP can be administered together in the form of a mixed composition or cocktail, however, there may be circumstances in which it is preferable to provide the peptides separately in the form of a kit for simultaneous, separate, sequential or combined administration.

[0084] For example, a kit may contain three peptides in separate containers. The contents of the containers may or may not be combined prior to administration.

[0085] The kit may also include mixing and / or administration means (e.g., a vapouriser for intranasal administration, or a syringe and needle or other medical device for subcutaneous / intradermal administration). The kit may also include instructions for use.

[0086] The compositions / kits of the present invention can be used to suppress or prevent the production of MBP autoantibodies in vivo. In particular, the compositions / kits can be used to treat and / or prevent uveitis in a subject.

[0087] Animal models The present invention also encompasses animal models of uveitis. As described in the Examples, the animal models according to the present invention offer the advantage of inducing uveitis without inducing encephalomyelitis (EAE).

[0088] Thus, in a further aspect, the present invention provides an animal model of uveitis, wherein the animal comprises elevated levels of myelin basic protein (MBP) and interphotoreceptor retinoid binding protein (IRBP) compared to control animals.

[0089] The term "elevated levels of MBP and IRBP" is intended to encompass elevated levels of either the full-length MBP or IRBP sequence (eg, MBP or IRBP peptides).

[0090] In one embodiment, the MBP is from or derived from human or guinea pig (gpMBP). The sequences of human and guinea pig MBP are set forth below: TIFF0007632996000001.tif35158

[0091] Other isoforms of MBP are also known, for example, UniProtKB, P02686-3, P02686-4, P02686-5 and P02686-6. TIFF0007632996000002.tif25157

[0092] In one embodiment of the invention, the IRBP is human IRBP or is derived from human IRBP. The sequence of human IRBP is set forth below (UniProtKB entry P10745): TIFF0007632996000003.tif92155

[0093] In one embodiment, the IRBP is bovine IRBP (SEQ ID NO:8) (UniProtKB entry P12661): TIFF0007632996000004.tif89121TIFF0007632996000005.tif125121

[0094] In one embodiment, the IRBP is a peptide derived from IRBP, preferably IRBP 1177-1191 ADGSSWEGVGVVPDV (SEQ ID NO: 9).

[0095] The levels of MBP and IRBP are elevated relative to suitable control animals, eg, animals to which exogenous or transgenic MBP and IRBP are not administered.

[0096] The levels of MBP and IRBP can be elevated in an animal by any suitable means, for example, this can be by immunizing the animal with MBP and IRBP, as described in the Examples herein.

[0097] Alternatively, the increase can be achieved by administering one or more vectors comprising one or more nucleic acid molecules encoding MBP and / or IRBP, hi a preferred embodiment, the vector is a viral vector.

[0098] In a further embodiment, the present invention provides a method for producing an animal model of uveitis, the method comprising increasing the levels of MBP and IRBP in an animal.

[0099] In a preferred embodiment, the method includes the step of increasing the levels of MBP and IRBP by administering MBP and IRBP to an animal, for example, by immunizing the animal with MBP and IRBP as described in the Examples herein.

[0100] The animal model has characteristics related to uveitis, for example, the animal model may exhibit increased clinical signs of uveitis compared to matched control animals that do not have elevated levels of MBP and IRBP.

[0101] The MBP and / or IRBP may be human MBP and / or IRBP.

[0102] As long as the required function is retained in the animal model, the MBP or IRBP may be a portion or fragment or part of MBP or IRBP. In one embodiment, the IRBP may be the IRBP1177-1191 peptide (ADGSSWEGVGVVPDV (SEQ ID NO: 9), also referred to as R16).

[0103] MBP and / or IRBP can be administered to the animal in any suitable manner, for example orally, intravenously (if water soluble), intramuscularly, subcutaneously, sublingually, intranasally, intradermally or via suppository routes or by implantation (e.g., using a sustained release molecule).

[0104] In one embodiment, the administration is subcutaneous.

[0105] A suitable dose of MBP administered to an animal may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 μg. In one embodiment, about 100 μg of MBP is administered.

[0106] A suitable dose of IRBP administered to an animal can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 μg. In one embodiment, about 20 μg of IRBP is administered.

[0107] In one embodiment, MBP and / or IRBP are administered with an adjuvant (eg, Freund's adjuvant).

[0108] Nucleic acids encoding MBP and / or IRBP can be provided using methods well known to those skilled in the art, for example, based on the MBP and / or IRBP sequences described herein.

[0109] The nucleic acid may be natural, synthetic or recombinant. The nucleic acid may be double-stranded or single-stranded, and may be DNA or RNA or a combination thereof. The nucleic acid may be, for example, a cDNA, a PCR product, a genomic sequence or an mRNA.

[0110] The nucleotide sequence may be codon optimized for production in a selected host / host cell.

[0111] The delivery of the nucleotide sequence encoding MBP and / or IRBP can be mediated by viral infection.Suitable viral vectors are well known in the art.For example, the viral vector can be adenovirus, retrovirus or lentivirus.

[0112] The generation of the animal model may involve multiple administrations of the vector.

[0113] The vector (e.g., adenovirus) can be administered at 3 week intervals. For example, the vector can be administered at 18-25, 18-23, 19-23, or 20-22 day intervals. The vector can be administered at 20, 21, or 22 day intervals.

[0114] The animal model involves administering the adenoviral vector at least two time points. For example, the adenoviral vector can be administered at two or three time points.

[0115] Vectors (e.g., adenovirus) can be administered at up to 10 9 ~10 11 In particular, the adenovirus can be administered in a dose of 10 viral particles per administration. 8 ~10 11 Adenovirus can be administered in doses of 10 viral particles per dose. 9 The vaccine can be administered in a dose of 100 viral particles.

[0116] The vector can be administered by any suitable means, for example, in the case of an adenoviral vector, the vector can be administered by intramuscular injection.

[0117] The animal can be a mammal. For example, the animal can be a mouse, a rat, a rabbit, a guinea pig or a primate. Preferably, the animal is a rat.

[0118] In one embodiment, the animal is a Lewis rat, preferably a female Lewis rat.

[0119] For example, the model described in the Examples herein was created by administering, via subcutaneous injection, 200 microliters of an emulsion containing 20 micrograms of IRBP1177-1191 and 100 micrograms of human (huMBP) or guinea pig myelin basic protein (gpMBP) in incomplete Freund's adjuvant supplemented with 5 mg / mL of Mycobacterium tuberculosis H37Ra. As discussed herein, the model according to the present invention offers the advantage of inducing uveitis with minimal induction of encephalomyelitis.

[0120] The invention will now be described with reference to the following examples. EXAMPLES

[0121] Example 1 The present study aimed to investigate the effect of immunization with guinea pig myelin basic protein (gpMBP), human myelin basic protein (huMBP) and / or interphotoreceptor retinoid-binding protein on the development of encephalomyelitis (EAE) and / or uveitis (EAU) in Lewis rats.

[0122] Experimental Overview Adult female Lewis rats were randomly assigned to experimental groups and allowed to acclimate for one week. On day 0, animals received 200 microliters of emulsion containing 20 micrograms of IRBP1177-1191 (Cambridge Bioscience, UK) and / or 100 micrograms of human (Fitzgerald Industries International, MA, US) or guinea pig myelin basic protein (gpMBP) in incomplete Freund's adjuvant (Sigma, UK) supplemented with 5 mg / mL Mycobacterium tuberculosis H37Ra (500 μg / rat) (Becton Dickenson, UK) by subcutaneous injection. Animals were weighed daily from day 0 until the end of the experiment. Animals were scored daily for clinical signs of experimental autoimmune encephalomyelitis, including tail, hindlimb and / or forelimb limpness, paresis and / or paralysis. Animals were scored twice weekly for clinical signs of uveitis from day 0 until the end of the experiment. On day 21 (groups 1, 2, and 3) or day 28 (groups 4 and 5), animals were culled and the following samples were dissected and preserved in tissue fixative for optional histopathological analysis: brain, spinal cord, right eye, and left eye.

[0123] Treatment Group and Dose Groups 1, 2 and 3: n=8 / group Groups 4 and 5: n=14 / group. TIFF0007632996000006.tif89169

[0124] Measurements Nonspecific clinical findings From day 0 until the end of the experiment, animals were checked daily for non-specific clinical signs, including abnormal posture (curling up), abnormal coat condition (piloerection) and abnormal activity levels (decreased or increased activity).

[0125] body weight Animals were weighed daily from day 0 until the end of the experiment.

[0126] Clinical score (uveitis) Animals were visually scored twice weekly for clinical signs of anterior uveitis from day 0 to the end of the experiment using the following scoring system, with an expected maximum score of 3. Data are graphed (mean ± SEM).

[0127] TIFF0007632996000007.tif35132

[0128] Animals were scored twice weekly for clinical signs of intermediate and posterior uveitis from day 0 until the end of the experiment. Retinal images were acquired using TEFI with restrained animals under non-anesthesia but after pupil dilation with 1% tropicamide followed by 2.5% phenylephrine hydrochloride. Retinal images were scored using the following system. The nominal maximum score was 6. Data were graphed (mean ± SEM). TEFI images show symptoms of both intermediate and posterior uveitis.

[0129] TIFF0007632996000008.tif61166

[0130] result Nonspecific clinical findings From day 0 to the end of the experiment, the animals did not show any non-specific clinical signs such as abnormal posture (curling), abnormal coat condition (piloerection) or abnormal activity levels (decreased or increased activity).A significant weight loss was observed in animals treated with gpMBP compared to the IRBP group from day 11 to the end of the experiment on day 21 (p<0.0001).

[0131] Significant weight loss was observed in animals receiving IRBP and gpMBP compared to the IRBP group from day 11 to the end of the experiment on day 21 (p<0.05 on day 11, p<0.001 on day 12, p<0.0001 on days 13 to 20, p<0.001 on day 21). There was no difference in weight loss between animals receiving gpMBP alone and animals receiving IRBP and gpMBP. No significant weight loss was observed in animals receiving IRBP alone, human MBP, or human MBP+IRBP.

[0132] Clinical score: Uveitis Animals with a mean score for both eyes of >0.5 for at least two consecutive TEFI sessions (see Figure 2) or a score of >1.0 at any time point were considered to have developed clinical signs of posterior uveitis.

[0133] Based on this criteria, the incidence of EAU was as follows: TIFF0007632996000009.tif42118

[0134] Due to the lack of pigmentation in the retina of Lewis rats, the following scoring system was used. This was due to the inability to identify areas of infiltration causing opacity and cellular infiltration on a light background. Inflammation of the optic disc was not clearly identifiable and therefore was not scored, and no structural damage was observed. Retinal vascular infiltration could be identified by its congested appearance, with a score of 0 to 3 ranging from mild to severe. Retinal inflammation was identified by the appearance of small to large areas of redness caused by increased angiogenesis, also with a score of 0 to 3, giving a notional total score of 6.

[0135] TIFF0007632996000010.tif60111

[0136] The incidence of clinical signs of posterior uveitis was greatest in all experimental groups receiving IRBP alone or in combination with gpMBP or huMBP. Clinical signs of EAU were also observed in 50% of animals receiving gpMBP and 100% of animals receiving huMBP without IRBP (see FIG. 1).

[0137] The onset of clinical signs of EAU was not statistically different between the experimental groups.

[0138] The severity of clinical signs of EAU was significantly lower in gpMBP-treated animals compared to the experimental groups receiving IRBP (p<0.001), IRBP and gpMBP (p<0.01), IRBP and huMBP (p<0.01) or huMBP (p<0.05).

[0139] Clinical signs of anterior uveitis were also observed during the study.

[0140] Data collected up to day 21 were analyzed by two-way analysis of variance followed by Dunnett's post-test for multiple comparisons between experimental groups.

[0141] In IRBP-treated animals, clinical signs were observed starting from day 11. Signs were significantly higher than in the gpMBP-treated group on days 11, 14, and 18 (p<0.0001) (Figure 3).

[0142] In IRBP & gpMBP treated animals, signs of anterior uveitis were also observed from day 11 compared to the gpMBP treated group (p<0.0001 on day 11, p<0.01 on days 14 and 18, p<0.05 on day 21).

[0143] In IRBP & huMBP treated animals, signs of anterior uveitis were observed on days 11 and 14 compared to the gpMBP treated group (p<0.0001).

[0144] In huMBP-treated animals, signs of anterior uveitis were observed on days 14 (p<0.0001) and 18 (p<0.01) compared to the gpMBP-treated group.

[0145] Guinea pig MBP induces experimental autoimmune encephalomyelitis (EAE) in Lewis rats, a model of multiple sclerosis (MS). In this study, Lewis rats immunized with gpMBP alone (incidence 8 / 8 rats; mean maximum score 3.5) or in combination with IRBP peptide (incidence 8 / 8 rats; mean maximum score 3.9) developed severe EAE, scored using the following scoring system for paralytic symptoms: (0) no abnormality, (0.5) partial tail weakness, (1) complete tail weakness, (2) paresthesia of one hind limb, (2.5) paresthesia of two hind limbs, (3) paralysis of one hind limb, (4) paralysis of two hind limbs, (5) incontinence, and (6) moribundity. Unlike the gpMBP-immunized rats, the other groups showed very mild symptoms: immunized with IRBP alone (incidence 2 / 8, mean maximum score 1.0); immunized with huMBP (incidence 3 / 14, mean maximum score 1.0) and combined huMBP and IRBP peptides (incidence 3 / 14, mean maximum score 1.0).

[0146] conclusion Administration of huMBP produced signs of both anterior and posterior uveitis.

[0147] When administered concomitantly with IRBP, it increased clinical signs of both anterior and intermediate / posterior uveitis, providing a robust model to test for efficacy against autoimmune uveitis.

[0148] In addition to the very low incidence and severity of EAE that developed after immunization with human MBP, it was decided to use a model of uveitis involving immunization with human MBP alone or with additional IRBP peptide to study the effects of the MBP peptides according to the invention, as described in Example 2.

[0149] Example 2 This example details a study of the effects of ATX-UV-3 in the Lewis rat model of human myelin basic protein (huMBP) and interphotoreceptor retinoid binding protein (IRBP)-induced uveitis, as described in Example 1.

[0150] ATX-UV-3 contains the following sequence: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (sequence number 4).

[0151] method Experimental Overview Adult female Lewis rats were randomly assigned to experimental groups and allowed to acclimate for one week. On day 0, animals received 200 microliters of an emulsion containing 100 micrograms of human myelin basic protein (MBP) (Fitzgerald Industries International, MA, US) in incomplete Freund's adjuvant (Sigma, UK) supplemented with 5 mg / mL Mycobacterium tuberculosis H37Ra (500 μg / rat) (Becton Dickenson, UK) and 20 micrograms of IRBP 1177-1191 (Cambridge Bioscience, UK) via subcutaneous injection. From day 0 until the end of the experiment, animals were scored twice weekly for clinical signs of uveitis. On day 21, clinical scores were analyzed and the study was extended until day 35. On day 35, animals were culled and eyes (right and left) were dissected and preserved in tissue fixative for histopathological analysis.

[0152] [Table 1]

[0153] Nonspecific clinical findings From day 0 until the end of the experiment, animals were checked daily for non-specific clinical signs, including abnormal posture (curling up), abnormal coat condition (piloerection) and abnormal activity levels (decreased or increased activity).

[0154] Clinical score (uveitis) From day 0 to the end of the experiment, animals were scored twice weekly for clinical signs of ocular inflammation (anterior uveitis) using the following scoring system: (0) normal, (1) slight iris vascular dilation and thickening of the iris stroma, a few scattered inflammatory cells, or both, (2) vascular congestion within the iris, abnormal pupillary constriction, and (3) anterior chamber opacification. Data are graphed (mean ± SEM).

[0155] [Table 2]

[0156] Animals were scored twice weekly for clinical signs of uveitis from day 0 until the end of the experiment. Retinal images were acquired using TEFI with restrained animals under non-anesthesia but after pupil dilation with 1% tropicamide followed by 2.5% phenylephrine hydrochloride. Retinal images were scored using a modified scoring system below with a nominal maximum score of 10. Data are graphed (mean ± SEM).

[0157] [Table 3]

[0158] result Nonspecific clinical findings From day 0 to the end of the experiment, the animals did not show any non-specific clinical signs such as abnormal posture (curling up), abnormal coat condition (piloerection) or abnormal activity levels (decreased or increased activity).

[0159] Clinical score (uveitis) Posterior uveitis Animals with a mean score for both eyes >0.5 for at least two consecutive TEFI sessions or a score >1.0 at any time point were considered to have developed clinical signs of posterior uveitis (Figure 5).

[0160] [Table 4]

[0161] Clinical scores were analyzed by two-way analysis of variance followed by Tukey's post-test for multiple comparisons between experimental groups on each experimental day (Figure 5).

[0162] In the huMBP and IRBP challenged groups, the ATX peptide significantly reduced the clinical scores compared to the vehicle-treated group on days 10 (p<0.0001), 14 (p<0.01) and 17 (p<0.05).

[0163] Clinical scores were further analyzed by calculating area under the curve (AUC) values ​​and comparing the AUC for each experimental group by one-way analysis of variance followed by Sidak's post-test for multiple comparisons among all experimental groups (Figure 6).

[0164] In the huMBP and IRBP challenged groups, the ATX peptide significantly reduced the clinical scores compared to the vehicle-treated group (p<0.001).

[0165] Example 3 Further studies were performed to examine the efficacy of ATX-UV-3 in an animal model of uveitis. Cyclosporine A was included as a positive control (using a high dose of cyclosporine A that would not be a physiological dose for humans but was chosen for the purposes of validating the model).

[0166] Experimental Overview Adult female Lewis rats were randomly assigned to experimental groups and allowed to acclimate for one week. Treatments were performed according to the following schedule: On day 0, animals received 200 microliters of an emulsion containing 100 micrograms of human myelin basic protein (MBP) in incomplete Freund's adjuvant supplemented with 5 mg / mL Mycobacterium tuberculosis H37Ra (500 μg / rat) and / or 20 micrograms of IRBP1177-1191 by subcutaneous injection. From day 0 until the end of the experiment, animals were scored twice weekly for clinical signs of anterior and posterior uveitis. On day 28, animals were culled and eyes (right and left) were dissected and transferred into tissue fixative for optional histopathological analysis, then processed for paraffin embedding and preserved in paraffin blocks.

[0167] Treatment Group and Dose All experimental groups had an n=12. The dosing volume was 5 mL / kg for PO dosing and a fixed volume of 100 μL per rat for SC dosing.

[0168] [Table 5]

[0169] Measurements Nonspecific clinical findings Animals were checked daily from day 0 to the end of the experiment for non-specific clinical signs, including abnormal posture (curling), abnormal coat condition (piloerection) and abnormal activity levels (decreased or increased activity). If non-specific clinical signs were deemed too severe, animals were culled prior to the scheduled end of the study.

[0170] body weight Animals were weighed daily from day 0 until the end of the experiment.

[0171] Clinical score (uveitis) From day 0 to the end of the experiment, animals were scored twice weekly by a single individual blinded to treatment for clinical signs of ocular inflammation (anterior uveitis) using the following scoring system: (0) normal, (1) slight iris vascular dilation and thickening of the iris stroma, a few scattered inflammatory cells, or both, (2) vascular congestion within the iris, abnormal pupillary constriction, and (3) anterior chamber opacification. Data are graphed (mean ± SEM).

[0172] [Table 6]

[0173] Animals were scored twice weekly for clinical signs of posterior uveitis from day 0 to the end of the experiment by a single individual blinded to the treatment. Retinal images were acquired using TEFI with restrained animals under non-anesthesia but after pupil dilation with 1% tropicamide followed by 2.5% phenylephrine hydrochloride. Retinal images were scored using a modified scoring system below with a nominal maximum score of 10. Data are graphed (mean ± SEM).

[0174] [Table 7]

[0175] result Nonspecific clinical findings From day -22 until the end of the experiment, no animals showed any non-specific clinical signs.

[0176] body weight Body weights for the huMBP and huMBP+IRBP models, expressed as a percentage of initial (day -22) body weights, were analyzed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental and vehicle groups. Two-way ANOVA revealed significant effects of time and treatment in both the huMBP and huMBP+IRBP models (all p<0.0001). In the huMBP model, Dunnett's post-test revealed significant increases in body weights at days 22, 24 and 28 in the cyclosporine A group (all p<0.05) and the ATX peptide group (p<0.05, p<0.05 and p<0.01, respectively) versus the vehicle group. In the huMBP+IRBP model, Dunnett's post-hoc tests revealed significant increases in body weight on days 13, 15, 22, 24 and 28 in the cyclosporine A group versus the vehicle group (p<0.05, p<0.05, p<0.05, p<0.01 and p<0.01, respectively).

[0177] Anterior uveitis clinical score Animals with a mean total score (average of left eye + right eye) of 1 at two consecutive time points or a mean total score >1 after any TEFI session were considered to have developed clinical signs of anterior uveitis. Anterior uveitis clinical scores were analyzed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental and vehicle groups. In the huMBP model, all mice showed no clinical signs of anterior uveitis throughout the study period. In the huMBP+IRBP model, two-way ANOVA revealed significant effects of time and treatment (both p<0.0001). Dunnett's post-hoc test revealed significant improvements in clinical scores in the cyclosporine A group at days 7, 10, 14, 17, 21 and 28 (p<0.05, p<0.0001, p<0.0001, p<0.0001, p<0.0001 and p<0.05, respectively) and in the ATX peptide group at day 10 (p<0.0001) versus the vehicle group (Figure 7A). Unpaired two-tailed two-sample Student's t-test of area under the curve (AUC) analysis of the anterior uveitis clinical score data revealed significant effects of cyclosporine A (p<0.0001) and ATX peptide (p<0.05) versus vehicle (Figure 7B).

[0178] Posterior / Intermediate Uveitis Clinical Score Animals with a mean total score (average of left eye + right eye) of 1 at two consecutive time points or a mean total score >1 after any TEFI session were considered to have developed clinical signs of posterior uveitis. Posterior uveitis clinical scores were analyzed by two-way ANOVA followed by Dunnett's post-test for multiple comparisons between experimental and vehicle groups. In the huMBP model, two-way ANOVA revealed significant effects of time and treatment (both p<0.0001). Dunnett's post-hoc test revealed significant improvements in clinical scores in the cyclosporine A group at days 7, 10, 14, 17, 21, 24 and 28 (p<0.05, p<0.0001, p<0.0001, p<0.0001, p<0.0001, p<0.01 and p<0.05, respectively) and in the ATX peptide group at days 7, 10, 14, 17, 21, 24 and 28 (p<0.05, p<0.0001, p<0.0001, p<0.0001, p<0.01 and p<0.05, respectively) versus the vehicle group (Figure 8A). In the huMBP model, unpaired two-tailed two-sample Student's t-test of area under the curve (AUC) analysis of posterior uveitis clinical score data revealed significant effects of cyclosporine A (p<0.0001) and ATX peptide (p<0.0001) versus vehicle (Figure 8B).

[0179] In the huMBP+IRBP model, two-way ANOVA revealed significant effects of time and treatment (both p<0.0001). Dunnett's post-hoc test revealed significant improvements in clinical scores in the cyclosporine A group at days 7, 10, 14, 17 and 21 (p<0.05, p<0.0001, p<0.0001, p<0.0001 and p<0.05, respectively) and in the ATX peptide group at day 10 (p<0.01) versus the vehicle group (Figure 8C). Unpaired two-tailed two-sample Student's t-test of area under the curve (AUC) analysis of the posterior uveitis clinical score data revealed a significant effect of cyclosporine A (p<0.0001) versus vehicle (Figure 8D). An unpaired, one-tailed, two-sample Student's t test of the area under the curve (AUC) analysis of the posterior uveitis clinical score data revealed a significant effect of the ATX peptide (p<0.05) versus vehicle (FIG. 8D).

[0180] Incidence of posterior / intermediate uveitis Animals with a mean sum score (left eye + right eye) of 1 for two consecutive time points or a mean sum score >1 after any TEFI session were considered to have developed clinical signs of posterior uveitis.

[0181] In the huMBP model, animals treated with cyclosporine A and ATX peptide had a reduced incidence compared to vehicle-treated animals (FIG. 9A). Only one animal treated with cyclosporine A and three animals treated with ATX peptide developed clinical signs of EAU (see Table 8).

[0182] [Table 8] In the huMBP+IRBP model, cyclosporine A significantly delayed the onset of posterior uveitis compared to the vehicle-treated group (p<0.0001) (FIG. 9B). ATX peptide significantly delayed the onset of posterior / intermediate uveitis compared to the vehicle-treated group (p<0.05) (FIG. 9B).

[0183] conclusion Administration of huMBP alone or in combination with IRBP induced clinical signs of posterior uveitis, resulting in vasodilation, retinal inflammation and cellular infiltration, as expected in these models of EAU. Clinical signs of posterior uveitis were less severe in the huMBP model compared to animals receiving huMBP+IRBP.

[0184] Clinical signs of anterior uveitis were observed in the huMBP+IRBP model but not in the huMBP model, where clinical signs included increased vascularization of the iris and cellular infiltration of the anterior chamber, as well as abnormal pupil constriction.

[0185] Overall, in the huMBP+IRBP model, cyclosporine A and the ATX peptide significantly reduced clinical signs of anterior uveitis.

[0186] In the huMBP model, cyclosporine A and the ATX peptide reduced the incidence of posterior uveitis compared to vehicle-treated animals.

[0187] Cyclosporine A and ATX peptide significantly delayed the onset of posterior uveitis in the huMBP+IRBP model when compared to the vehicle-treated group.

[0188] Overall, in the huMBP and huMBP+IRBP models, cyclosporine A and the ATX peptide significantly reduced clinical signs of posterior / intermediate uveitis.

[0189] All publications mentioned above are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of molecular biology, cellular immunology, or related fields are intended to be within the scope of the following claims. The present invention also provides the following aspects. [1] A composition comprising a myelin basic protein peptide selected from the following peptides for use in treating or preventing uveitis in a subject: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (sequence number 4). [2] A composition for use according to [1], comprising the following peptide: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (sequence number 4). [3] The composition for use according to [1], wherein the uveitis is posterior uveitis, anterior uveitis or intermediate uveitis. [4] Use of a composition as defined in [1] or [2] in the manufacture of a medicament for the treatment and / or prevention of uveitis in a subject. [5] The use according to [4], wherein the uveitis is posterior uveitis, anterior uveitis or intermediate uveitis. [6] A method for treating or preventing uveitis in a subject, comprising administering to the subject a composition as defined in [1] or [2]. [7] The method according to [8], wherein the uveitis is posterior uveitis, anterior uveitis, posterior uveitis or intermediate uveitis. [8] A kit comprising a myelin basic protein peptide selected from the following peptides for use in treating or preventing uveitis in a subject: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (sequence number 4). [9] A kit for use according to [8], comprising the following peptide: MBP 30-44 (SEQ ID NO:1); MBP 83-99 (SEQ ID NO:2); MBP 131-145 (SEQ ID NO:3); and MBP 140-154 (sequence number 4).

[10] The kit for use according to [9], wherein the MBP peptide is for simultaneous administration, separate administration or sequential administration.

[11] An animal model of uveitis, wherein the animal is immunized with human MBP and interphotoreceptor retinoid binding protein (IRBP) peptides.

[12] The animal model described in

[11] , wherein the uveitis is posterior uveitis, anterior uveitis, posterior uveitis or intermediate uveitis.

[13] The animal model according to

[11] or

[12] , wherein the MBP is human MBP and the IRBP is bovine IRBP.

[14] The animal model described in any one of

[11] to

[13] , wherein MBP and IRBP are administered by subcutaneous injection.

[15] The animal model according to any one of

[11] to

[14] , wherein the animal is a rat.

[16] The animal model described in

[15] , wherein the rats are Lewis rats.

[17] The animal model described in

[16] , where Lewis rats are female Lewis rats.

Claims

1. 1. A composition comprising a myelin basic protein peptide for use in treating or preventing uveitis in a subject: MBP 30-44 consisting of SEQ ID NO:1; MBP 83-99 consisting of SEQ ID NO:2; MBP 131-145 consisting of SEQ ID NO:3; and MBP 140-154 consisting of sequence number 4.

2. 2. The composition for use according to claim 1, wherein the uveitis is posterior uveitis, anterior uveitis or intermediate uveitis.

3. 2. Use of a composition as defined in claim 1 in the manufacture of a medicament for the treatment and / or prevention of uveitis in a subject.

4. 4. The use according to claim 3, wherein the uveitis is posterior uveitis, anterior uveitis or intermediate uveitis.

5. 1. A kit for use in treating or preventing uveitis in a subject comprising the following myelin basic protein peptides: MBP 30-44 consisting of SEQ ID NO:1; MBP 83-99 consisting of SEQ ID NO:2; MBP 131-145 consisting of SEQ ID NO:3; and MBP 140-154 consisting of sequence number 4.

6. The kit for use according to claim 5, wherein the MBP peptides are for simultaneous, separate or sequential administration.

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