Compositions containing S-arrestin peptides

Abstract

The present invention relates to compositions comprising peptides derived from S-arrestin (retinal arrestin, S-antigen, S-Ag). The compositions or peptides may be useful for preventing and / or suppressing S-Ag autoimmunity and are useful for treating and / or preventing uveitis.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising peptides derived from S-arrestin (retinal arrestin, S-antigen, S-Ag), which may be useful in preventing and / or suppressing S-Ag autoimmunity, which in turn may be useful in treating and / or preventing 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; adjacent structures such as the retina, optic nerve, lens, vitreous, and sclera can also be affected by the onset 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 that uveitis was responsible for 10% of these cases (Global data on visual impairments, The World Health Report, WHO (2010) http: / / www.who.int / blindness / GLOBALDATAFINALforweb.pdf). Current treatments for uveitis include the use of glucocorticoid steroids and other immunosuppressants such as methotrexate. Summary of the Invention [Problem to be solved by the invention]

[0004] However, there is a need in the art for alternative treatments for uveitis. The present invention addresses this need. [Means for solving the problem]

[0005] The present inventors have found that a "cocktail" of three S-Ag peptides is particularly effective in suppressing or preventing S-Ag-specific T cell activation ex vivo. Thus, in a first aspect, the present invention provides a composition comprising the following S-Ag peptide: a peptide comprising all or part of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 60% sequence identity to SEQ ID NO: 1; and / or a peptide comprising all or part of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 60% sequence identity to SEQ ID NO: 2; and / or A peptide comprising all or part of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 60% sequence identity to SEQ ID NO:3.

[0006] KKKAFVEQVANVVLKKK (SEQ ID NO: 1) is also referred to herein as 9K1K. KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) is also referred to herein as 17JK. KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) is also referred to herein as 15N3K. Compositions according to the present invention can be used in the therapeutic aspects of the invention described herein.

[0007] In a second aspect, the present invention provides a composition of the invention as described herein for use in the treatment and / or prevention of uveitis in a subject. In a third aspect, the present invention relates to the use of a composition of the invention as described herein in the manufacture of a medicament for treating and / or preventing uveitis.

[0008] In a fourth aspect, the present invention relates to a method for treating uveitis in a subject, comprising the step of administering to the subject all or part of a peptide of SEQ ID NO: 1 or a peptide having at least 60% sequence identity thereto, and / or all or part of a peptide of SEQ ID NO: 2 or a peptide having at least 60% sequence identity thereto, and / or all or part of a peptide of SEQ ID NO: 3 or a peptide having at least 60% sequence identity thereto.

[0009] In one aspect, the invention relates to a combination of all or part of a peptide of SEQ ID NO: 1 or a peptide having at least 60% sequence identity thereto, and / or all or part of a peptide of SEQ ID NO: 2 or a peptide having at least 60% sequence identity thereto.

[0010] In one aspect, the invention relates to a combination of all or part of a peptide of SEQ ID NO: 2 or a peptide having at least 60% sequence identity thereto, and / or all or part of a peptide of SEQ ID NO: 3 or a peptide having at least 60% sequence identity thereto.

[0011] In one aspect, the present invention relates to a combination of all or part of a peptide of SEQ ID NO: 1 or a peptide having at least 60% sequence identity thereto, and / or all or part of a peptide of SEQ ID NO: 3 or a peptide having at least 60% sequence identity thereto.

[0012] A peptide composition in accordance with the invention can comprise an amino acid sequence in accordance with the invention as described herein. In one embodiment, the peptide composition comprises only an amino acid sequence in accordance with the invention as described herein. The subject may be HLA-DR3. The subject may be HLA-DR2. The peptide of the invention as defined herein, or the composition of the invention, may be administered according to a dose escalation protocol.

[0013] In a fifth aspect, the present invention relates to a kit for simultaneous, separate or sequential administration comprising the following S-Ag peptides: the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1), or a portion thereof, or all or part of a sequence having at least 60% sequence identity to SEQ ID NO: 1; and / or the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2), or a portion thereof, or all or a portion of a sequence having at least 60% sequence identity to SEQ ID NO: 2; and / or The amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3), or a portion thereof, or all or a portion of a sequence having at least 60% sequence identity to SEQ ID NO:3. The kit can be used for the treatment of uveitis. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows that peptide 9K1K is properly loaded onto MHC class II in vivo. The response of peptide-specific CD4+ T cells to the peptide loaded on dendritic cells in vivo is shown. HLA-DR2 transgenic mice were subcutaneously injected with 100 μg of 9K1K peptide. Two hours after injection, spleens were dissected and CD11c+ cells (dendritic cells) were isolated. These CD11c+ cells were co-cultured with peptide-specific CD4+ cells at 37°C for 72 hours. IFNγ was measured in the supernatants of these cultures to assess the response of CD4+ T cells to the peptide (*p<0.05 Mann-Whitney U test). [Figure 2]Figure 1 shows that peptide 17JK is properly loaded onto MHC class II in vivo. The response of peptide-specific CD4+ T cells to the peptide loaded on dendritic cells in vivo is shown. HLA-DR3 transgenic mice were subcutaneously injected with 100 μg of 17JK peptide. Two hours after injection, spleens were dissected and CD11c+ cells (dendritic cells) were isolated. These CD11c+ cells were co-cultured with peptide-specific CD4+ cells at 37°C for 72 hours. IFNγ was measured in the supernatants of these cultures to assess the response of CD4+ T cells to the peptide (***p<0.001 Mann-Whitney U test). [Figure 3] Figure 1 shows that peptide 15N3K is properly loaded onto MHC class II in vivo. The response of peptide-specific CD4+ T cells to the peptide loaded on dendritic cells in vivo is shown. HLA-DR3 transgenic mice were subcutaneously injected with 100 μg of 15N3K peptide. Two hours after injection, spleens were dissected and CD11c+ cells (dendritic cells) were isolated. These CD11c+ cells were co-cultured with peptide-specific CD4+ cells at 37°C for 72 hours. IFNγ was measured in the supernatants of these cultures to assess the response of CD4+ T cells to the peptide (***p<0.001 Mann-Whitney U test). [Figure 4]Figure 1 shows that 9K1K alone induces tolerance to S-Ag protein in DR2tg mice. Mice were subcutaneously injected with 0.1 μg / mL, 1 μg / mL, and 10 μg / mL of 9K1K into the flank on days -15, -13, and -11, followed by three 100 μg / mL injections on days -8, -6, and -4 (escalating dose regimen). On day 0, mice were subcutaneously immunized at the base of the tail with antigen / CFA. Ten days after immunization, mice were sacrificed to measure the activation of LN cells and splenocytes in response to S-Ag restimulation. IFNγ concentrations were measured in the culture supernatants as an indicator of cell activation. Data represent the mean ± SEM of IFNγ concentrations for PBS-treated mice (black line) and 9K1K-treated mice (gray line). A two-way analysis of variance was used to measure the overall effect of treatment on T cell activation. Dunnett's multiple comparison test was used and significant differences are indicated in the graphs (**p<0.01; ****p<0.0001). The % inhibition of IFNγ production compared to the control group is indicated in the graphs. (A) Tolerization to S-Ag in LN. IFNγ production expressed as IFNγ concentration (pg / mL). (B) Tolerization to S-Ag in spleen. IFNγ production expressed as IFNγ concentration (pg / mL). LN, lymph node. Data representative of three independent experiments. [Figure 5]Figure 1 shows that 17JK alone induces tolerance to S-Ag protein in DR3tg mice. Mice were subcutaneously injected with 0.1 μg / mL, 1 μg / mL, and 10 μg / mL of 17JK into the flank on days -15, -13, and -11, followed by three 100 μg / mL injections on days -8, -6, and -4 (escalating dose regimen). On day 0, mice were subcutaneously immunized at the base of the tail with antigen / CFA. Ten days after immunization, mice were sacrificed to measure the activation of LN cells and splenocytes in response to S-Ag restimulation. IFNγ concentrations were measured in the culture supernatants as an indicator of cell activation. Data represent the mean ± SEM of IFNγ concentrations for PBS-treated mice (black line) and 17JK-treated mice (gray line). Two-way ANOVA was used to measure the overall effect of treatment on T cell activation. Dunnett's multiple comparison test was used and significant differences are indicated in the graphs (**p<0.01; ***p<0.001). The % inhibition of IFNγ production compared to the control group is indicated in the graphs. (A) Tolerization to S-Ag in LN. IFNγ production expressed as IFNγ concentration (pg / mL). (B) Tolerization to S-Ag in spleen. IFNγ production expressed as IFNγ concentration (pg / mL). LN, lymph node. Data representative of three independent experiments. [Figure 6]Figure 1 shows that 15N3K alone induces tolerance to S-Ag protein in DR3tg mice. Mice were subcutaneously injected with 0.1 μg / mL, 1 μg / mL, and 10 μg / mL of 15N3K into the flank on days -15, -13, and -11, followed by three 100 μg / mL injections on days -8, -6, and -4 (escalating dose regimen). On day 0, mice were subcutaneously immunized at the base of the tail with antigen / CFA. Ten days after immunization, mice were sacrificed to measure the activation of LN cells and splenocytes in response to S-Ag restimulation. IFNγ concentrations were measured in the culture supernatants as an indicator of cell activation. Data represent the mean ± SEM of IFNγ concentrations for PBS-treated mice (black line) and 15N3K-treated mice (gray line). A two-way analysis of variance was used to measure the overall effect of treatment on T cell activation. Dunnett's multiple comparison test was used and significant differences are indicated in the graphs (*p<0.05; ****p<0.0001). The % inhibition of IFNγ production compared to the control group is indicated in the graphs. (A) Tolerization to S-Ag in LN. IFNγ production expressed as IFNγ concentration (pg / mL). (B) Tolerization to S-Ag in spleen. IFNγ production expressed as IFNγ concentration (pg / mL). LN, lymph node. Data representative of three independent experiments. [Figure 7]This figure shows that treatment with the peptide cocktail ATX975 reduces S-Ag-induced immune cell activation in DR3tg mice more efficiently than treatment with 17JK or 15N3K alone. Mice were subcutaneously injected with 0.015 nmol, 0.15 nmol, and 1.5 nmol of peptide into the flank on days -15, -13, and -11, respectively, followed by three injections of 15 nmol peptide on days -8, -6, and -4 (dose-escalation schedule). For treatment with the cocktail containing three peptides (ATX975), these doses were given per peptide (reaching a total maximum dose of 45 nmol peptide). Mice were immunized subcutaneously at the base of the tail with antigen / CFA on day 0. Mice were sacrificed 10 days after immunization, and splenocyte activation in response to S-Ag restimulation was measured. Data represent the mean ± SEM of IFNγ concentrations for control-treated mice (black line) and peptide-treated mice (gray line). A two-way analysis of variance was used to measure the overall effect of treatment on T cell activation. Dunnett's multiple comparison test was used and significant differences are indicated in the graphs (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 when compared to the control group; #p<0.05, ####p<0.0001 when compared to the ATX975 group). The percentage inhibition of IFNγ production compared to the control group is indicated in the graphs. (A) Tolerization to S-Ag in the spleen. IFNγ production expressed as IFNγ concentration (pg / mL). (B) Tolerization to S-Ag in the spleen. IFNγ production expressed as IFNγ concentration (pg / mL). [Figure 8]This figure shows that treatment with the peptide cocktail ATX975 reduces S-Ag-induced immune cell activation in DR2tg mice more efficiently than treatment with 9K1K alone. Mice were subcutaneously injected with 0.015 nmol, 0.15 nmol, and 1.5 nmol of peptide into the flank on days -15, -13, and -11, respectively, followed by three injections of 15 nmol peptide on days -8, -6, and -4 (dose-escalation schedule). For treatment with the cocktail containing three peptides (ATX975), these doses were given per peptide (reaching a total maximum dose of 45 nmol peptide). Mice were immunized subcutaneously at the base of the tail with antigen / CFA on day 0. Ten days after immunization, mice were sacrificed, and splenocyte activation in response to S-Ag restimulation was measured. Data represent the mean ± SEM of IFNγ concentrations for control-treated mice (black line) and peptide-treated mice (gray line). Two-way ANOVA was used to measure the overall effect of treatment on T cell activation. Dunnett's multiple comparison test was used and significant differences are indicated in the graphs (*p<0.05; **p<0.01). The % inhibition of IFNγ production compared to the control group is indicated in the graphs. Tolerization to S-Ag in the spleen. IFNγ production expressed as IFNγ concentration (pg / mL). [Figure 9] Figure 1 shows the diverse patterns of in vitro peptide-MHC II binding for peptides 9K1, 17JK, and 15N3K. The binding of peptides 9K1, 17JK, and 15N3K to recombinant HLA-DRA1*0101, DRB1*0101 (DR1), DRB1*1501 (DR2), DRB1*0301 (DR3), DRB1*0401 (DR4), DRB1*1101 (DR11), DRB1*0405 (DR4*05), and DRB1*0901 (DR9) was assessed in vitro. IC50 values ​​(μM) are presented and color-coded for each HLA-DR molecule. Low values ​​(green) indicate strong binding, while high values ​​(red) indicate (relatively) weak binding. [Figure 10]Figure 1 shows that variants of peptide 15N3K are apitopes. The ability of variants of the 15N3K peptide to behave as apitopes (i.e., bind to MHC II molecules without antigen processing and be presented to T cells by antigen-presenting cells) was tested using an APIPS assay. DR3tg mice were immunized with SAg to generate hybridomas. 5 x 10 SAg-specific hybridoma cells were cultured with 5 x 10 fresh or fixed commercially available APC (VAVY) cells. Cultures were stimulated with 10 or 25 μg / mL of peptide, as indicated in the graph. T cell activation was measured by IL-2 ELISA on supernatants collected 48 hours later. Graphs represent the mean ± SEM of duplicate measurements. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a novel and alternative therapeutic option for the treatment and / or prevention of uveitis. As demonstrated in the present application, the combination of peptides of SEQ ID NOs: 1, 2 and 3 induces tolerance to S-Ag in a tolerance model in HLA-DR transgenic mice.

[0016] Thus, a first aspect of the present invention relates to a composition comprising a plurality of peptides derived from S-Ag, i.e. the peptides of the invention as defined herein, preferably the peptides of SEQ ID NO: 1, 2 and / or 3.

[0017] S-arrestin S-arrestin (also known as retinal arrestin, S-antigen, or S-Ag) is a soluble photoreceptor protein expressed in the retina and pineal gland. This molecule is known to be involved in desensitization of the light-activated transduction cascade and was originally isolated for its binding to activated rhodopsin. The crystal structure reveals two domains of antiparallel β-sheet connected by a hinge region and a short α-helix behind the amino-terminal fold.

[0018] The light-activated form of the visual pigment rhodopsin (Rh*) interacts with the retinal G protein transducin, thereby initiating the conversion of a GDP molecule to GTP at the transducin α subunit. In its GTP-bound form, transducin activates cyclic GMP phosphodiesterase (PDE) by dissociating from Rh* and binding to its two inhibitory subunits, PDEγ. This results in a rapid decrease in the concentration of the endogenous transmitter cyclic GMP. Because the interaction between Rh* and transducin takes only about 1 ms, a single Rh* can subsequently interact with hundreds of transducin molecules. The turnover rate of PDEs can be on the order of thousands of hydrolyzed cGMP molecules per second per PDE. Thus, rapid and effective removal of Rh* before it can activate too many PDE molecules is essential for limiting the light response and rapid recovery. This inactivation of Rh* is accomplished in two steps: phosphorylation of Rh* reduces its ability to catalyze nucleotide conversion of transducin, and subsequent binding of arrestin to P-Rh* completely blocks its further interaction with transducin.

[0019] The amino acid sequence of mature human S-Ag is shown below (SEQ ID NO: 4). UniProt database P10523 (https: / / www.uniprot.org / ) TIFF0007778696000001.tif130144

[0020] 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 (panuveitis uveitis).

[0021] Anterior uveitis is the most common form of uveitis and includes iridocyclitis and iritis. Iritis is inflammation of the anterior chamber and iris, while iridocyclitis involves inflammation within the ciliary body. Intermediate uveitis (pars planitis) generally refers to vitritis, an inflammation of cells within the vitreous cavity, accompanied by deposition of inflammatory material on the pars plana. Posterior uveitis (chorioretinitis) is inflammation of the retinal and choroidal areas. Panuveitis uveitis is a general term that refers to inflammation affecting all layers of the uvea.

[0022] Uveitis can also be classified as either infectious or noninfectious, as well as uveitis associated with autoimmune disease (i.e., primarily noninfectious), which is more common in developed countries. Common animal models used to study uveitis are also autoimmune-driven, demonstrating a clear correlation between the two. It is estimated that 25-30% of uveitis cases are associated with systemic autoimmune or autoinflammatory disease. In one aspect of the invention, the uveitis is non-infectious uveitis.

[0023] 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 and transplant rejection) has been demonstrated through the use of experimental models. Autoimmune or allergic diseases can be induced by injection of synthetic peptides (based on the structure of T cell epitopes) combined with adjuvants.

[0024] In contrast, it has been shown that administering soluble forms of peptide epitopes can induce immunogenic tolerance to specific antigens.The administration of soluble peptides has been demonstrated as an effective means of inhibiting disease in experimental autoimmune encephalomyelitis (EAE - a 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).

[0025] 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 disease. The adaptive immune system must maintain the ability to respond to a vast variety of infectious agents while avoiding autoimmune attack of self-antigens contained within its own tissues. This is controlled to a large extent by negative selection of high-affinity T lymphocytes in the thymus (central tolerance). However, not all self-antigens are expressed in the thymus, and therefore death of self-reactive thymocytes remains incomplete. Therefore, there is also a mechanism 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 provided by Anderton et al. (1999) (Immunological Reviews 169: 123-137). See also Wraith (2016) Nature 530:422-423.

[0026] Available data suggest that uveitis may result from autoreactive T cells generated by retinal proteins, including S-Ag, that drive inflammation and lead to chronic disease. The compositions of the present invention can induce tolerance to autoantigens, such as S-Ag, thereby restoring tolerance to S-Ag proteins and reducing pathogenic immune responses when administered to a subject.

[0027] Apitope In the adaptive immune response, T lymphocytes are capable of recognizing epitopes of protein antigens. APCs ingest protein antigens and degrade them into short peptide fragments. Peptides can bind to major histocompatibility complexes (MHC) inside the cell and be transported to the cell surface. When presented on the cell surface together with MHC molecules, peptides can be recognized by T cells (via T cell receptors (TCRs)), in which case the peptides are T cell epitopes. Briefly, an epitope is a peptide derivable from an antigen that is capable of binding to the peptide-binding groove of an MHC molecule and being recognized by a T cell.

[0028] 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, one can typically generate a "nested set" of overlapping peptides that act as epitopes, all of which contain the minimal epitope but differ in their flanking regions.

[0029] Similarly, by measuring responses to truncated peptides, it is possible to identify minimal epitopes for particular MHC molecule:T cell combinations. For example, if responses are obtained to peptides containing residues 1-15 in an overlapping library, sets 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 minimal epitopes.

[0030] The present inventors have previously found that there is a correlation between the ability of a peptide to bind to an MHC molecule and be presented to T cells without further processing and the ability of the peptide to induce tolerance in vivo (WO 02 / 16410). If a peptide is too long or binds in an inappropriate conformation to bind to the peptide-binding groove of an MHC molecule without further processing (e.g., trimming), it will not be tolerogenic in vivo. On the other hand, if a peptide is of the appropriate size and conformation to bind directly to the MHC peptide-binding groove and be presented to T cells, the peptide can be predicted to be useful for tolerance induction.

[0031] Thus, the tolerogenic potential of a peptide can be determined by examining whether it can bind to MHC molecules and be presented to T cells in vitro without further antigen processing.

[0032] S-Ag apitopes (antigen processing-independent epitopes) are capable of binding to MHC class II molecules and stimulating responses from S-Ag-specific T cells without further antigen processing. Such apitopes can be predicted to induce tolerance to S-Ag according to the rule-based method described in WO 02 / 16410.

[0033] Peptides that bind to MHC class I molecules are typically 7-13 amino acids long, more commonly 8-10 amino acids long. Peptide binding is stabilized at its two ends by contacts between atoms in the peptide backbone and invariant sites in the peptide-binding groove of all MHC class I molecules. Invariant sites are located at either end of the groove, binding to the amino and carboxy termini of the peptide. Variation in peptide length is often accommodated by twisting the peptide backbone at proline or glycine residues, which allow flexibility.

[0034] 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 exist in an elongated conformation along the MHC II peptide-binding groove, which (unlike the MHC class I peptide-binding groove) is free at both ends. The peptide is held in place primarily by contacts of main-chain atoms with conserved residues lining the peptide-binding groove. In a preferred embodiment, peptides derived from S-Ag are capable of binding to MHC class II molecules without further processing.

[0035] peptide The term "peptide" is used in its conventional sense to refer to a series of residues (typically L-amino acids) joined together by peptide bonds, typically between the α-amino and carboxyl groups of adjacent amino acids. The term includes modified peptides and synthetic peptide analogs.

[0036] The peptides of the present invention can be prepared 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) according to the manufacturer's instructions.

[0037] Alternatively, peptides can be produced by recombinant means or by cleavage from a longer polypeptide. For example, peptides can be obtained by cleavage from an S-antigen 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., the Edman degradation procedure).

[0038] For practical purposes, there are various other characteristics that a peptide can exhibit. For example, it is important that the peptide be sufficiently stable in vivo to be therapeutically useful. The half-life of the peptide in vivo can be at least 10 minutes, 30 minutes, 4 hours, or 24 hours.

[0039] The peptides used in the present invention are as follows: a peptide comprising all or part of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 60% sequence identity to SEQ ID NO: 1; and / or a peptide comprising all or part of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 60% sequence identity to SEQ ID NO: 2; and / or A peptide comprising all or part of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 60% sequence identity to SEQ ID NO:3.

[0040] A peptide in accordance with the invention can comprise or consist of an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a peptide of SEQ ID NO: 1, 2 or 3. In one embodiment, the peptide has at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 1, 2 or 3.

[0041] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising all or part of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 80% sequence identity to SEQ ID NO: 1; and / or a peptide comprising all or part of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 80% sequence identity to SEQ ID NO: 2; and / or A peptide comprising all or part of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 80% sequence identity to SEQ ID NO:3.

[0042] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising all or part of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising all or part of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 90% sequence identity to SEQ ID NO: 2; and / or A peptide comprising all or part of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 90% sequence identity to SEQ ID NO:3.

[0043] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising all or part of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising all or part of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 95% sequence identity to SEQ ID NO: 2; and / or A peptide comprising all or part of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 95% sequence identity to SEQ ID NO:3.

[0044] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 80% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 80% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 80% sequence identity to SEQ ID NO:3.

[0045] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 90% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 90% sequence identity to SEQ ID NO:3.

[0046] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 95% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 95% sequence identity to SEQ ID NO:3.

[0047] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3).

[0048] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 80% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 80% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 60% sequence identity to SEQ ID NO: 3.

[0049] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 90% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 60% sequence identity to SEQ ID NO: 3.

[0050] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 90% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 80% sequence identity to SEQ ID NO:3.

[0051] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 95% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 60% sequence identity to SEQ ID NO: 3.

[0052] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 90% sequence identity to SEQ ID NO: 1; and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 95% sequence identity to SEQ ID NO: 2; and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 80% sequence identity to SEQ ID NO:3.

[0053] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 60% sequence identity to SEQ ID NO: 3.

[0054] In some embodiments, the composition comprises the following S-Ag peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and / or a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and / or A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 80% sequence identity to SEQ ID NO:3.

[0055] In a preferred embodiment, the peptide comprises SEQ ID NO: 1, 2 and / or 3. In a further embodiment, the peptide consists of SEQ ID NO: 1, 2 and / or 3.

[0056] Sequence identity can be evaluated by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that perform multiple alignment of 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. Additionally, 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).

[0057] Multiple sequence alignments and percent identity calculations can be determined using standard BLAST parameters (sequences from all available organisms, matrix Blosum 62, gap costs: existence 11, extension 1).

[0058] 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.

[0059] That is, variants of a specified or given sequence are included within the scope of the present invention so long as the variant retains the functional activity of the parent, i.e., so long as the variant is a functional equivalent, in other words, so long as they have or exhibit the activity of the parent peptide as defined herein. Such variants can include, for example, one or more amino acid substitutions, additions, or deletions (including truncations at either or both termini) of the parent sequence, e.g., of 1 to 14 amino acids. Also included are functionally equivalent derivatives in which one or more of the amino acids have been chemically derivatized, eg, substituted with a chemical group.

[0060] That is, the peptides of the present invention can comprise a portion, part, or fragment of SEQ ID NOs: 1 to 3, provided that the peptide retains the required activity. The portion, part, or fragment of SEQ ID NOs: 1 to 3 can be, for example, 6 to 14 residues in length, such as 6, 7, 8, 9, 10, 11, 12, or 13 residues in length.

[0061] The peptides of the present invention can contain 8 to 30 amino acids, for example, 8 to 25 amino acids, 8 to 20 amino acids, 8 to 15 amino acids, or 8 to 12 amino acids. That is, in one embodiment, the peptides of the present invention can 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.

[0062] part The peptides of the present invention may comprise all or part of the S-Ag derived peptides shown as SEQ ID NOs: 1, 2 and / or 3. The term "portion" means a peptide derived from SEQ ID NOs: 1 to 3 and including at least the minimal epitope of the peptide.

[0063] Such peptides can contain one or more mutations, typically amino acid substitutions, in the S-Ag-derived sequence. The amino acids can be substituted with amino acids such as glycine, lysine, or glutamic acid. The peptides can contain up to three, up to two, or one amino acid substitutions from the S-Ag-derived sequence.

[0064] Such peptides may contain amino acids at one or both termini that are not from the S-Ag sequence, for example, the peptides may have one or more glycine and / or lysine and / or glutamic acid residues at one or both termini.

[0065] Peptides containing non-S-Ag derived amino acids are apitopes, ie, capable of binding to MHC molecules and being presented to T cells in vitro and in vivo without antigen processing.

[0066] solubility Solubility may be an important consideration in peptide-mediated tolerance induction. Solubility can be improved by the incorporation of additional amino acids, which can be glycine (G), lysine (K) and / or glutamic acid (E), at both the N- and C-termini.

[0067] In one embodiment, a peptide according to the invention can have, for example, one, two, or three additional amino acids at the N-terminus and / or C-terminus. The additional amino acids can be selected from glycine (G), lysine (K), and / or glutamic acid (E). Various combinations of these amino acids can be added to a peptide according to the invention.

[0068] For example, a peptide according to the invention can have one, two or three lysine (K) residues at both the N-terminus and the C-terminus. Peptides according to the invention can have one, two or three glycine (G) residues at both the N-terminus and the C-terminus. Peptides according to the invention can have one, two or three glutamic acid (E) residues at both the N-terminus and the C-terminus.

[0069] In one embodiment, a peptide according to the invention may have one glycine and one lysine residue at both the N-terminus and the C-terminus. In one embodiment, a peptide according to the invention may have one glycine and two lysine residues at both the N-terminus and C-terminus. In one embodiment, a peptide according to the invention may have one glutamic acid and one lysine residue at both the N-terminus and the C-terminus. In one embodiment, a peptide according to the invention may have one glutamic acid and two lysine residues at both the N-terminus and C-terminus.

[0070] For example, the additional amino acids can include a glycine or lysine spacer at one or both termini followed by an amino acid pair KK, KE, EK, or EE.

[0071] In one embodiment, the peptide can have a glycine spacer at either end followed by a combination of two additional amino acids at both the N- and C-termini, which can be lysine (K) and / or glutamic acid (E). Possible combinations at a given terminus can thus be GKK, GKE, GEK, or GEE.

[0072] The peptide may have the following general formula: XXG-parent peptide-GXX. In one embodiment, a peptide according to the invention may have three additional lysine (K) residues at both the N-terminus and the C-terminus. A modified peptide according to the invention may therefore have six additional amino acids (three at each end) relative to the parent peptide.

[0073] Alternatively, the peptides of the invention can have the following general formula: KKK-parent peptide-KKK KK-parent peptide-KK K - parent peptide-K GK-parent peptide-KG GKK-parent peptide-KKG KKG-parent peptide-GKK EK-parent peptide-KE EKK-parent peptide-KKE GKE-parent peptide-EKG GEK - parent peptide - KEG.

[0074] The modified peptides may be more soluble than the parent (unmodified) peptides. The modified peptides may have 2-fold, 3-fold, 4-fold, or 5-fold higher solubility than the parent peptides. The peptides may be soluble at concentrations of up to 0.5 mg / mL, 1 mg / mL, or 5 mg / mL.

[0075] As discussed herein, the modified peptides of the present invention may have 2, 4, or 6 additional amino acids (1, 2, or 3 at each end) relative to the parent peptide. In a most preferred embodiment, the modification is the inclusion of KKK at both the N- and C-termini.

[0076] The modified peptide may be more soluble than the parent (unmodified) peptide. The modified peptide may have 2-fold, 3-fold, 4-fold, or 5-fold higher solubility than the parent peptide. The peptide may be soluble at concentrations of up to 0.5 mg / mL, 1 mg / mL, or 5 mg / mL or more, for example, 8 mg / mL. In one embodiment, the modified peptide may be soluble at a concentration of 4 mg / mL.

[0077] composition The S-Ag peptide may be in the form of a composition, preferably a pharmaceutical composition. A peptide composition in accordance with the invention can comprise an amino acid sequence in accordance with the invention as described herein. In one embodiment, the peptide composition comprises only an amino acid sequence in accordance with the invention as described herein, i.e., does not comprise any additional peptides other than those in accordance with the invention. In one aspect of the invention discussed herein, as a first step, subjects having or at risk of developing uveitis are identified. The peptides or compositions according to the invention may be for prophylactic or therapeutic use.

[0078] When administered for prophylactic use, the peptide or composition can reduce or prevent the occurrence of an immune response to S-Ag. The level of the immune response is less than that which would be obtained if the subject had not been treated with the composition. The term "reduce" indicates a partial reduction in the immune response, for example, a 50%, 60%, 70%, 80% or 90% reduction in the response that would be observed if the subject had not been treated with the composition (or in the response observed in an untreated subject over the same period). The term "prevent" indicates that no appreciable immune response to S-Ag is observed.

[0079] When administered for therapeutic use, the peptides or compositions may suppress an already ongoing immune response to S-Ag, the term "suppress" indicating a reduction in the level of an ongoing immune response compared to the level before peptide treatment or compared to the level that would have been observed at the same time point if no treatment had been given.

[0080] Treatment with the compositions of the present invention may result in a reduction in the levels of any or all of the following: (i) S-Ag autoantibody (ii) S-Ag-specific inflammatory CD4+ T cells (iii) B cells secreting S-Ag autoantibodies. The detection of all these elements can be performed by techniques known in the art such as ELISA, flow cytometry, etc.

[0081] Treatment with the peptides or compositions of the invention may additionally or alternatively induce anergy in CD4+ T cells specific for S-Ag, which can be detected, for example, by subsequent administration of S-Ag in vitro.

[0082] formulation The composition may be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active compounds. Such a formulation may be in a form suitable for, for example, intradermal or subcutaneous administration.

[0083] The composition can be prepared as an injection, either as a liquid solution or suspension; solid forms suitable for solution or suspension in liquid before injection can also be prepared. Alternatively, the peptide can be encapsulated in a carrier (e.g., nanoparticles) or bound to the surface of a carrier. The active ingredient can be mixed with an additive that is pharmaceutically acceptable and compatible with the active ingredient. Suitable additives include, for example, water, saline (e.g., phosphate-buffered saline), dextrose, glycerol, ethanol, etc., and combinations thereof.

[0084] In addition, if desired, the composition can contain small amounts of auxiliary substances such as wetting agents or emulsifiers and / or pH buffering agents.Buffer salts include phosphate, citrate, and acetate.Hydrogen acid and / or sodium hydroxide can be used to adjust pH.Disaccharides such as sucrose or trehalose can be used for stabilization.

[0085] In the composition, the relative ratio of peptides to each other can be about 1:1:1, or the relative ratio of each peptide can be varied if, for example, one peptide is found to function better with a particular HLA type than another. 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.

[0086] Advantageously, the composition is prepared as lyophilized (freeze-dried) powder.Lyophilization allows long-term storage in a stable form.Lyophilization procedures are well known in the art, for example, see http: / / www.devicelink.com / ivdt / archive / 97 / 01 / 006.html.Bulking agents such as mannitol, dextran or glycine are generally used before lyophilization.

[0087] The compositions can be administered in any convenient manner, such as by oral, intravenous, intramuscular, subcutaneous, sublingual, intranasal, intradermal or suppository routes, or by implantation (eg, using a sustained release molecule). The compositions may be advantageously administered via intranasal, subcutaneous or intradermal routes, hi one aspect, administration may be via a transdermal patch.

[0088] A peptide or composition as described herein is typically administered in an "effective amount"; that is, in an amount effective to produce, inter alia, any one or more therapeutic or prophylactic effects. One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount to be included in a pharmaceutical composition or to be administered for a desired outcome. Generally, a peptide or composition disclosed herein can be administered in a manner compatible with the route of administration and the physical characteristics (including health status) of the recipient, and in a manner that produces the desired effect (i.e., therapeutically effective and / or prophylactic). For example, the appropriate dosage of a 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 one 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).

[0089] The peptides and compositions of the present invention can be used to treat human subjects. The subject can have uveitis. The subject can have S-Ag autoreactive T cells.

[0090] The subject may express an HLA haplotype that is associated with a tendency to overproduce T cells specific to S-Ag. Methods for determining an individual's HLA haplotype are known in the art. In one embodiment, the subject has HLA genes selected from the following genes: A29, B51, B27, DR8, DR4, DP5, DR4, DQA3, DR3, DR2, DR51, and DR17 (see Mattapallil et al. J Immunol 2011, 187:1977-1985).

[0091] Dose Escalation Protocol In a preferred embodiment, the peptides or compositions of the present invention can be administered to a subject using a "dose escalation" protocol, in which multiple doses of increasing concentration are given to the subject. Such an approach has been used, for example, for phospholipase A2 peptides in immunotherapy for 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).

[0092] In one aspect, the dose escalation protocol can include administering to the subject the tolerogenic peptide at the following doses: Day 1: First dose of about 15 to about 40 μg; Day 14±7: second dose of approximately 35–65 μg; Day 28±7: third dose of approximately 80–120 μg; Day 42±7: fourth dose of approximately 300–500 μg; Day 56±7: fifth dose of approximately 300–1800 μg; Day 70±7: sixth dose of approximately 300–1800 μg; Day 84±7: Dose 7, approximately 300–1800 μg; Day 98 ± 7: Eighth dose of approximately 300–1800 μg; Day 112 ± 7: Dose 9 of approximately 300–1800 μg; and Day 126±7: Dose 10, approximately 300–1800 μg. In one aspect of the invention as described herein, the fifth to tenth doses may be about 600 to 1500 μg.

[0093] Reference to "±7 days" is intended to mean the specified day (i.e., the specified day after the first administration of the peptide, taken as day 1) that can be administered either up to and inclusively 7 days before or up to and inclusively 7 days after the specified day. Thus, administration can occur 7, 6, 5, 4, 3, 2, or 1 day before, or 1, 2, 3, 4, 5, 6, or 7 days after, the specified day.

[0094] In one aspect of the invention described herein, reference to "±7 days" is preferably ±3 days. That is, administration can occur either up to and inclusively 3 days before or up to and inclusively 3 days after the specified date. Thus, administration can occur 3, 2, or 1 day before, or 1, 2, or 3 days after, the specified date.

[0095] The peptides can be administered in the following doses: Day 1: first dose of approximately 25 μg; Day 14: second dose of approximately 50 μg; Day 28: third dose of approximately 100 μg; Day 42: fourth dose of approximately 400 μg; Day 56: fifth dose of approximately 800 μg; Day 70: sixth dose of approximately 800 μg; Day 84: Dose 7 of approximately 800 μg; Day 98: Eighth dose of approximately 800 μg; Day 112: a ninth dose of approximately 800 μg; and Day 126: Dose 10 of approximately 800 μg. In one aspect, alternatively, the fifth to tenth doses may be about 400 μg. In an alternative embodiment, the fifth through tenth doses may alternatively be about 1600 μg.

[0096] Thus, the peptide may be administered in the following doses: Day 1: first dose of approximately 25 μg; Day 14: second dose of approximately 50 μg; Day 28: third dose of approximately 100 μg; Day 42: fourth dose of approximately 400 μg; Day 56: fifth dose of approximately 400 μg; Day 70: sixth dose of approximately 400 μg; Day 84: Dose 7 of approximately 400 μg; Day 98: Eighth dose of approximately 400 μg; Day 112: a ninth dose of approximately 400 μg; and Day 126: Dose 10 of approximately 400 μg.

[0097] Alternatively, the peptides can be administered in the following doses: Day 1: first dose of approximately 25 μg; Day 14: second dose of approximately 50 μg; Day 28: third dose of approximately 100 μg; Day 42: fourth dose of approximately 400 μg; Day 56: fifth dose of approximately 1600 μg; Day 70: sixth dose of approximately 1600 μg; Day 84: Dose 7 of approximately 1600 μg; Day 98: Eighth dose of approximately 1600 μg; Day 112: a ninth dose of approximately 1600 μg; and Day 126: Dose 10 of approximately 1600 μg.

[0098] In a further embodiment of the invention as described herein, an 11th dose of about 300 to 1800 μg, preferably about 600 to 1500 μg, preferably about 1200 μg, more preferably about 400, 800 or 1600 μg is administered on day 140±7, preferably on day 140±3, more preferably on day 140. In a preferred embodiment, the dose is about 800 μg.

[0099] In an even more preferred embodiment, a 12th dose of about 300 to 1800 μg, preferably about 600 to 1500 μg, preferably about 1200 μg, more preferably about 400, 800, or 1600 μg, is administered on day 154±7, preferably on day 154±3, more preferably on day 154. In a preferred embodiment, the dose is about 800 μg.

[0100] In a further preferred embodiment, a thirteenth dose of about 300 to 1800 μg, preferably about 600 to 1500 μg, preferably about 1200 μg, more preferably about 400, 800, or 1600 μg, is administered on day 168±7, preferably on day 168±3, more preferably on day 168. In a preferred embodiment, the dose is about 800 μg.

[0101] In a further preferred embodiment, a 14th dose of about 300 to 1800 μg, preferably about 600 to 1500 μg, preferably about 1200 μg, more preferably about 400, 800, or 1600 μg, is administered on day 182±7, preferably on day 182±3, more preferably on day 182. In a preferred embodiment, the dose is about 800 μg.

[0102] Additional doses as described above can be administered, if necessary, for example, over a period of 1 month to 20 years, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years or 20 years. Any of the teachings herein relating to the "method" of the present invention are equally applicable to the "use" encompassed by the present invention.

[0103] kit Peptides derived from S-Ag 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 kit form for simultaneous, separate, sequential or combined administration.

[0104] For example, a kit can contain multiple peptides in separate containers, the contents of which may or may not be combined prior to administration.

[0105] The kit may also include mixing and / or administration means (e.g., a vaporizer for intranasal administration, or a syringe and needle or other medical device for subcutaneous / intradermal administration). The kit may also include instructions for use. The pharmaceutical composition or kit of the present invention can be used to treat and / or prevent diseases such as uveitis as described herein.

[0106] In particular, the composition / kit can be used to suppress or block the production of S-Ag-specific CD4+ T cells (or S-Ag autoantibodies) in vivo. The composition / kit can be used to treat and / or prevent uveitis in a subject. [Example]

[0107] Example 1: Peptides 9K1K, 17JK and 15N3K are properly loaded onto MHC II molecules of dendritic cells in vivo When 100 μg of peptide 9K1K was injected into DR2tg mice or peptides 17JK or 15N3K were injected into DR3tg mice, these peptides stimulated the proliferation of dendritic cells (CD11c) in the spleens of these mice 2 hours after injection. + Isolated CD11c could be detected on the + Peptide-specific CD4 cells + T cell activation (IFNγ production) was used as a readout.

[0108] Figures 1, 2, and 3 show CD11c isolated from the spleens of mice injected with 9K1K, 17JK, or 15N3K, respectively. + The cells were CD11c derived from the spleens of PBS-injected control mice. + Significant CD4 + It is clearly understood that peptides 9K1K, 17JK, and 15N3K induce T cell activation. These results demonstrate that peptides 9K1K, 17JK, and 15N3K reach dendritic cells in secondary lymphoid organs. Furthermore, these peptides bind to MHC II molecules on these dendritic cells in the correct conformation. As a result, these peptides can induce tolerance.

[0109] Example 2: Single peptide treatment induces S-Ag-specific T cell tolerance We previously demonstrated the tolerogenic effects of the single peptides 9K1K, 17JK, and 15N3K, and these results were confirmed in these experiments. HLA-DR transgenic mice were treated with one of the peptides alone according to a dose-escalation schedule.

[0110] In the first experiment (Figure 4), DR2tg mice were administered the 9K1K peptide or PBS as described in the Methods section. Pretreatment with this modified apitope reduced S-Ag-induced cell activation by 77% and 70% in the lymph nodes (Figure 4A) and spleen (Figure 4B), respectively.

[0111] To confirm the tolerogenic potential of 17JK, DR3tg mice were treated with this modified apitope or PBS. This experiment demonstrated that pretreatment with 17JK reduced S-Ag-induced cell activation by 78% and 84% in the lymph nodes (Figure 5A) and spleen (Figure 5B), respectively.

[0112] Pretreatment of DR3tg mice with peptide 15N3K resulted in a reduction of S-Ag-induced cell activation in the lymph nodes (Fig. 6A) and spleen (Fig. 6B) by 61% and 72%, respectively, when compared with PBS-treated control mice.

[0113] Example 3: Combination peptide treatment induces S-Ag-specific T cell tolerance The finding that peptide treatment with individual peptides 9K1K, 17JK, or 15N3K reduced S-Ag-specific immune activation in DRtg mice led us to investigate whether combination treatment, administering peptides as a cocktail, could similarly reduce S-Ag-specific responses. DR3tg and DR2tg mice were treated with ATX975 according to a dose-escalation schedule and immunized with an emulsion containing all three antigens in CFA. The results for DR3tg and DR2tg mice are shown in Figures 7 and 8, respectively.

[0114] Upon ATX975 treatment in DR3tg mice (Fig. 7), S-Ag-induced cell activation was reduced by 95% and 89% in the spleen in two independent experiments. Tolerance induction by ATX975 was much more pronounced when compared with tolerance induction by treatment with peptide 17JK alone (80% and 52% reduction in S-Ag-induced cell activation in the spleen in two independent experiments) or peptide 15N3K alone (74% reduction in S-Ag-induced cell activation in the spleen).

[0115] When DR2tg mice were treated with ATX975 (Figure 8), S-Ag-induced cell activation was reduced by 78% in the spleens of treated mice compared with control mice. This tolerance induction was even more pronounced than the 51% reduction in S-Ag-induced cell activation seen after treatment with 9K1K alone. These data clearly demonstrate the additive effects of single peptides when combined in peptide cocktail treatment in DRtg mice.

[0116] Example 4: In vitro peptide-MHC II binding assays, peptides 9K1K, 17JK, and 15N3K demonstrate diverse binding patterns Figure 9 illustrates the IC50 (μM) values ​​of peptides 9K1K, 17JK, and 15N3K for binding to the indicated HLA-DR molecules and competing with known competitor peptides. Values ​​should be compared only within each HLA-DR molecule. The lowest IC50 value (green) indicates the strongest binder per HLA-DR molecule. The highest value (red) per HLA-DR molecule indicates the weakest binder for that HLA-DR molecule. Intermediate values ​​are shown in orange. These results demonstrate that the three peptides 9K1K, 17JK, and 15N3K have distinct binding profiles for the HLA-DR molecules investigated. Therefore, combining peptides into a cocktail treatment reduces the treatment's restriction to specific HLA-DR types.

[0117] conclusion The present inventors have identified a peptide cocktail, ATX975, which can contain three peptides derived from the S-Ag protein and can induce tolerance to S-Ag in HLA-DR transgenic mice.

[0118] Example 5: Variants of peptide 15N3K behave as apitopes Apitopes (antigen processing-independent epitopes) can bind to MHC II molecules without further antigen processing and stimulate responses from SAg-specific T cells. Mutants of peptide 15N3K (SEQ ID NO: 3) were tested for their ability to bind to MHC II molecules without further antigen processing and be presented to T cell hybridomas in an in vitro antigen processing-independent presentation system (APIPS) assay. In other words, peptides consisting of part of peptide 15N3K and peptides with various degrees of sequence identity to peptide 15N3K were tested for their ability to act as apitopes.

[0119] The peptides tested are presented in the table below: TIFF0007778696000002.tif78169

[0120] The T cell response, as measured by IL-2 secretion, to each of these peptides in an APIPS assay is shown in Figure 10. All but three of the peptides are apitopes.

[0121] material and method mouse Throughout peptide identification and apitope development, HLA-DR transgenic mice were used to confirm that the peptide-MHC class II binding motifs were indeed as required for tolerizing therapy in uveitis patients.

[0122] DR3tg mice were maintained under specific pathogen-free conditions externally by Charles River (UK) or Innoser (Belgium). The DR3tg strain was originally generated by Strauss et al. (Strauss et al., 1994, Immunogenetics 3, 104-108). Briefly, the genomic constructs used were a 6-kb NdeI fragment of the HLA-DRA genomic clone in pUC13 and a 24-kb ClaI × SalI fragment of cos4.1, a cosmid (pTCF) containing the B gene of DRB1*0301. Solutions containing 1–2 μg / mL of each construct were used for co-injection into fertilized eggs from an F1 donor mated with C57BL / 6 male mice (C57BL / 6 × DBA / 2). The offspring were then bred onto an IA-β knockout C57BL / 6 genetic background (AB0 mice), which lacks mouse MHC class II molecule expression. These DR3tg mice express HLA-DRB1*0301 molecules but not mouse MHC-II molecules. Mice were maintained by backcrossing to C57BL / 6 and to B10.Q. Transgenic mice were identified by Southern blot analysis of tail DNA digested with EcoRI and probed with a 1.35 kb BamHI fragment of the DRA cDNA and a 1.25 kb BamHI fragment of the DRB1*0301 cDNA.

[0123] DR2tg mice were maintained under specific pathogen-free conditions externally at Charles River (UK) or Innoser (Belgium). HLA-DR2 transgenic (DR2tg) mice were originally obtained from Lars Fugger (Madsen et al., 1999). Briefly, DRα and DRβ chain cDNAs (DRA*0101 and DRB1*1501) were expressed using the pDOI-5 expression vector, which contains a mouse MHC II promoter. The constructs were injected into fertilized eggs derived from a (DBA / 2 × C57BL / 6) F1 mating. Mice were backcrossed onto an IA-β knockout C57BL / 6 genetic background (AB0 mice), which lacks mouse MHC class II molecule expression. DR2tg mice express HLA-DRB1*1501 molecules but not mouse MHC molecules. Animal studies were approved by the Ethical Committee on Animal Experiments (ECD) of Hasselt University and were performed using the highest standards of care in pathogen-free facilities.

[0124] antigen All single peptides were synthesized by Genscript (Piscataway, USA) and stored at -80°C as stock solutions of 20 mg / mL in DMSO (Sigma-Aldrich) or 4 mg / mL in PBS (Lonza). Peptides were synthesized with an N-terminal free amine and a C-terminal amide. Human S-Ag (S-arrestin) was produced in HEK293F cells (QBiologicals, Eurofins Amatsigroup, Ghent, Belgium).

[0125] In vivo MHC class II loading assay DR3tg or DR2tg mice were administered 100 μg of peptide subcutaneously (sc) in the flank in 100 μL of PBS. Control animals were injected sc with 100 μL of PBS. Two hours later, spleens were harvested and single cell suspensions were prepared. +Cells were positively selected using CD11c microbeads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). An average purity of >92% was reached. 1 × 10 5 CD11c + Cells were cultured at 1 × 10 in round-bottom 96-well plates in X-vivo 15 medium (supplemented with 2 mM L-glutamine, 50 U / mL penicillin, and 50 U / mL streptomycin; Lonza; and 50 mM β-mercaptoethanol; Gibco). 5 CD4 + These CD4 + Cells were isolated from DR3tg or DR2tg mice immunized subcutaneously at the base of the tail with 50 μg peptide emulsified in CFA (peptide / CFA). Ten days after immunization, draining lymph nodes (LN) and spleens were harvested. LN cells and splenocytes were isolated and CD4 + T cells were isolated by negative selection using the Magnisort Mouse CD4 Isolation Kit (ThermoFisher Scientific) according to the manufacturer's instructions. After 72 hours, the supernatants of these co-cultures were collected and CD4 + T cell activation was analyzed by IFNγ ELISA (R&D Systems, Abingdon, UK). In parallel experiments, T cells were transfected with CD11c + To confirm that the cells recognized the peptides presented by the cells, we performed in vitro immunoassays of CD4 against the added peptides. + T cell responses were assessed.

[0126] Ex vivo tolerization experiments DR3tg or DR2tg mice were subcutaneously injected with 0.015 nmol, 0.15 nmol, and 1.5 nmol of peptide in the flank region on days -15, -13, and -11, respectively (dose-escalation schedule), followed by three injections of 15 nmol peptide on days -8, -6, and -4. The doses shown are for single peptide treatments; for treatments using a cocktail containing three peptides in ATX975, these doses were given per peptide (reaching a total maximum dose of 45 nmol peptide). Control mice received the same dose of an irrelevant peptide capable of binding to HLA-DR2 or HLA-DR3, depending on the mouse strain used. On day 0, mice were subcutaneously immunized at the base of the tail with 150 μg antigen emulsified in CFA (50 μg of each 30-mer peptide containing the respective epitope) (peptide / CFA). Ten days after immunization, draining lymph nodes (LN) and spleens were harvested. LN cells and splenocytes were isolated and cultured in 96-well round-bottom plates in X-vivo 15 medium (supplemented with 2 mM L-glutamine, 50 U / mL penicillin, and 50 U / mL streptomycin; Lonza; and 50 mM β-mercaptoethanol; Gibco). To examine antigen-induced cell activation, 0.5 × 10 6 Cells / well were cultured for 72 hours with different antigen concentrations (0–25 μg / mL) or 12.5 μg / mL purified protein derivative (PPD; priming control; AJ Vaccines, Copenhagen, Denmark) (200 μL / well). After 72 hours, supernatants were collected and stored at -80°C until further analysis. IFN-γ concentrations in the supernatants were assessed by cytokine ELISA (R&D Systems, Abingdon, UK) to measure cell activation.

[0127] Peptide sequence TIFF0007778696000003.tif35154

[0128] Peptide-MHC class II binding assay Binding of peptides 9K1, 17JK and 15N3K to recombinant HLA-DRA1*0101, DRB1*0101 (DR1), DRB1*1501 (DR2), DRB1*0301 (DR3), DRB1*0401 (DR4), DRB1*1101 (DR11), DRB1*0405 (DR4*05) and DRB1*0901 (DR9) was performed by ProImmune (Oxford, UK) using the cell-free MHC class II REVEAL binding assay.

[0129] Antigen Processing-Independent Presentation System (APIPS) assay Antigen-specific hybridoma clones were tested for their reactivity to peptides presented by fixed or unfixed (fresh) cells (APC). 4 Cells were treated with 10 μg / mL and 25 μg / mL peptide and 5 × 10 4 Cells were cultured with fixed or fresh APCs. To fix APCs, cells were incubated with 0.5% paraformaldehyde (Merck, Darmstadt, Germany) (pH 7) for 5 min at room temperature (RT). The fixation reaction was stopped by adding 0.4 M glycine (Sigma-Aldrich) and washing the cells in RPMI-10% FCS. After 48 h, antigen-induced IL-2 production was measured by ELISA (R&D Systems, Abingdon, UK). The present application provides the following: 1. A composition comprising the following S-arrestin (S-Ag) peptide: A peptide comprising all or part of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 60% sequence identity to SEQ ID NO: 1; and A peptide comprising all or part of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 60% sequence identity to SEQ ID NO: 2; and A peptide comprising all or part of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 60% sequence identity to SEQ ID NO:3. 2. A composition comprising the following S-arrestin (S-Ag) peptide: a peptide comprising all or part of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 1; and a peptide comprising all or a portion of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2; and A peptide comprising all or part of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:3. 3. The composition according to claim 1, comprising the following S-arrestin (S-Ag) peptide: a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a sequence having at least 60% sequence identity to SEQ ID NO: 1; and a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a sequence having at least 60% sequence identity to SEQ ID NO: 2; and A peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3) or a sequence having at least 60% sequence identity to SEQ ID NO:3. 4. The composition according to any one of 1 to 3 above, comprising the following S-arrestin (S-Ag) peptide: A peptide consisting of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and A peptide consisting of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and A peptide consisting of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3). 5. The composition described in any one of 1 to 4 above, wherein the peptide is capable of binding to an MHC molecule in vitro and being presented to T cells without antigen processing. 6. A composition according to any one of 1 to 5 above for use in therapy. 7. A composition described in any of 1 to 5 above for use in treating and / or preventing uveitis in a subject. 8. Use of a composition described in any of 1 to 5 above in the manufacture of a medicament for treating and / or preventing uveitis. 9. A method for treating uveitis in a subject, comprising the step of administering to the subject a composition described in any one of 1 to 5 above. 10. The composition for use according to 6 or 7, the use according to 8 or the method according to 9, wherein the subject is, but is not limited to, HLA-DR3. 11. The composition for use according to 6 or 7, the use according to 8 or the method according to 9, wherein the subject is, but is not limited to, HLA-DR2. 12. A composition, use or method for use according to any one of 6 to 11 above, wherein the composition is administered according to a dose escalation protocol. 13. The dose escalation protocol comprises the following doses: Day 1: First dose of about 15 to about 40 μg; Day 14±7: second dose of approximately 35–65 μg; Day 28±7: third dose of approximately 80–120 μg; Day 42±7: fourth dose of approximately 300–500 μg; Day 56±7: fifth dose of approximately 600–1500 μg; Day 70±7: sixth dose of approximately 600–1500 μg; Day 84±7: Dose 7, approximately 600–1500 μg; Day 98 ± 7: Eighth dose of approximately 600–1500 μg; Day 112 ± 7: a ninth dose of approximately 600–1500 μg; and Day 126±7: 10th dose of approximately 600-1500 μg 13. A composition, use or method for use according to claim 12, comprising: 14. the first dose is about 25 μg; and / or the second dose is about 50 μg; and / or the third dose is about 100 μg; and / or the fourth dose is about 400 μg; 14. A composition, use or method for use as defined in claim 13. 15. An 11th dose of approximately 600-1500 μg is administered on day 140 ± 7; and / or a 12th dose of approximately 600 to 1500 μg administered on day 154 ± 7; and / or A 13th dose of approximately 600 to 1500 μg is administered on day 168 ± 7. 15. A composition, use or method for use as defined in 13 or 14 above. 16. A composition, use or method for use according to any one of claims 13 to 15 above, wherein the fifth, sixth, seventh, eighth, ninth and tenth, and optionally the eleventh, twelfth and thirteenth doses are each about 800 μg. 17. A composition, use or method for use according to any one of 6 to 16 above, wherein the administration of the composition is intradermal. 18. A composition, use or method for use according to any one of 6 to 17 above, wherein the composition is administered to a human. 19. A kit comprising an S-Ag peptide as defined in any one of 1 to 5 above. 20. The kit according to claim 19, for simultaneous, separate or sequential administration in the prevention or treatment of uveitis.

Claims

1. A composition comprising the following S-arrestin (S-Ag) peptides: A peptide consisting of the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and A peptide consisting of the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and A peptide consisting of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3).

2. The composition of claim 1, wherein the peptide is capable of binding to an MHC molecule and being presented to T cells in vitro without antigen processing.

3. 3. The composition of claim 1 or 2 for use in therapy.

4. 4. A composition according to any one of claims 1 to 3 for use in the treatment and / or prevention of uveitis in a subject.

5. 5. A composition according to any one of claims 1 to 4 for use in the treatment of uveitis.

6. The composition of claim 4, wherein the subject is HLA-DR3.

7. The composition of claim 4, wherein the subject is HLA-DR2.

8. The composition of any one of claims 3 to 7, wherein the composition is administered according to a dose escalation protocol.

9. The dose escalation protocol comprises the following doses: Day 1: First dose of 15-40 μg; Day 14 ± 7: second dose of 35–65 μg; Day 28 ± 7: third dose of 80–120 μg; Day 42 ± 7: fourth dose of 300–500 μg; Day 56 ± 7: fifth dose of 600–1500 μg; Day 70 ± 7: sixth dose of 600–1500 μg; Day 84 ± 7: Dose 7, 600–1500 μg; Day 98 ± 7: Dose 8, 600–1500 μg; Day 112 ± 7: Dose 9 of 600–1500 μg; and Day 126 ± 7: 10th dose of 600–1500 μg 9. The composition of claim 8, comprising:

10. the first dose is 25 μg; and / or the second dose is 50 μg; and / or the third dose is 100 μg; and / or the fourth dose is 400 μg; 10. The composition of claim 9.

11. an 11th dose of 600–1500 μg administered on day 140 ± 7; and / or A 12th dose of 600 to 1500 μg is administered on day 154 ± 7; and / or A 13th dose of 600-1500 μg is administered on day 168 ± 7; 11. The composition according to claim 9 or 10.

12. 12. The composition of any one of claims 9 to 11, wherein the fifth, sixth, seventh, eighth, ninth and tenth, and optionally the eleventh, twelfth and thirteenth doses are each 800 μg.

13. The composition of any one of claims 3 to 12, wherein administration of the composition is intradermal.

14. The composition of any one of claims 3 to 13, wherein the composition is administered to a human.

15. A kit comprising an S-Ag peptide as defined in claim 1 or 2.

16. 16. The kit of claim 15 for simultaneous, separate or sequential administration in the prevention or treatment of uveitis.

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