A composition comprising an s-repressin peptide

Abstract

The present invention relates to a composition comprising a peptide derived from S- Retinol dehydrogenase (S-antigen, S-Ag). The composition or peptide can be used to prevent and / or inhibit S-Ag autoimmunity, can be used to treat and / or prevent uveitis.

Description

Technical Field

[0001] This invention relates to a composition comprising a peptide derived from S-repressor protein (retinal repressor protein, S-antigen, S-Ag). This composition or peptide can be used to prevent and / or inhibit S-Ag autoimmunity, and can be used to treat and / or prevent uveitis. Background Technology

[0002] Uveitis refers to a group of diseases associated with 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. Related diseases are not limited to those directly affecting the uvea; clinical manifestations of the uvea can also affect adjacent structures such as the retina, optic nerve, lens, vitreous body, and sclera.

[0003] All forms of uveitis are characterized by inflammatory cell infiltration, typically observed under a biological microscope. In 2010, approximately 285 million people suffered from vision impairment; of these, 39 million were blind, and about 10% of cases were caused by 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 glucocorticoids and other immunosuppressants, such as methotrexate.

[0005] However, there remains a need for alternative treatments for uveitis in existing technologies. This invention addresses this need. Invention Overview

[0007] The inventors have discovered that a “mixture” of three S-Ag peptides is particularly effective in inhibiting or preventing S-Ag-specific T cell activation in vitro.

[0008] Therefore, a first aspect of the present invention provides a composition comprising the following S-Ag peptide:

[0009] 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 with SEQ ID NO:1; and / or

[0010] 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 with SEQ ID NO:2; and / or

[0011] a peptide comprising all or a portion of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 60% sequence identity to SEQ ID NO: 3.

[0012] KKKAFVEQVANVVLKKK (SEQ ID NO: 1) also referred to herein as 9K1K. KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) also referred to herein as 17JK. KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) also referred to herein as 15N3K.

[0013] The composition according to the application can be used in the therapeutic aspects of the application described herein.

[0014] In a second aspect, the present application provides a composition of the application as described herein for use in the treatment and / or prevention of uveitis in a subject.

[0015] In a third aspect, the present application relates to the use of a composition of the application as described herein for the manufacture of a medicament for the treatment and / or prevention of uveitis.

[0016] In a fourth aspect, the present application relates to a method of treating uveitis in a subject comprising the step of administering to the subject a peptide of SEQ ID NO: 1 in whole or in part or a peptide having at least 60% sequence identity thereto, and / or a peptide of SEQ ID NO: 2 in whole or in part or a peptide having at least 60% sequence identity thereto, and / or or a peptide of SEQ ID NO: 3 in whole or in part or a peptide having at least 60% sequence identity thereto.

[0017] In one aspect, the present application relates to a combination of a peptide of SEQ ID NO: 1 in whole or in part or a peptide having at least 60% sequence identity thereto, and a peptide of SEQ ID NO: 2 in whole or in part or a peptide having at least 60% sequence identity thereto.

[0018] In one aspect, the present application relates to a combination of a peptide of SEQ ID NO: 2 in whole or in part or a peptide having at least 60% sequence identity thereto, and a peptide of SEQ ID NO: 3 in whole or in part or a peptide having at least 60% sequence identity thereto.

[0019] In one aspect, the present application relates to a combination of a peptide of SEQ ID NO: 1 in whole or in part or a peptide having at least 60% sequence identity thereto, and a peptide of SEQ ID NO: 3 in whole or in part or a peptide having at least 60% sequence identity thereto.

[0020] The peptide composition according to the application can comprise an amino acid sequence according to the application as described herein. In one aspect, the peptide composition comprises only an amino acid sequence according to the application as described herein.

[0021] The subject can be HLA-DR3. The subject can be HLA-DR2.

[0022] The peptide of the application or the composition of the application as defined herein can be administered according to a dose escalation regimen.

[0023] In a fifth aspect, the present application relates to a kit comprising the following S-Ag peptides for simultaneous, separate or sequential administration:

[0024] the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1) or a portion of this sequence or all or a portion of a sequence having at least 60% sequence identity to SEQ ID NO: 1; and / or

[0025] the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2) or a portion of this sequence or all or a portion of a sequence having at least 60% sequence identity to SEQ ID NO: 2; and / or

[0026] the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a portion of this sequence or all or a portion of a sequence having at least 60% sequence identity to SEQ ID NO: 3.

[0027] The kit can be used for the treatment of uveitis. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 : Peptide 9K1 K is correctly loaded on MHC class II in vivo. Peptide-specific CD4+ T cells were shown to respond to the peptide loaded on dendritic cells in vivo. HLA-DR2 transgenic mice were injected subcutaneously with 100 pg of 9K1 K peptide. Two hours after injection, the spleen was dissected and CD11c+ cells (dendritic cells) were isolated. These CD11c+ cells were co-cultured with peptide-specific CD4+ cells for 72 hours at 37°C. IFNy was measured in the supernatant of these cultures to assess the response of CD4+ T cells to the peptide. (*p<0.05 Mann-Whitney U test)

[0029] Figure 2Peptide 17JK is correctly loaded onto MHC class II in vivo. Peptide-specific CD4+ T cells were shown to respond to the peptide loaded on dendritic cells in vivo. HLA-DR3 transgenic mice were injected subcutaneously with 100 μg of 17JK peptide. Two hours after injection, the spleen was dissected and CD11c+ cells (dendritic cells) were isolated. These CD11c+ cells were co-cultured with peptide-specific CD4+ cells for 72 hours at 37°C. IFNy was measured in the supernatant of these cultures to assess the response of CD4+ T cells to the peptide. (***p < 0.001 Mann-Whitney U test)

[0030] Figure 3 Peptide 15N3K is correctly loaded onto MHC class II in vivo. Peptide-specific CD4+ T cells were shown to respond to the peptide bound on dendritic cells in vivo. HLA-DR3 transgenic mice were injected subcutaneously with 100 μg of 15N3K peptide. Two hours after injection, the spleen was dissected and CD11c+ cells (dendritic cells) were isolated. These CD11c+ cells were co-cultured with peptide-specific CD4+ cells for 72 hours at 37°C. IFNy was measured in the supernatant of these cultures to assess the response of CD4+ T cells to the peptide. (***p < 0.001 Mann-Whitney U test)

[0031] Figure 4 9K1K alone induces tolerance to S-Ag protein in DR2tg mice. Mice were injected subcutaneously in the flank with 0.1, 1 and 10 μg / ml of 9K1K at days -15, -13 and -11, then 3 times with 100 μg / ml at days -8, -6 and -4 (dose escalation regimen). On day 0, mice were immunized subcutaneously in the base of the tail with antigen / CFA. Mice were sacrificed 10 days post-immunization to measure LN and spleen cell activation upon re-stimulation with S-Ag. IFNy concentration in culture supernatant was measured as an indicator of cell activation. Data represent the mean ± SEM of IFNy concentration for PBS (black line) and 9K1K (grey line) treated mice. Two-way ANOVA was used to measure the overall treatment effect on T cell activation. Dunnett’s multiple comparison test was used and significant differences are shown in the graph (**p < 0.01; ****p < 0.0001). The percentage of inhibition on IFNy production compared to the control group is shown in the graph. (A) LN tolerance to S-Ag. IFNy production is expressed in IFNy concentration (pg / ml). (B) Spleen tolerance to S-Ag. IFNy production is expressed in IFNy concentration (pg / ml). LN, lymph node. Data are representative of 3 independent experiments.

[0032] Figure 5:17JK alone induces tolerance to S-Ag protein in DR3tg mice. Mice were injected subcutaneously in the flank with 0.1 pg / ml, 1 pg / ml and 10 pg / ml of 17JK on days -15, -13 and -11, followed by 3 injections of 100 pg / ml on days -8, -6 and -4 (dose escalation regimen). On day 0, mice were immunized subcutaneously in the base of the tail with antigen / CFA. Mice were sacrificed 10 days post-immunization to measure LN and splenocyte activation upon re-stimulation with S-Ag. IFNy concentration in culture supernatant was measured as an indicator of cell activation. Data represent the mean ± SEM of IFNy concentration for PBS (black line) and 17JK (grey line) treated mice. Two-way ANOVA was used to measure the overall treatment effect on T cell activation. Dunnett’s multiple comparison test was used and significant differences are shown in the graph (**p<0.01; ****p<0.001). The percentage of inhibition on IFNy production compared to the control group is shown in the graph. (A) LN tolerance to S-Ag. IFNy production is expressed in pg / ml. (B) Spleen tolerance to S-Ag. IFNy production is expressed in pg / ml. LN, lymph node. Data are representative of 3 independent experiments.

[0033] Figure 6 :15N3K alone induces tolerance to S-Ag protein in DR3tg mice. Mice were injected subcutaneously in the flank with 0.1 pg / ml, 1 pg / ml and 10 pg / ml of 15N3K on days -15, -13 and -11, followed by 3 injections of 100 pg / ml on days -8, -6 and -4 (dose escalation regimen). On day 0, mice were immunized subcutaneously in the base of the tail with antigen / CFA. Mice were sacrificed 10 days post-immunization to measure LN and splenocyte activation upon re-stimulation with S-Ag. IFNy concentration in culture supernatant was measured as an indicator of cell activation. Data represent the mean ± SEM of IFNy concentration for PBS (black line) and 15N3K (grey line) treated mice. Two-way ANOVA was used to measure the overall treatment effect on T cell activation. Dunnett’s multiple comparison test was used and significant differences are shown in the graph (**p<0.05; ****p<0.0001). The percentage of inhibition on IFNy production compared to the control group is shown in the graph. (A) LN tolerance to S-Ag. IFNy production is expressed in pg / ml. (B) Spleen tolerance to S-Ag. IFNy production is expressed in pg / ml. LN, lymph node. Data are representative of 3 independent experiments.

[0034] Figure 7: Peptide cocktail ATX975 treatment was able to reduce S-Ag induced immune cell activation in DR3tg mice more efficiently compared to treatment with 17JK or 15N3K alone. Mice were injected subcutaneously in the flank with 0.015 nmol, 0.15 nmol and 1.5 nmol of peptides on days -15, -13 and -11, respectively, followed by 3 injections of 15 nmol of peptides on days -8, -6 and -4 (dose escalation regimen). For treatment with the cocktail containing 3 peptides (ATX975), these doses were administered per peptide (total maximum dose of 45 nmol of peptides was reached). On day 0, mice were immunized subcutaneously in the base of the tail with antigen / CFA. Mice were sacrificed 10 days post-immunization to measure activation of splenocytes upon re-stimulation with S-Ag. Data represent mean ± SEM of IFNy concentration for PBS treated mice (black lines) and peptide treated mice (grey lines). Two-way ANOVA was used to measure overall treatment effect on T cell activation. Dunnett’s multiple comparison test was used and significant differences are shown in the graph (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to control; #p<0.05, ####p<0.0001 compared to ATX975). Percentage of inhibition on IFNy production compared to control is shown in the graph. (A) Tolerance to S-Ag in the spleen. IFNy production is expressed in IFN-g concentration (pg / ml). (B) Tolerance to S-Ag in the spleen. IFNy production is expressed in IFN-g concentration (pg / ml).

[0035] Figure 8: Peptide mixture ATX975 treatment was able to reduce S-Ag induced immune cell activation in DR2tg mice more effectively compared to treatment with 9K1K alone. Mice were injected subcutaneously in the flank with 0.015 nmol, 0.15 nmol and 1.5 nmol of peptides on days -15, -13 and -11 respectively, followed by 3 injections of 15 nmol of peptides on days -8, -6 and -4 (dose escalation regimen). For treatment with the mixture containing 3 peptides (ATX975), these doses were administered per peptide (total maximum dose of 45 nmol of peptides was reached). On day 0, mice were immunized subcutaneously in the base of the tail with antigen / CFA. Mice were sacrificed 10 days post-immunization to measure activation of splenocytes upon re-stimulation with S-Ag. Data represent mean ± SEM of IFNy concentration for PBS treated mice (black line) and peptide treated mice (grey line). Two-way ANOVA was used to measure overall treatment effect on T cell activation. Dunnett's multiple comparison test was used and significant differences are shown in the graph (*p<0.05; **p<0.01). Percent inhibition of IFNy production compared to control is shown in the graph. Tolerance to S-Ag in the spleen. IFN-γ production is expressed in pg / ml.

[0036] Figure 9 : Different patterns of in vitro peptide-MHC II binding for peptides 9K1, 17JK and 15N3K. Peptide 9K1, 17JK and 15N3K binding 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 (pM) are shown for each HLA-DR molecule and color-coded. Low values (green) indicate strong binding, high values (red) indicate weak (or relatively weak) binding.

[0037] Figure 10 : Variants of peptide 15N3K are apitopes. 15N3K peptide variants were tested for their ability to act as apitopes (i.e. to bind to MHC II molecules and be presented to T cells by antigen presenting cells without antigen processing) using the APIPS assay. DR3tg mice were immunized with SAg and hybridomas were generated. 5*10 4 SAg specific hybridoma cells were incubated with 5*10 4Fresh or fixed commercial APC (VAVY) cells were cultured together. Cultures were stimulated with 10 or 25 pg / ml peptide as indicated. Supernatants were collected after 48 hours and T cell activation was measured by IL-2 ELISA. The graph represents the mean of duplicate measurements ± SEM. DETAILED DESCRIPTION

[0039] The present application provides a new alternative treatment option for the treatment and / or prevention of uveitis. As demonstrated in the present application, the combination of the peptides of SEQ ID NO: 1, 2 and 3 induced tolerance to S-Ag in a tolerance model in HLA-DR transgenic mice.

[0040] Thus, a first aspect of the present application relates to a composition comprising a plurality of peptides from S-Ag, i.e. the peptides of the present application as defined herein, preferably the peptides of SEQ ID NO: 1, 2 and / or 3.

[0041] S-arrestin

[0042] S-arrestin (also known as retinal arrestin, S-antigen or S-Ag) is a soluble photoreceptor protein expressed in the retina and pineal gland. It is known to be involved in the desensitization of the light-activated transduction cascade and was first isolated from its association with activated rhodopsin. The crystal structure shows two anti-parallel beta-sheet domains connected by a hinge region and a short alpha-helix at the back of the amino-terminal fold.

[0043] The light-activated form of the visual pigment rhodopsin (RhhhoG) interacts with the retinal G protein transducin, initiating the exchange of a GDP molecule for GTP on the alpha subunit of the transducin. In its GTP-bound form, the transducin dissociates from Rh* and activates the cyclic GMP phosphodiesterase (PDE) by binding to two inhibitory subunits PDE gamma. The result is a rapid decrease in the concentration of the intracellular transmitter cyclic GMP. Because the interaction of Rh* and transducin requires only about 1 ms, a single Rh* can subsequently interact with several hundred transducin molecules. The turnover rate of the PDE can be several thousand hydrolyzed cGMP per PDE per second. Therefore, to limit the photoreaction and to recover rapidly, Rh* must be eliminated quickly and efficiently before too many PDE molecules are activated. This inactivation of Rh* is accomplished in two steps: phosphorylation of Rh* reduces its ability to catalyze the nucleotide exchange of transducin, and subsequent binding of arrestin to P-Rh* completely shields it from further interaction with transducin.

[0044] The amino acid sequence of the mature human S-Ag is shown below (SEQ ID NO: 4).

[0045] UniProt database, P10523 https: / / www.uniprot.org / )

[0046]

[0047] Uveitis

[0048] Clinically, uveitis is usually classified into the following categories according to the part of the eye it primarily affects: anterior uveitis, intermediate uveitis, posterior uveitis or panuveitis.

[0049] 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 includes inflammation of the ciliary body.

[0050] Intermediate uveitis (pars planitis) usually refers to vitreous inflammation - inflammation of the cells within the vitreous cavity, associated with deposition of inflammatory material on the pars plana.

[0051] Posterior uveitis (choroidoretinitis) is inflammation of the retinal and choroidal regions.

[0052] Panuveitis refers to inflammation affecting all levels of the uvea.

[0053] Uveitis can also be classified as infectious or non-infectious, with autoimmune disease-related uveitis (i.e. primarily non-infectious) being more common in developed countries. Animal models commonly used to study uveitis are also initiated by autoimmune processes, and there is a clear link between the two. It is estimated that 25-30% of uveitis is associated with systemic autoimmune or autoinflammatory diseases.

[0054] In one aspect of the application, the uveitis is non-infectious uveitis.

[0055] Tolerance

[0056] 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 allergic disease (including allergy and transplant rejection) has been demonstrated through the use of experimental models. Autoimmune or allergic disease can be induced by injection of synthetic peptides (based on the structure of T cell epitopes) in combination with adjuvants.

[0057] In contrast, it has been shown that immunogenic tolerance to specific antigens can be induced by administration of soluble forms of peptide epitopes. Administration of soluble peptides has proven to be an effective means of suppressing disease in experimental models of autoimmune encephalomyelitis (EAE - a model of multiple sclerosis) (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 arthritis, diabetes and experimental models of uveoretinitis (reviewed in Anderton and Wraith (1998), supra). It has also been shown to be a means of treating progressive disease in EAE (Anderton and Wraith (1998), supra).

[0058] Tolerance is the failure to respond to an antigen. Tolerance to self-antigens is an essential feature of the immune system, and when this tolerance is lost, autoimmune disease results. The adaptive immune system must maintain the ability to respond to a wide variety of infectious agents, while avoiding autoimmune attack on self-antigens contained in self-tissues. This is controlled in large part 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 the death of self-reactive thymocytes is incomplete. Thus, mature self-reactive T lymphocytes in the periphery can also acquire tolerance by certain mechanisms (peripheral tolerance). A review of central and peripheral tolerance mechanisms is given in Anderton et al (1999) Immunological Reviews 169: 123-137. See also Wraith (2016) Nature 530: 422-423.

[0059] Current data suggests that uveitis can be caused by self-reactive T cells to self-activity of retinal proteins including S-Ag, leading to inflammation and causing chronic disease. The compositions of the present invention are capable of inducing tolerance to self-antigens such as S-Ag, such that when administered to a subject, it can restore tolerance to the S-Ag protein and reduce the pathogenic immune response.

[0060] Apitope

[0061] In the adaptive immune response, T lymphocytes are able to recognise epitopes of protein antigens. Antigen presenting cells (APCs) take up protein antigens and degrade them into short peptide fragments. The peptides can bind to major histocompatibility complex (MHC) class I or class II molecules in the cell and be transported to the cell surface. When presented to the cell surface together with MHC molecules, the peptides can be recognised by T cells (via the T cell receptor (TCR)), in which case the peptides are T cell epitopes.

[0062] Thus, an epitope is a peptide derived from an antigen which is able to bind to the peptide binding groove of an MHC molecule and be recognised by a T cell.

[0063] A minimal epitope is the shortest fragment derived from an epitope which is able to bind to the peptide binding groove of an MHC class I or class II molecule and be recognised by a T cell. For a given immunogenic region, it is often possible to generate a set of "nested" overlapping peptides as epitopes, all of which contain the minimal epitope but differ in their flanking regions.

[0064] For the same reason, the minimal epitope for a particular MHC molecule: T cell binding can be identified by measuring the response to truncated peptides. For example, if a response is generated to a peptide containing residues 1-15 in an overlapping library, then 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 the minimal epitope.

[0065] The present inventors have previously determined that there is a link between the ability of a peptide to bind to an MHC molecule and be presented to a T cell without further processing and the ability of the peptide to induce tolerance in vivo (WO 02 / 16410). If a peptide is too long to bind to the peptide binding groove of an MHC molecule without further processing (e.g. cleavage) or binds in an inappropriate conformation, then it will not be tolerogenic in vivo. On the other hand, if a peptide has the appropriate size and conformation to bind directly to the MHC peptide binding groove and be presented to a T cell, then it can be expected that the peptide can be used to induce tolerance.

[0066] Thus, the tolerogenic potential of a peptide can be investigated by studying whether the peptide can bind to an MHC molecule and be presented to a T cell in vitro without further antigen processing.

[0067] An S-Ag apitope (antigen processing independent epitope) is able to bind to an MHC class II molecule and stimulate a response by S-Ag specific T cells without the need for further antigen processing. Such apitopes can be expected to lead to tolerance to S-Ag according to the rule-based approach described in WO 02 / 16410.

[0068] Peptides bound to MHC class I molecules are usually 7 to 13, more usually 8 to 10 amino acids in length. Their binding is stabilised at both ends of the peptide by contacts between atoms in the peptide backbone and invariant sites in the peptide binding groove of all MHC class I molecules. Invariant sites exist at both the amino and carboxy termini of the bound peptide. Variations in peptide length are accommodated by twists in the peptide backbone, usually at flexible proline or glycine residues.

[0069] Peptides bound to MHC class II molecules are usually between 8 and 20 amino acids in length, more usually between 10 and 17 amino acids, and can be longer (e.g. up to 40 amino acids). These peptides lie in an extended conformation along the MHC class II peptide binding groove, which (unlike the MHC class I peptide binding groove) is open at both ends. The peptides are held in place primarily by contacts between backbone atoms and conserved residues lining the peptide binding groove.

[0070] In a preferred embodiment, the peptide derived from S-Ag is capable of binding to an MHC class II molecule without the need for further processing.

[0071] Peptides

[0072] The term "peptide" is used in accordance with its standard meaning to refer to a series of residues (usually L-amino acids) linked to one another, usually by peptide bonds between the a-amino and carboxy groups of adjacent amino acids. The term includes modified peptides and synthetic peptide analogues.

[0073] Peptides of the application can be prepared using chemical methods (Peptide Chemistry, A utility Textbook. Mikos Bodansky, Springer-Verlag, Berlin.). For example, peptides can be synthesised 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 431 A peptide synthesiser (Perkin Elmer) according to the manufacturer's instructions.

[0074] Peptides can alternatively be prepared recombinantly or by cleavage from longer polypeptides. For example, a peptide can be obtained by cleavage from an S-antigen protein, followed by modification of one or both ends. The composition of the peptide can be confirmed by amino acid analysis or sequencing (e.g. the Edman degradation procedure).

[0075] For practical purposes, the peptide can exhibit various other characteristics. For example, it is important that the peptide is sufficiently stable in vivo to be useful for therapy. The in vivo half-life of the peptide can be at least 10 minutes, 30 minutes, 4 hours or 24 hours.

[0076] The peptides used in the present application are shown below:

[0077] a peptide comprising all or a portion 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

[0078] a peptide comprising all or a portion 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

[0079] a peptide comprising all or a portion of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 60% sequence identity to SEQ ID NO: 3.

[0080] The peptide according to the present application 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 the peptide of SEQ ID NO: 1, 2 or 3. In one aspect, the peptide has at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 1, 2 or 3.

[0081] In some embodiments, the composition comprises the following S-Ag peptides:

[0082] a peptide comprising all or a portion 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

[0083] a peptide comprising all or a portion 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

[0084] a peptide comprising all or a portion of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 80% sequence identity to SEQ ID NO: 3.

[0085] In some embodiments, the composition comprises the following S-Ag peptide:

[0086] a peptide comprising all or a portion 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

[0087] a peptide comprising all or a portion 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

[0088] a peptide comprising all or a portion of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 90% sequence identity to SEQ ID NO: 3.

[0089] In some embodiments, the composition comprises the following S-Ag peptide:

[0090] a peptide comprising all or a portion 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

[0091] a peptide comprising all or a portion 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

[0092] a peptide comprising all or a portion of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3) or a sequence having at least 95% sequence identity to SEQ ID NO: 3.

[0093] In some embodiments, the composition comprises the following S-Ag peptide:

[0094] 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

[0095] 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

[0096] 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.

[0097] In some embodiments, the composition comprises the following S-Ag peptides:

[0098] 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

[0099] 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

[0100] 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.

[0101] In some embodiments, the composition comprises the following S-Ag peptides:

[0102] 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

[0103] 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

[0104] 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.

[0105] In some embodiments, the composition comprises the following S-Ag peptides:

[0106] a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and / or

[0107] a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and / or

[0108] a peptide comprising the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO: 3).

[0109] In some embodiments, the composition comprises the following S-Ag peptides:

[0110] 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

[0111] 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

[0112] 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.

[0113] In some embodiments, the composition comprises the following S-Ag peptides:

[0114] 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

[0115] 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

[0116] 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.

[0117] In some embodiments, the composition comprises the following S-Ag peptides:

[0118] 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

[0119] 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

[0120] 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.

[0121] In some embodiments, the composition comprises the following S-Ag peptides:

[0122] 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

[0123] 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

[0124] 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.

[0125] In some embodiments, the composition comprises the following S-Ag peptides:

[0126] 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

[0127] 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

[0128] 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.

[0129] In some embodiments, the composition comprises the following S-Ag peptides:

[0130] a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and / or

[0131] a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and / or

[0132] 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.

[0133] In some embodiments, the composition comprises the following S-Ag peptides:

[0134] a peptide comprising the amino acid sequence KKKAFVEQVANVVLKKK (SEQ ID NO: 1); and / or

[0135] a peptide comprising the amino acid sequence KKKLTFRRDLYFSRVQVYKKK (SEQ ID NO: 2); and / or

[0136] 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.

[0137] In preferred aspects, the peptide comprises SEQ ID NO: 1, 2 and / or 3. In further aspects, the peptide consists of SEQ ID NO: 1, 2 and / or 3.

[0138] Sequence identity can be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that perform multiple alignments of sequences are useful, such as Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22: 4673-4680). 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) can also be used for this purpose. In addition, the Dali server of the European Bioinformatics Institute provides structure-based alignment 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).

[0139] Multiple sequence alignment and percentage identity calculation can be determined using standard BLAST parameters (using sequences from all available organisms, matrix Blosum 62, gap cost: existence 11, extension 1).

[0140] Alternatively, the following program 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, extended gap penalty = 1. Amino acid comparison: global comparison, BLOSUM 62 scoring matrix.

[0141] Accordingly, the scope of the present application includes variants of the recited or given sequences, provided that the variant retains the functional activity of the parent peptide, i.e. the variant is functionally equivalent, in other words, they have or exhibit the activity of the parent peptide as defined herein. Such variants can comprise substitution, addition or deletion of one or more amino acids (e.g. 1 to 14 amino acids) of the parent sequence, including truncation at one or both ends.

[0142] Also included are functionally equivalent derivatives in which one or more amino acids are chemically derivatized, e.g., amino acids substituted with chemical groups.

[0143] Thus, the peptides of the application can comprise a portion, region or fragment of SEQ ID NOs: 1-3, as long as the peptide retains the desired activity. The portion, region or fragment of SEQ ID NOs: 1-3 can be, for example, 6 to 14 residues in length, e.g., 6, 7, 8, 9, 10, 11, 12 or 13 residues in length.

[0144] The peptides of the application can comprise 8 to 30 amino acids, e.g., 8 to 25 amino acids, 8 to 20 amino acids, 8 to 15 amino acids or 8 to 12 amino acids. In one aspect, the peptides of the application can thus 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.

[0145] Portions

[0146] The peptides of the application can comprise all or part of an S-Ag derived peptide as set forth in SEQ ID NOs: 1, 2 and / or 3.

[0147] The term "portion" refers to a peptide derived from SEQ ID NOs: 1-3 and comprising at least one minimal epitope of the peptide.

[0148] Such peptides can comprise one or more mutations, typically amino acid substitutions within the S-Ag derived sequence. The amino acids can be substituted with amino acids such as glycine, lysine or glutamic acid. The peptides can comprise up to three, up to two or one amino acid substitution from the S-Ag derived sequence.

[0149] Such peptides can comprise non-S-Ag sequence derived amino acids at one or both termini. For example, the peptides can have one or more glycine and / or lysine and / or glutamic acid residues at one or both termini

[0150] The peptides comprising non-S-Ag derived amino acids are apitopes, i.e., capable of binding to MHC molecules in vitro and in vivo and being presented to T cells without antigen processing.

[0151] Solubility

[0152] Solubility can be an important consideration in peptide-mediated tolerance induction.

[0153] Solubility can be improved by adding additional amino acids at the N and C termini, which can be glycine (G), lysine (K) and / or glutamic acid (E).

[0154] In one aspect, the peptide according to the application can have one, two or three additional amino acids at the N- and / or C-terminus. The additional amino acids can be selected from glycine (G), lysine (K) and / or glutamic acid (E). Different combinations of these amino acids can be added to the peptide according to the application.

[0155] For example, the peptide according to the application can have one, two or three lysine (K) residues at the N- and C-terminus.

[0156] The peptide according to the application can have one, two or three glycine (G) residues at the N- and C-terminus.

[0157] The peptide according to the application can have one, two or three glutamic acid (E) residues at the N- and C-terminus.

[0158] In one aspect, the peptide according to the application can have one glycine and one lysine residue at the N- and C-terminus.

[0159] In one aspect, the peptide according to the application can have one glycine and two lysine residues at the N- and C-terminus.

[0160] In one aspect, the peptide according to the application can have one glutamic acid and one lysine residue at the N- and C-terminus.

[0161] In one aspect, the peptide according to the application can have one glutamic acid and two lysine residues at the N- and C-terminus.

[0162] For example, the additional amino acids can comprise a glycine or lysine spacer at one or both ends, followed by the amino acid pair KK, KE, EK or EE.

[0163] In one aspect, the peptide can have a glycine spacer at both ends, followed by a combination of two additional amino acids at the N- and C-terminus, which can be lysine (K) and / or glutamic acid (E). Thus, the possible combinations for a given end can be GKK, GKE, GEK or GEE.

[0164] The peptide can have the following general formula:

[0165] XXG - parent peptide - GXX

[0166] In one aspect, the peptide according to the application can have three additional lysine (K) residues at the N- and C-terminus.

[0167] The modified peptide according to the application can thus have 6 additional amino acids (3 at each end) relative to the parent peptide.

[0168] Alternatively, the peptide of the application can also have the following general formula:

[0169] KKK - parent peptide - KKK

[0170] KK - parent peptide - KK

[0171] K - parent peptide - K

[0172] GK - parent peptide - KG

[0173] GKK - parent peptide - KKG

[0174] KKG - parent peptide - GKK

[0175] EK - parent peptide - KE

[0176] EKK - parent peptide - KKE

[0177] GKE - parent peptide - EKG

[0178] GEK - parent peptide - KEG

[0179] The modified peptide can be more soluble than the parent (unmodified) peptide. The modified peptide can have 2-fold, 3-fold, 4-fold or 5-fold solubility compared to the parent peptide. The peptide can be soluble at a concentration of up to 0.5 mg / ml, 1 mg / ml or 5 mg / ml.

[0180] As discussed herein, the modified peptide of the application can have 2, 4 or 6 additional amino acids (1, 2 or 3 per end) compared to the parent peptide.

[0181] In the most preferred aspect, the modification comprises KKK at both the N and C terminus.

[0182] The modified peptide can be more soluble than the parent (unmodified) peptide. The modified peptide can have 2-fold, 3-fold, 4-fold or 5-fold solubility compared to the parent peptide. The peptide can be soluble at a concentration of up to 0.5 mg / ml, 1 mg / ml, 5 mg / ml or higher, for example 8 mg / ml. In one aspect, the modified peptide can be soluble at a concentration of 4 mg / ml.

[0183] Composition

[0184] The S-Ag peptide can be in the form of a composition, preferably a pharmaceutical composition.

[0185] The peptide composition according to the application can comprise an amino acid sequence according to the application as described herein. In one aspect, the peptide composition comprises only an amino acid sequence according to the application as described herein, i.e. it does not comprise other peptides than the peptide according to the application.

[0186] In one aspect of the application discussed herein, a first step is to identify a subject having uveitis or at risk of developing uveitis.

[0187] The peptide or composition according to the application can be used for prophylactic or therapeutic use.

[0188] When administered for prophylactic use, the peptide or composition can reduce or prevent the development of an immune response to S-Ag. The level of the immune response is lower than the level that would be obtained by the subject if not treated with the composition. The term "reduce" means that a partial reduction in the immune response is observed, for example a reduction of 50%, 60%, 70%, 80% or 90% relative to the response that would be observed by the subject if not receiving the composition treatment (or the response observed in a non-treated subject over the same time period). The term "prevent" means that no significant immune response to S-Ag is observed.

[0189] When administered for therapeutic use, the peptide or composition can inhibit an already developed immune response to S-Ag. The term "inhibit" means that the level of the already developed immune response is reduced compared to the level before treatment with the peptide or the level that would be observed at the same time point if no treatment was given.

[0190] Treatment with the composition of the application can result in a reduction of any or all of the following levels:

[0191] i) S-Ag autoantibodies;

[0192] ii) S-Ag specific pro-inflammatory CD4+ T cells;

[0193] iii) B cells secreting S-Ag autoantibodies.

[0194] Detection of all factors can be performed by techniques known in the art, for example ELISA, flow cytometry, etc.

[0195] Treatment with the peptide or composition of the application can also or alternatively induce anergy of S-Ag specific CD4+ T cells. The anergy can be detected by, for example, subsequent challenge in vitro with S-Ag.

[0196] Formulations

[0197] The composition can be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition can optionally comprise one or more other pharmaceutically active compounds. Such formulations can be, for example, in a form suitable for intradermal or subcutaneous administration.

[0198] The composition can be prepared as injectable liquid solutions or suspensions; it can also be prepared as solid forms suitable for dissolving or suspending in liquid prior to injection. Alternatively, the peptide can be encapsulated in a carrier or bound to the surface of a carrier such as a nanoparticle. The active ingredients can be mixed with pharmaceutically acceptable and compatible with the active ingredient excipients. Suitable excipients are, for example, water, saline (e.g. phosphate buffered saline), dextrose, glycerol or ethanol, etc. and combinations thereof.

[0199] In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents and / or pH buffering agents. Buffer salts include phosphate, citrate, and tris. Sodium chloride and / or potassium chloride can be used to adjust the pH. To stabilize, disaccharides such as sucrose or trehalose can be used.

[0200] In the composition, the relative proportions of the peptides can be about 1 : 1 : 1. Alternatively, the relative proportions of each peptide can be varied, for example, if one peptide is found to be more effective in a particular HLA type than the others.

[0201] After formulation, the composition can be placed in a sterile container, which is then sealed and stored at low temperature, for example 4°C, or it can be freeze-dried.

[0202] Conveniently, the composition is prepared as a lyophilized (freeze-dried) powder. Lyophilization allows long-term storage in a stable form. Lyophilization procedures are well known in the art, see for example http: / / www.devicelink.com / ivdt / archive / 97 / 01 / 006.html. Typically a bulking agent such as mannitol, dextran or glycine is used prior to freeze-drying.

[0203] The composition can be administered in a convenient way, for example, by oral, intravenous, intramuscular, subcutaneous, sublingual, intranasal, intradermal or suppository routes or by implantation (for example using slow release molecules).

[0204] The composition can be preferably administered by intranasal, subcutaneous or intradermal routes. In one aspect, it can be administered by a transdermal patch.

[0205] The peptides or compositions described herein are generally administered in an "effective amount"; that is, in an amount effective to elicit any one or more therapeutic or prophylactic effects. One of skill in the art would be able to determine an effective, non-toxic amount to include in a pharmaceutical composition or to administer for a desired result through routine experimentation. Generally, a peptide or composition as disclosed herein can be administered in a manner compatible with the dosage route and the physiological condition of the recipient (including health conditions) in such a way as to elicit the desired effect (i.e., therapeutically effective and / or protective). For example, a suitable dose of a composition can depend on a variety of factors, including, but not limited to, the physiological characteristics of the subject (e.g., age, weight, sex) and other factors identifiable by one of skill in the art. Other exemplary examples of common considerations that need to be taken into account in determining, for example, a suitable dose 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).

[0206] The peptides and compositions of the present application can be used to treat a human subject. The subject can have uveitis. The subject can have S-Ag autoreactive T cells.

[0207] The subject can express an HLA haplotype that is associated with a predisposition to produce excess S-Ag specific T cells. Methods for determining the HLA haplotype of an individual are known in the art. In one aspect, the subject has an HLA gene selected from the group consisting of A29, B51, B27, DR8, DR4, DP5, DR4, DQA3, DR3, DR2, DR51, and DR17 (see Mattapallil et al. J Immunol 2011, 187: 1977-1985).

[0208] Dose escalation regimen

[0209] In a preferred embodiment, the peptides or compositions of the application can be administered to a subject using a "dose escalation" regimen, in which multiple doses are administered to the subject at increasing concentrations. Such an approach has been used, for example, in the immunotherapeutic application of phospholipase A2 peptides 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).

[0210] In one aspect, the dose escalation regimen can comprise administering to the subject a tolerogenic peptide at:

[0211] Day 1 : first dose, about 15 to about 40 μg;

[0212] Day 14 ± 7 days: second dose, about 35-65 μg;

[0213] Day 28 ± 7 days: third dose, about 80-120 μg;

[0214] Day 42 ± 7 days: fourth dose, about 300-500 μg;

[0215] Day 56 ± 7 days: fifth dose, about 300-1800 μg;

[0216] Day 70 ± 7 days: sixth dose, about 300-1800 μg;

[0217] Day 84 ± 7 days: seventh dose, about 300-1800 μg;

[0218] Day 98 ± 7 days: eighth dose, about 300-1800 μg;

[0219] Day 112 ± 7 days: ninth dose, about 300-1800 μg; and

[0220] Day 126 ± 7 days: tenth dose, about 300-1800 μg.

[0221] In one aspect of the application described herein, the fifth through tenth doses can be about 600-1500 μg.

[0222] "± 7 days" means that the specified date (i.e. the specified date after the first administration of the peptide as day 1), the administration can occur up to 7 days before (including 7 days), or up to 7 days after (including 7 days) the specified date. Thus, the administration can be performed 7, 6, 5, 4, 3, 2 or 1 day before the specified date, or 1, 2, 3, 4, 5, 6 or 7 days after the specified date.

[0223] In one aspect of the application described herein, "±7 days" preferably means ±3 days. That is, the administration can be performed up to three days before (inclusive) or up to three days after (inclusive) the specified date. Thus, the administration can be performed 3, 2 or 1 day before the specified date, or 1, 2 or 3 days after the specified date.

[0224] The peptide can be administered in the following doses:

[0225] Day 1 : first dose, about 25 μg;

[0226] Day 14: second dose, about 50 μg;

[0227] Day 28: third dose, about 100 μg;

[0228] Day 42: fourth dose, about 400 μg;

[0229] Day 56: fifth dose, about 800 μg;

[0230] Day 70: sixth dose, about 800 μg;

[0231] Day 84: seventh dose, about 800 μg;

[0232] Day 98: eighth dose, about 800 μg;

[0233] Day 112: ninth dose, about 800 μg; and

[0234] Day 126: tenth dose, about 800 μg.

[0235] In one aspect, the fifth through tenth doses can also be about 400 μg.

[0236] In an alternative aspect, the fifth through tenth doses can also be about 1600 μg.

[0237] Thus, the peptide can be administered in the following doses:

[0238] Day 1 : first dose, about 25 μg;

[0239] Day 14: second dose, about 50 μg;

[0240] Day 28: third dose, about 100 μg;

[0241] Day 42: fourth dose, about 400 μg;

[0242] Day 56: fifth dose, about 400 μg;

[0243] Day 70: sixth dose, about 400 μg;

[0244] Day 84: Seventh dose, about 400 μg;

[0245] Day 98: Eighth dose, about 400 μg;

[0246] Day 112: Ninth dose, about 400 μg; and

[0247] Day 126: Tenth dose, about 400 μg.

[0248] Alternatively, the peptide can be administered in the following doses:

[0249] Day 1 : First dose, about 25 μg;

[0250] Day 14: Second dose, about 50 μg;

[0251] Day 28: Third dose, about 100 μg;

[0252] Day 42: Fourth dose, about 400 μg;

[0253] Day 56: Fifth dose, about 1600 μg;

[0254] Day 70: Sixth dose, about 1600 μg;

[0255] Day 84: Seventh dose, about 1600 μg;

[0256] Day 98: Eighth dose, about 1600 μg;

[0257] Day 112: Ninth dose, about 1600 μg; and

[0258] Day 126: Tenth dose, about 1600 μg.

[0259] In another aspect of the application as described herein, an eleventh dose of about 300-1800 μg, preferably about 600-1500 μg, preferably about 1200 μg, more preferably about 400, 800 or 1600 μg is administered on day 140 ± 7 days, preferably day 140 ± 3 days, more preferably day 140. In a preferred aspect the dose is about 800 μg.

[0260] In a more preferred aspect, a twelfth dose of about 300-1800 μg, preferably about 600-1500 μg, preferably about 1200 μg, more preferably about 400, 800 or 1600 μg is administered on day 154 ± 7 days, preferably day 154 ± 3 days, more preferably day 154. In a preferred aspect the dose is about 800 μg.

[0261] In a further preferred aspect, the thirteenth dose of about 300-1800 μg, preferably about 600-1500 μg, preferably about 1200 μg, more preferably about 400, 800 or 1600 μg is administered on day 168 ± 7 days, preferably day 168 ± 3 days, more preferably day 168. In a preferred aspect the dose is about 800 μg.

[0262] In a further preferred aspect, the fourteenth dose of about 300-1800 μg, preferably about 600-1500 μg, preferably about 1200 μg, more preferably about 400, 800 or 1600 μg is administered on day 182 ± 7 days, preferably day 182 ± 3 days, more preferably day 182. In a preferred aspect the dose is about 800 μg.

[0263] Additional doses as described above can be given as required, for example, for a period of one month to twenty years, for example, for a period of one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, ten years, eleven years, twelve years, thirteen years, fourteen years, fifteen years, sixteen years, seventeen years, eighteen years, nineteen years or twenty years.

[0264] Any teaching herein in relation to a "method" of the application equally applies to a "use" encompassed by the application.

[0265] Kit

[0266] Peptides derived from S-Ag can be administered together in a mixed composition or mixture. However, in certain cases, it is preferred to provide the peptides separately in a kit for simultaneous, separate, sequential or combined administration.

[0267] For example, the kit can contain the peptides in separate containers. The contents of the containers can or can not be combined prior to administration.

[0268] The kit can also contain mixing and / or administration devices (e.g. a nebulizer for intranasal administration; or a syringe and needle or other medical device for subcutaneous / intradermal administration). The kit can also include instructions for use.

[0269] The pharmaceutical composition or kit of the application can be used in the treatment and / or prevention of a disease, for example uveitis as discussed herein.

[0270] In particular, the composition / kit can be used to inhibit or prevent 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. Examples

[0271] Example 1 - Peptides 9K1K, 17JK and 15N3K are correctly loaded on MHC II molecules on dendritic cells in vivo.

[0272] When 100 μg of peptide 9K1K was injected into DR2tg mice or peptides 17JK or 15N3K were injected into DR3tg mice, these peptides could be detected on dendritic cells (CD11c+ cells) in the spleen of these mice 2 hours after injection. Activation (production of IFNy) of peptide-specific CD4+ T cells by isolated CD11c+ cells was used as readout.

[0273] In Figure 1 , Figure 2 and Figure 3 , it is clear that CD11c+ cells isolated from the spleen of mice injected with 9K1K, 17JK or 15N3K, respectively, induced a significant CD4+ T cell activation compared to CD11c+ cells in the spleen of control mice injected with PBS. These results indicate that peptides 9K1K, 17JK and 15N3K reached dendritic cells in the secondary lymphoid organs. Moreover, these peptides bound to MHC II molecules on these dendritic cells in the correct conformation. Therefore, these peptides were able to induce tolerance.

[0274] Example 2 - Single peptide treatment induces S-Ag specific T cell tolerance

[0275] We previously demonstrated the tolerogenic effect of single peptides 9K1K, 17JK and 15N3K, which results were confirmed in these experiments. HLA-DR transgenic mice were treated with one peptide alone according to a dose escalation regimen.

[0276] In a first experiment ( Figure 4 ), DR2tg mice received 9K1K peptide or PBS as described in the methods section. Pre-treatment with this modified apitope reduced S-Ag induced cell activation in the lymph nodes ( Figure 4 A) and spleen ( Figure 4 B) by 77% and 70%, respectively.

[0277] To confirm the tolerogenic capacity of 17JK, DR3tg mice were treated with this modified apitope or PBS. This experiment showed that pre-treatment with 17JK reduced S-Ag induced cell activation in the lymph nodes ( Figure 5 A) and spleen ( Figure 5 B) by 78% and 84%, respectively.

[0278] Pre-treatment of DR3tg mice with peptide 15N3K resulted in a reduction of S-Ag induced cell activation in the lymph nodes ( Figure 6 A) and spleen (Figure 6 B) S-Ag-induced cell activation was reduced by 61% and 72%, respectively.

[0279] Example 3 - Combination peptide treatment induces S-Ag-specific T cell tolerance

[0280] The results of the study investigating the reduction of S-Ag-specific immune activation in DR3tg mice with peptide treatment with the individual peptides 9K1K, 17JK or 15N3K led to the investigation whether a combination treatment with the peptides as a mixture could also reduce S-Ag-specific responses. DR3tg and DR2tg mice were treated with ATX975 according to a dose escalation regimen and immunized with an emulsion containing all three antigens in CFA. The results in DR3tg and DR2tg mice are shown in Figure 7 and Figure 8

[0281] After ATX975 treatment of DR3tg mice (Figure 2), S-Ag-induced cell activation in the spleen was reduced by 95% and 89% in two independent experiments. The tolerance induced by ATX975 was more pronounced compared to the tolerance induced by peptide 17JK alone (reduction of S-Ag-induced cell activation in the spleen by 80% and 52% in two independent experiments) or peptide 15N3K alone (reduction of S-Ag-induced cell activation in the spleen by 74%). Figure 7

[0282] When DR2tg mice were treated with ATX975 (Figure 3), S-Ag-induced cell activation in the spleen of the treated mice was reduced by 78% compared to control mice. This tolerance induction was more pronounced than the reduction of S-Ag-induced cell activation by 51% after treatment with 9K1K alone. Figure 8

[0283] These data clearly indicate an additive effect of the single peptides when treated as a peptide mixture in DRtg mice.

[0284] Example 4 - Peptides 9K1K, 17JK and 15N3K show different binding patterns in an in vitro peptide-MHC II binding assay

[0285] Figure 9 ​​​IC50 (pM) values are described for peptides 9K1K, 17JK and 15N3K binding to and competing with known competitor peptides for the indicated HLA-DR molecules. The values should only be compared within each HLA-DR molecule. Low IC50 values (green) indicate the strongest binder for each HLA-DR molecule. The highest value for each HLA-DR molecule (red) indicates the lowest binder for that HLA-DR molecule. Intermediate values are indicated in orange. These results show that the three peptides 9K1K, 17JK and 15N3K have different binding profiles for the HLA-DR molecules studied. Therefore, combining these peptides into a cocktail therapy can allow treatment to be not restricted to a certain HLA-DR type.

[0286] SUMMARY

[0287] We have identified a peptide mixture, ATX975, which can comprise three peptides from the S-Ag protein and is able to induce tolerance to S-Ag in HLA-DR transgenic mice.

[0288] Example 5 - Variants of peptide 15N3K as apitopes

[0289] Apitopes (antigen processing independent epitopes) are able to bind to MHC II molecules and stimulate the response of SAg-specific T cells without the need for further antigen processing. Variants of peptide 15N3K (SEQ ID NO: 3) were tested in an in vitro antigen processing independent presentation system (APIPS) assay for their ability to bind to MHC II molecules and be presented to T cell hybridomas without the need for further antigen processing. In other words, peptides consisting of parts of peptide 15N3K and peptides with varying degrees of sequence identity to peptide 15N3K were tested for their ability to act as apitopes.

[0290] The peptides tested are shown in the table below:

[0291]

[0292]

[0293] The response of T cells to each peptide in the APIPS assay was measured by IL-2 secretion as shown in the table below. Figure 10 All peptides except the three peptides are apitopes.

[0294] Materials and Methods

[0295] Mice

[0296] Throughout the peptide identification and epitope development process, HLA-DR transgenic mice were used to ensure that the peptide-MHC class II binding motif is the therapy required for tolerising therapy for patients with uveitis.

[0297] DR3tg mice were bred by an external facility under specific pathogen-free conditions at Charles River, UK or Innoser, Belgium. The DR3tg strain was originally established by Strauss et al. (Strauss et al., 1994, Immunogenetics 3, 104-108). Briefly, the genomic construct used was a 6 kb Ndel fragment of the HLA-DRA genomic clone in pUC 13 and a 24 kb ClalxSaII fragment of cos 4.1, a cosmid containing the B gene of DRB1*0301 (pTCF). Solutions containing 1-2 pg / mL of each construct were used for coinjection into fertilized eggs of (C57BL / 6 x DBA / 2) Fl donors mated to C57BL / 6 males. The offspring were subsequently bred into an IA-beta knockout C57BL / 6 genetic background (AB0 mice) that lacks expression of mouse MHC class II molecules. These DR3tg mice express the HLA-DRB1*0301 molecule, but not the mouse MHC-II molecule. Mice were maintained by backcrossing to C57BL / 6 and B10.Q. Transgenic mice were identified by Southern blot analysis of EcoRI-digested tail DNA and probing with a 1.35 kb BamHI fragment of DRA cDNA and a 1.25 kb BamHI fragment of DRB1*0301 cDNA.

[0298] DR2tg mice were bred by an external facility under specific pathogen-free conditions at Charles River, UK or Innoser, Belgium. HLA-DR2 transgenic (DR2tg) mice were originally obtained from Lars Fugger (Madsen et al., 1999). Briefly, the DRa and DRb chain cDNAs (DRA*0101 and DRB1*1501) were expressed using the pDOI-5 expression vector containing the mouse MHC II promoter. The construct was injected into fertilized eggs from (DBA / 2 x C57BL / 6) Fl matings. Mice were backcrossed into an IA-beta knockout C57BL / 6 genetic background (AB0 mice) that lacks expression of mouse MHC class II molecules. DR2tg mice express the HLA-DRB1*1501 molecule, but not the mouse MHC molecule.

[0299] Animal studies were approved by the “Ethical Committee for Animal Experiments” (ECD) of Hasselt University and were performed in a pathogen-free facility with the highest standard of care.

[0300] Antigen

[0301] All peptides were synthesized using Genscript (Piscataway, USA) and stored at -80°C in stock solutions of 20 mg / ml DMSO (Sigma-Aldrich) or 4 mg / ml PBS (Lonza). Peptides were synthesized using N-terminal free amines and C-terminal amides. Human S-Ag (S-repressor protein) was produced in HEK293F cells (QBiologicals, Eurofins Amatsigroup, Ghent, Belgium).

[0302] In vivo MHC Class II loading test

[0303] 100 μg of peptide in 100 μl PBS was subcutaneously (sc) injected into the flank of DR3tg or DR2tg mice. Control animals received a subcutaneous injection of 100 μl PBS. Two hours later, spleens were harvested and single-cell suspensions were prepared. CD11c+ cells were positively selected using CD11c microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). A mean purity of >92% was achieved. Cells were incubated in 96-well round-bottom plates with 1x10 cells 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+ cells were co-cultured together at 1x10 5 CD11c+ cells. These CD4+ cells were isolated from DR3tg or DR2tg mice subcutaneously immunized at the tail base with 50 μg of peptide emulsified in CFA (peptide / CFA). Ten days post-immunization, draining lymph nodes (LNs) and spleens were harvested. LN cells and spleen cells were isolated, and CD4+ T cells were isolated by negative selection using the Magnisort Mouse CD4 Isolation Kit (ThermoFisherScientific) according to the manufacturer's instructions. After 72 hours, the supernatant of these co-cultures was collected, and CD4+ T cell activation was analyzed by IFNγ ELISA (R&D Systems, Abingdon, UK). The response of CD4+ T cells to in vitro added peptides was evaluated in parallel experiments to ensure that T cells recognized peptides presented by CD11c+ cells.

[0304] In vitro tolerance experiment

[0305] DR3tg or DR2tg mice were subcutaneously injected with 0.015 nmol, 0.15 nmol, and 1.5 nmol peptides (dose escalation schedule) into the flank region on days -15, -13, and -11, respectively, followed by three 15 nmol peptide injections on days -8, -6, and -4. Indicative doses were used for single-peptide treatment; for treatment using a mixture of three peptides contained in ATX975, these doses were administered per peptide (up to a total maximum dose of 45 nmol peptide). Control mice received the same dose of an unrelated peptide capable of binding HLA-DR2 or HLA-DR3, depending on the mouse strain used. On day 0, mice were subcutaneously immunized at the tail base with 150 μg of antigen emulsified in CFA (50 μg of each 30-mer peptide containing its respective epitope) (peptide / CFA). Ten days post-immunization, draining lymph nodes (LNs) 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 mL M glutamine, 50 U / mL penicillin and 50 U / mL streptomycin; Lonza and 50 mM β-mercaptoethanol; Gibco). To investigate antigen-induced cell activation, 0.5 x 10⁻⁶ cells were cultured in 96-well round-bottom plates. 6 Cells / well (200 μl / 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 vaccine, Copenhagen, Denmark. After 72 hours, the supernatant was collected and stored at -80°C until further analysis. IFN-γ concentration in the supernatant was assessed and cell activation was measured by cytokine ELISA (R&D Systems, Abingdon, UK).

[0306] peptide sequence

[0307] Peptide Sequence SEQ ID NO: 9K1K KKKAFVEQVANVVLKKK SEQ ID NO: 1 17JK KKKLTFRRDLYFSRVQVYKKK SEQ ID NO: 2 15N3K KKKVIFKKISRDKSVTIYLGKKK SEQ ID NO: 3

[0308] Peptide-MHC Class II Binding Assay

[0309] 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 performed by ProImmune (Oxford, UK) using their cell-free MHC class II REVEAL binding assay.

[0310] Antigen Processing Independent Presentation System (APIPS) Assay

[0311] Antigen-specific hybridoma clones were tested for reactivity to peptides presented by fixed or non-fixed (fresh) cells (APC). 5 x 105cells were incubated with 10 μg / ml and 25 μg / ml of peptide and 5 x 105 4 Fixed or fresh APC were incubated for 5 x 105 4 cells. To fix the APC, 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 the addition of 0.4 M glycine (Sigma-Aldrich) and washing the cells in RPMI-10% FCS. 48 h later, the antigen-induced IL-2 production was measured by ELISA (R&D Systems, Abingdon, UK).

Claims

1. A composition comprising the following S-repressor peptide: A peptide composed 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 composed of the amino acid sequence KKKVIFKKISRDKSVTIYLGKKK (SEQ ID NO:3); In the composition, the relative ratio of peptides is 1:1:

1.

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

3. The composition according to claim 1, used for treatment.

4. The composition according to claim 1, used in subjects for the treatment and / or prevention of uveitis.

5. Use of the composition according to claim 1 in the preparation of a medicament for treating and / or preventing uveitis in a subject.

6. The use as described in claim 5, wherein the subject is HLA-DR3.

7. The use as described in claim 5, wherein the subject is HLA-DR2.

8. The use as claimed in claim 5, wherein the composition is administered in a dose-escalation regimen.

9. The use as described in claim 8, wherein the dose escalation scheme comprises the following doses: Day 1: First dose, 15 to 40 μg; Day 14 ± 7: Second dose, 35-65 μg; Day 28 ± 7: Third dose, 80-120 μg; Day 42 ± 7: Fourth dose, 300-500 μg; Day 56 ± 7: Fifth dose, 600-1500 μg; Day 70 ± 7: Sixth dose, 600-1500 μg; Day 84 ± 7: Seventh dose, 600-1500 μg; Day 98 ± 7: Eighth dose, 600-1500 μg; Day 112 ± 7: Ninth dose, 600-1500 μg; and Day 126 ± 7: Tenth dose, 600-1500 μg.

10. The use as described in claim 9, Wherein 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.

11. The use as described in claim 9 or 10, The eleventh dose, administered at 600-1500 μg, was given on day 140 ± 7; and / or The twelfth dose, consisting of 600-1500 μg, was administered on day 154 ± 7; and / or The thirteenth dose, consisting of 600-1500 μg, was administered on day 168 ± 7.

12. The use as described in claim 11, wherein the fifth, sixth, seventh, eighth, ninth, and tenth doses, and optionally the eleventh, twelfth, and thirteenth doses, are each 800 μg.

13. The use as claimed in claim 5, wherein the composition is administered intradermally.

14. The use as claimed in claim 5, wherein the composition is administered to a human.

15. A kit comprising the composition defined in claim 1.

16. The kit of claim 15, for the prevention or treatment of uveitis by simultaneous, individual or sequential administration of the peptide consisting of SEQ ID NO:1, the peptide consisting of SEQ ID NO:2, and the peptide consisting of SEQ ID NO:3.

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