Tolerogenic peptide

Abstract

The present disclosure is based in part on research related to novel tolerogenic peptides derived from proteins expressed by pancreatic cells, developed for use in antigen-specific immunotherapy for type 1 diabetes. Disclosed are tolerogenic peptides that can bind to MHC class II molecules without relying on antigen processing, and are derived from proteins expressed by pancreatic cells, for use in the treatment of type 1 diabetes.

Description

Technical Field

[0001] The present disclosure relates to tolerogenic peptides and their use in the treatment or prevention of autoimmune diseases.

Background Art

[0002] Type 1 diabetes is a chronic autoimmune condition resulting from the destruction of insulin-secreting pancreatic β-cells (Atkinson et al., 2014). This is evidenced by studies in mouse models in which type 1 diabetes can be reproduced through the transfer of T cell clones specific for β-cell proteins (Peterson and Haskins, 1996). Similarly, in humans, the type 1 diabetic pancreas is characterized by the infiltration of CD8 and CD4 T cells targeting specific β-cell-derived proteins (Babon et al., 2016). Therapies for type 1 diabetes exist in the form of insulin injection therapy, but it is difficult and incomplete to titrate the amount of insulin replacement therapy to reproduce the physiological insulin secretion profile. Another therapeutic strategy attempts to regulate the function of β-cell antigen-specific T cells to delay the progression of type 1 diabetes. Initial trials with non-specific immunosuppressive agents resulted in improved diabetes management (Feutren et al., 1986). In more recent studies where T cells are targeted with non-antigen-specific immunomodulatory monoclonal antibodies against CD3, β-cell function has been maintained in newly diagnosed type 1 diabetes patients, where it has been demonstrated that residual β-cell function is preserved, leading to recent US FDA approval (Herold et al. 2019, Mullard 2022). However, non-antigen-specific approaches are associated with side effects related to systemic immunosuppression, such as the risk of reactivation of latent viral infections in T1D patients after B cell depletion (Kroll et al., 2013). Therefore, there is a need to develop more selective immunosuppressive therapies that target β-cell-specific autoreactive T cells.

[0003] Previous studies have shown that injecting precisely identified T cell epitopes into animal models can generate T cells that can migrate to target organs and suppress autoimmune processes at those sites to regulate the onset of clinical disease (Burton et al., 2014). A variety of immunomodulatory peptides for use in the treatment of autoimmune diseases such as type 1 diabetes have been disclosed in the art. WO1994004557A1 discloses immunomodulatory peptides comprising segments of naturally occurring human proteins that bind to human major histocompatibility complex class II allotypes. US20070026465A1 discloses a method for detecting GAD65-specific T cells for predicting the onset of type 1 diabetes. US20040234531A1 discloses that a chimeric molecule comprising an IILA-DR element linked to an epitope of GAD65, an antigen associated with autoimmune diabetes, stimulated the secretion of the inhibitory cytokine IL-10 from CD4 T cells of type I diabetic patients. US20090305340A1 discloses a modified GAD65 composition that antagonizes the activity of islet-specific T cells that contribute to the progression of one or more autoimmune disorders. GB2454687A discloses MHC class II-binding peptides derived from GAD65, where these peptides can be used to produce MHC peptide complexes or to generate antibodies or CD4+ cell lines specific for these peptides. However, not all T cell epitopes derived from an antigen will induce tolerance in autoreactive T cells against the autoantigen. In order to use peptides in antigen-specific immunotherapy, it is very important to develop peptides that accurately mimic naturally processed antigens such that the peptides can bind to MHC class II molecules without further antigen processing and associate with autoreactive T cells against the autoantigen.

[0004] Among the objects of the present disclosure is to develop novel tolerogenic peptides that mitigate one or more of the aforementioned disadvantages of existing treatments for type 1 diabetes. SUMMARY OF THE INVENTION

[0005] The present disclosure is based in part on research related to novel tolerogenic peptides derived from proteins expressed by pancreatic cells, developed for use in antigen-specific immunotherapy for type 1 diabetes.

[0006] In a first aspect, there is provided a tolerogenic peptide capable of binding to MHC class II molecules without dependence on antigen processing, for use in the treatment of type 1 diabetes, said peptide being derived from a protein expressed by splenocytes.

[0007] Type 1 diabetes is a chronic autoimmune disease primarily mediated by the destruction of pancreatic β-cells by autoreactive CD4 and CD8 T cells. Treatment of type 1 diabetes typically includes insulin replacement therapy and, optionally, other therapies that attempt to control the immune response in a non-specific manner to restore β-cell function or prevent the destruction of β-cells. However, there is a need to develop immunotherapies that selectively target autoreactive immune cells while not affecting other aspects of immune function.

[0008] The principle underlying antigen-specific immunotherapy is that exposure in the environment to antigens that are tolerogenic (rather than inflammatory) will educate T cells such that subsequent T cell responses to those antigens will be downregulated (rather than activated). Downregulated T cell responses include the secretion of anti-inflammatory cytokines (Burton et al., 2014), which can act locally to downregulate other nearby T cells. This is the concept recognized as bystander suppression (Wraith, 2016). The inventors have developed novel tolerogenic peptides derived from GAD65 that bind to major histocompatibility complex (MHC) class II molecules and induce T cell-mediated immune tolerance without depending on antigen processing. Importantly, these tolerogenic peptides of the present disclosure bind to MHC class II molecules of antigen-presenting cells without the need for further antigen processing. If a peptide is too long to bind to the peptide-binding groove of the MHC molecule without further processing, or binds in an inappropriate conformation, it will not be tolerogenic in vivo. Since not all T cell epitopes induce tolerance to self-antigens, it is very important to identify specific T cell epitopes that mimic the conformation of native processed antigens that can be presented on MHC class II molecules in order to downregulate autoreactive CD4+ T cells.

[0009] The term "tolerogenic", as used herein, means capable of inducing tolerance to a specific antigen. Immune tolerance refers to a range of host processes that prevent potentially harmful immune responses and result in a non-responsive state of the immune system to substances and / or tissues (s) capable of inducing an immune response. Immune tolerance is a highly regulated process that enables the discrimination of self from non-self, the suppression of allergic reactions, and the prevention of reactive immune responses by the maternal immune system to fetal antigens.

[0010] Self-tolerance is the ability to prevent an immune response against self-generated antigens. When the immune system induces an abnormal immune response against self-antigens, autoimmune diseases can occur. For example, in autoimmune conditions such as type 1 diabetes, the individual lacks immune tolerance to their own self-antigens, and the body does not accurately distinguish between self-antigens and non-self antigens.

[0011] The inventors assume that administration of soluble peptide analogs can render it possible to induce immunological tolerance to specific T cell epitopes. In a normal adaptive immune response, T cells recognize internal epitopes of protein antigens. Antigen-presenting cells (APCs) internalize protein antigens and degrade them into short fragments (antigen processing). Peptides can bind to MHC class I or II molecules inside the cell for presentation on the cell surface. Peptides presented on MHC molecules can be recognized by T cells, in which case the peptide is a T cell epitope. Using such epitopes in the form of peptides, it is possible to induce immunological tolerance, and these are referred to throughout the present disclosure as tolerogenic peptides.

[0012] Tolerance can result from, or be characterized by, induction of anergy in at least a subset of CD4+ T cells. To activate a T cell, the peptide must associate with a professional APC capable of delivering two signals to the T cell. The first signal is delivered by the MHC-peptide complex on the cell surface of the APC and is received by the T cell through the T cell receptor. The second signal is delivered by costimulatory molecules on the APC surface, such as CD80 and CD86, and is received by CD28 on the T cell surface. When a T cell receives the first signal in the absence of the second signal, it is thought to become inactivated and anergic. Anergic T cells are resistant to subsequent antigen exposure and may be able to suppress other immune responses. Anergic T cells are thought to be involved in mediating T cell tolerance. Peptides that can bind to class II MHC molecules without antigen processing would be able to bind to MHC molecules on immature APCs. Thus, they are likely to be presented to T cells without costimulation, leading to T cell anergy and tolerance.

[0013] Peptides that require processing before they can be presented in combination with MHC molecules do not induce tolerance because they must be processed by mature APCs. Mature APCs (e.g., macrophages, B cells, and dendritic cells) can process antigens but can also deliver both the first and second signals to T cells, resulting in T cell activation. Peptides of the correct size and conformation that can bind to MHC molecules without antigen processing, such as those described herein, are likely to bind to MHC molecules on immature APCs and induce T cell anergy.

[0014] When used herein, the term "peptide" can refer to a fragment of a wild-type sequence or a reference sequence, such as a fragment up to 40 amino acids in length. In one embodiment, a peptide as described herein for use in the treatment of type 1 diabetes can include a fragment of a wild-type sequence or a reference sequence that is at least 5 amino acids in length. In some embodiments, a peptide as described herein can be between 5 and 40 amino acids in length. In some embodiments, a peptide as described herein can be between 5 and 20, 25, 30, or 35 amino acids in length. In any of these embodiments, the peptide is derived from GAD65 and includes at least a minimal epitope. An epitope is a peptide that can bind to the peptide-binding groove of an MHC class I or II molecule and can be recognized by T cells. A minimal epitope is a peptide that can bind to the peptide-binding groove of an MHC class I or II molecule and can be recognized by T cells.

[0015] The term "protein expressed by pancreatic cells" refers to any protein expressed by pancreatic cells that is found to be expressed intracellularly and / or can be membrane-bound. The protein can be differentially expressed (e.g., overexpressed or underexpressed) in pancreatic cells from a subject with type 1 diabetes or by a subject with type 1 diabetes, as compared to the same pancreatic cells from a subject without type 1 diabetes. In some cases, the protein expressed by pancreatic cells can refer to a protein synthesized by pancreatic cells for secretion. The term "pancreatic cells" refers to any cells found in the pancreas, such as α or β cells found in pancreatic islets.

[0016] In one embodiment, a tolerogenic peptide derived from a protein expressed by pancreatic cells can include a fragment of glutamic acid decarboxylase 65 (GAD65) or a variant thereof.

[0017] In one teaching, the present disclosure provides a tolerogenic peptide that binds to MHC class II molecules without further antigen processing, where the peptide comprises a fragment of GAD65, and presentation of the tolerogenic peptide-MHC class II complex to CD4+ T cells induces a tolerogenic response. In one embodiment, the tolerogenic peptide can comprise a wild-type sequence or a fragment of a reference GAD65 sequence that is at least 5 amino acids in length. In one embodiment, the tolerogenic peptide can comprise a fragment of GAD65 that is 5 to 40 amino acids in length. In some embodiments, the fragment can be between 5 and 20, 25, 30, or 35 amino acids in length.

[0018] The wild-type sequence or reference sequence of GAD65 from which the tolerogenic peptide is derived may correspond to the species of the subject to which the tolerogenic peptide is provided. In one embodiment, the preferred wild-type or reference GAD65 sequence from which the tolerogenic peptide is derived is mammalian, such as the human GAD65 sequence. In another embodiment, the wild-type or reference GAD65 sequence from which the tolerogenic peptide is derived may be, for example, from a mouse, rat, dog, cat, pig, sheep, or horse GAD65 sequence.

[0019] "Peptide," as used herein, may also include variants of the fragment(s) of the wild-type sequence or reference sequence, and the variant may include one or more amino acid modifications. In one embodiment, the variant may include up to 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modifications. In one embodiment, the variant may include at least 2, 3, 4, or 5 modifications. The term "peptide," as used herein, includes peptides obtained from peptides derived from naturally occurring proteins, or may be a peptide synthesized by synthetic methods. The term "peptide derived from a protein" refers to a peptide obtained by cleavage of a naturally occurring protein.

[0020] In some embodiments, the tolerogenic peptide can include a variant peptide that exhibits at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence found in the wild-type or reference sequence of GAD65. Any of the peptides of GAD65 as described herein that are capable of binding to MHC class II molecules and inducing immune tolerance without relying on antigen processing can be provided for use in the treatment of type 1 diabetes.

[0021] Amino acid modifications can refer to substituting a wild-type or reference amino acid with another amino acid. Such substitutions may be conservative in that they exchange the wild-type residue with another residue of the same or similar structural, chemical and / or physicochemical properties. "Conservative" amino acid substitutions can be made based on the similarity of the polar, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0022] Naturally occurring peptides or proteins contain L-configured amino acids. In some instances, the amino acid modifications of the present disclosure may include substitution with the equivalent D-configured amino acid or conservative amino acid substitution with D-amino acids. In other examples, the amino acid modification may include substitution with non-conventional amino acids, such as citrulline, hydroxyproline, β-alanine, ornithine, norleucine, 3-nitrotyrosine, pyroglutamic acid, nitroarginine, etc., provided that the tolerogenic peptide retains the ability to bind to MHC class II molecules independent of antigen processing. Alternatively, the peptide sequence may include modified amino acids, such as homoamino acids, β-homoamino acids, N-methyl amino acids and / or α-methyl amino acids.

[0023] Substitutions may also be "non-conservative" in that the wild-type residue is replaced with a different class of amino acids, such as amino acids that are structurally dissimilar, chemically distinct, and / or physicochemically different or dissimilar.

[0024] Amino acid modifications may include deletion of amino acid residues from the wild-type sequence or reference sequence. Other amino acid modifications may include insertion of one or more amino acids into the wild-type sequence or reference sequence. Amino acid modifications may further include inversion of a particular portion or part of the wild-type / reference sequence.

[0025] Optionally, or in addition to the above modifications, the peptides as described herein may be appropriately modified by those skilled in the art using methods known in the art, for example, one or more pharmacokinetic properties of the tolerogenic peptide may be modified. In one embodiment, the tolerogenic peptide may be modified to increase its solubility. Subsequently, hydrophilic lysine residues may be added to the peptide to increase the solubility of the core epitope. In one embodiment, the amino acid modification may include the addition or substitution of wild-type or reference residues with one or more amino acids selected from lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine and / or glutamine, which are one or more amino acid modifications that increase the solubility of the peptide. In some embodiments, the amino acid modification for increasing the solubility of any one or more tolerogenic peptides as disclosed herein may include the addition or substitution of wild-type residues with lysine. In one embodiment, the modified tolerogenic peptide with improved solubility has a grand average of hydropathy (GRAVY) score lower than 0.

[0026] The peptide may also exhibit excellent bioavailability in vivo. The peptide may maintain in vivo a conformation that allows it to bind to MHC molecules at the cell surface without the corresponding interference.

[0027] In summary, the tolerogenic peptide may comprise any one or more of the disclosed sequences in Tables 1-3. In one embodiment, a tolerogenic peptide for use in the treatment of type 1 diabetes comprises, consists essentially of, or consists of a sequence that is identical to any one of the "minimal" peptide sequences in Tables 1-3, where the peptide may further comprise one or more additional modifications according to any of the modifications described herein. "Consisting essentially of" or "consisting of" means that as long as the peptide retains the ability to bind to MHC class II molecules independently of antigen processing, any peptide sequences from GAD65 as disclosed herein do not necessarily form a part of the peptide that functions as a core epitope sequence, and may contain additional stretches of amino acids located at the N and / or C termini.

[0028] Table 1. Set I of Peptide Sequences Derived from GAD65 [Table 1]

[0029] Table 2. Set II of Peptide Sequences Derived from GAD65 [Table 2]

[0030] Table 3. Set III of Peptide Sequences Derived from GAD65 [Table 3]

[0031] In certain embodiments, the tolerogenic peptide can comprise the amino acid sequences IFSPGGAISNMYAMMIARFKMFPEVKEKGMA, DLERRILEAKQKGFVPFLVSATAGTTVYGA, or DAAWGGGLLMSRKHKWKLSGVERANSVTWN for use in the treatment of type 1 diabetes. In one embodiment, the tolerogenic peptide can consist essentially of a fragment derived from IFSPGGAISNMYAMMIARFKMFPEVKEKGMA, DLERRILEAKQKGFVPFLVSATAGTTVYGA, or DAAWGGGLLMSRKHKWKLSGVERANSVTWN, where the fragment can optionally be modified to modify one or more pharmacokinetic properties of the tolerogenic peptide as described above and can comprise at least 5 amino acids in length. In one embodiment, the fragment can comprise 5 to 20 amino acids in length.

[0032] In one embodiment, the tolerogenic peptide can be derived from glutamic acid decarboxylase 65 (GAD65) and can comprise the sequences WKLSGVER, ISNMYAMMIA, and / or ERRILEAKQKGFVP that are capable of binding to MHC class II molecules independent of antigen processing for use in the treatment of type 1 diabetes. In one embodiment, the MHC class II molecule-binding portion of the GAD65-derived peptide can have a maximum length of 14 amino acids, and / or optionally, the peptide can further comprise up to 3 (e.g., 1, 2, or 3) additional amino acids at the N- or C-terminus, or both termini, that modify one or more pharmacokinetic properties of the peptide, such as the solubility of the peptide in an aqueous environment (e.g., blood).

[0033] The present disclosure also provides a method of treating or preventing type 1 diabetes using any one or more of the peptides as described herein, the method comprising administering a therapeutically effective amount of any of the tolerogenic peptides as described herein to a subject in need of treatment. In another embodiment, the method can comprise the step of administering a plurality of the peptides described herein.

[0034] Subjects to whom the tolerance-inducing peptides of the present disclosure are to be administered can include any human or animal subject with type 1 diabetes. The subject can also be any human or animal subject that has a predisposition and / or is susceptible to developing type 1 diabetes, where type 1 diabetes can be treated, alleviated, or prevented by using one or more tolerance-inducing peptides as described herein.

[0035] The present disclosure also relates to the use of the tolerance-inducing peptide(s) as described herein in the manufacture of a medicament for use in treating or preventing type 1 diabetes. The peptide(s) can be produced according to any method of peptide synthesis known in the art, such as techniques based on liquid-phase peptide synthesis (LPPS) or solid-phase peptide synthesis (SPSS). For example, the peptide can be synthesized by SPSS (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). For example, automated synthesis can be achieved using an ABI 431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer. Alternatively, the peptide can be made by recombinant means or by cleavage from a longer polypeptide. For example, the peptide can be obtained by cleavage from the GAD65 protein, and subsequently one or both ends thereof can be modified. The composition of the peptide(s) can be confirmed by amino acid analysis or sequencing. The peptide(s) can be produced with or subsequently combined with any substance(s) known in the art that increase the stability and / or solubility of the peptide(s).

[0036] In one teaching, a tolerogenic peptide as described herein may be provided as a pharmaceutical composition, which may optimally be formulated with at least one pharmaceutically acceptable excipient thereof. In one embodiment, acceptable excipients may be selected from water, saline (e.g., phosphate buffered saline), human serum albumin, dextrose, trehalose, sucrose, mannitol, sorbitol, polysorbate 20, polysorbate 80, glycerol, ethanol, polyethylene glycol, etc. and combinations thereof. The pharmaceutical composition may contain a therapeutically or prophylactically effective amount of one or more tolerogenic peptides as described herein. In one embodiment, the pharmaceutical composition may contain an effective amount of any one or more tolerogenic peptides or combinations thereof of the present disclosure.

[0037] The effective amount of the tolerogenic peptide refers to an amount of peptide sufficient to induce immune tolerance to an epitope and / or antigen of interest, or to minimize and / or reduce autoreactive T cell activation thereto. Typically, the tolerogenic peptide can reduce autoreactive T cell activation by at least 20%, such as 30%, 50%, 60%, 70%, 80%, or more. Using the tests described herein, and techniques known in the art, it is possible to count and test the T cell number or T cell percentage. Alternatively, a decrease in cytokine induction may be correlated with a decrease in T cell activation. The tolerogenic peptide of the pharmaceutical composition can bind to MHC class II molecules without depending on antigen processing. In some examples, the tolerogenic peptide may also bind to MHC class I molecules.

[0038] In one embodiment, the pharmaceutical composition may optionally further contain one or more pharmaceutically acceptable stabilizers, wetting agents, emulsifiers, salts, buffers and / or adjuvants known in the art.

[0039] The tolerogenic peptide may be formulated into a composition in its non-ionic form or in the form of a salt. A pharmaceutically acceptable salt refers to a salt of a compound that is pharmaceutically acceptable and possesses or can be converted into a form possessing the desired pharmacological activity of the parent compound. Such salts include, for example, acid addition salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as acetic acid, citric acid, glucoheptonic acid, lactic acid, etc.

[0040] In one embodiment, provided is a method for the treatment of type 1 diabetes, comprising administering to a subject suffering from or predisposed to developing type 1 diabetes an effective amount of a tolerogenic peptide or a pharmaceutical composition thereof as described herein.

[0041] It may also be possible to provide a tolerogenic peptide or a pharmaceutical composition as a combination therapy in combination with other types of treatment of type 1 diabetes. In one embodiment, the tolerogenic peptide of the present disclosure may be combined with one or more active ingredients and / or treatments for type 1 diabetes, such as insulin. In one embodiment, the tolerogenic peptide of the present disclosure may be combined with one or more tolerogenic peptides derived from other proteins.

[0042] In one teaching, the pharmaceutical composition may be provided in the form of a kit for use as described in the present disclosure, in which some or each of the peptide(s) is provided simultaneously, separately or in sequential administration. Alternatively or in addition, when attempting to administer the pharmaceutical composition in multiple doses, each dose may be packaged separately.

[0043] Induction of tolerance to GAD65 may be monitored in vivo by using techniques known in the art to search for a decrease in the levels of GAD65 autoantibodies, CD4+ T cells specific for GAD65, and / or B cells capable of secreting GAD65 autoantibodies.

[0044] Tolerance induction can be monitored by a variety of techniques that include anergy (detectable by continuous antigen exposure in vitro) in CD4+ T cells and / or induction of changes in the CD4+ T cell population. Changes in the CD4+ T cell population can include decreased proliferation, down-regulation of the production of IL-2, IFN-γ and / or IL-4, and increased production of IL-10.

[0045] In another teaching, the tolerogenic peptides of the present disclosure may be in the form of a kit comprising one or more tolerogenic peptides or modified tolerogenic peptides as disclosed herein. For example, these kits may include tolerogenic peptides for use in in vitro assays for the generation or detection of antigen-specific T cells. In one embodiment, the peptide(s) may be further labeled with one or more moieties selected from, for example, radionuclides, peptide tags, luminescent molecules, fluorescent molecules, quencher molecules, pH-sensitive molecules, oxygen-sensitive molecules, or combinations thereof.

[0046] Detailed Description The present disclosure will be further described by way of example and with reference to the figures. Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0054] [Method] In silico prediction of pan-HLA-binding peptides In silico MHC II binding prediction was performed using the ProPred and NetMHCII-2.3 programs (Singh and Raghava 2001, Jensen et al., 2018) to predict pan-HLA-DRB1-binding 30-mer peptides for the GAD65 human protein.

[0055] Peptide / protein antigen Peptides were synthesized by GL Biochem (Shanghai) Ltd or Genscript (Leiden, The Netherlands). Peptides were >90% pure and resuspended from lyophilized powder either in 100% v / v dimethyl sulfoxide (DMSO) for 30-mers or in PBS for soluble peptides. The GAD65 protein was synthesized by Biologics Corporation. Purified protein derivative (PPD; Prionics; 7600060) was used at 300 IU / mL. Keyhole limpet hemocyanin (KLH; ThermoFisher Scientific; 77600) was used at 20 μg / mL.

[0056] Human PBMC isolation and 3 H-thymidine incorporation assay Fresh blood samples were collected into CPDA tubes. PBMCs were isolated from lymphocyte cones or fresh blood by Ficoll gradient centrifugation and frozen in 40% RPMI-1640, 50% heat-inactivated fetal bovine serum (Sigma; F9665), and 10% DMSO, and stored in liquid nitrogen until needed. Thawed PBMCs were cultured at 1.5x10 6 cells / mL in X-VIVO-15 medium (Lonza BE02-061Q) supplemented with 5% v / v human AB serum (Sigma H4522), 1X penicillin / streptomycin (Gibco 15140122) in the presence or absence of 25 - 50 μg / mL of the GAD65 P10 30-mer antigen (negative control). As previously described (Mazza et al., 2002), on days 5 and 7, 3 the peptide response was measured by 3 3H-thymidine incorporation by pulse treating the cell culture with 3H-thymidine (Perkin Elmer). A positive response had a corrected count per minute (ccpm) >1000 and a stimulation index (SI) ≥3, calculated as the fold-change in peptide-stimulated conditions over the negative control.

[0057] HLA-DR Typing Genomic DNA was extracted from 1 - 5x10 6 PBMCs (Qiagen; 69504). After PCR analysis, low-resolution HLA-DR serotypes were interpreted from positive lanes using the reagents and result sheets of the HLA-DR low typing kit (Olerup; 101.101-12u). Medium-resolution HLA-DRB1 genotyping was provided by VH Bio.

[0058] Mouse HLA-DR4 transgenic mice express HLA-DRA*0101 and -DRB1*0401, and human CD4 has been described previously (Fugger et al., 1994). B cells derived from the peripheral blood of HLA-DR4 mice were phenotyped by flow cytometry for HLA (clone TUE39) and murine MHCII (clone M5 / 114.15.2). Male and female mice aged 6 - 12 weeks were used. Animals were housed in specific pathogen-free conditions in the Biomedical Services Unit of the University of Birmingham. Experiments were conducted in accordance with the regulations of the local ethics committee and the UK Home Office.

[0059] Generation and screening of T cell hybridomas HLA-DR4 transgenic mice were injected subcutaneously with complete Freund's adjuvant (CFA) and 100 μg of GAD65 P10 30-mer. Ten days later, spleen cells were isolated, restimulated with GAD65 P10 30-mer for an additional 4 - 5 days, and then fused with hypoxanthine-aminopterin-thymidine (HAT)-sensitive BW5147 cells using polyethylene glycol (PEG). The fused hybridomas were grown in HAT selection medium and then subjected to antigen-specific screening. 1x10 5 Hybridomas and 2x10 as antigen-presenting cells (APCs) 5 The co-culture of Priess cells (Epstein - Barr virus (EBV)-transformed DR4 (DRB1*04:01)-expressing human cell line (ECACC 86052111)) with peptide, whole GAD65 protein, or medium alone was incubated in 96-well plates for 48 hours. Antigen-specific responses were determined by IL-2 secretion into the culture supernatant using an ELISA assay (BioLegend 431004). Hybridomas that grew in response to both GAD65 protein and GAD65 30-mer were cloned to single cells by limiting dilution.

[0060] Peptide Six 15-mers spanning the parental 30-mer amino acid sequence were generated by a 3-amino acid shift from the N-terminus to the C-terminus. Twelve truncated peptides were generated by removing 1 amino acid from each peptide in either direction up to 6 amino acids from the N and C termini of the hybridoma-reactive 15-mer peptides.

[0061] Hydrophilic amino acid lysine (K) was added to the N and C termini of the core epitope to generate peptides that are more soluble as determined by the GRAVY score (http: / / www.gravy-calculator.de / ).

[0062] Fixed APC T cell hybridoma screening Spleen cells from HLA-DR4 transgenic mice were fixed with formaldehyde by the following protocol performed at room temperature; 1x10 6 cells / mL, incubation with 0.5% w / v formaldehyde in PBS for 5 minutes, quenching the reaction for an additional 5 minutes by addition of 0.4M glycine in an equal volume of PBS solution, followed by 3 washes with cold PBS. 1x10 5 hybridoma and 2x10 5 either formaldehyde-fixed APC or 2x10 5 non-fixed APC were used and a T cell hybridoma screening was set up where stimulator or control was co-cultured for 48 hours and the response was measured by IL-2 secretion.

[0063] Steady state CD11c + Peptide binding assay After subcutaneous injection of 80 μg of GAD65 P10.5.c+1.6K (GAD65 P10Sol) peptide into HLA-DR4 transgenic mice, CD11c + spleen cells were isolated 1 hour later with a positive selection kit (Miltenyi; 130-125-835). 0.5x10 + CD11c 5 spleen cells were isolated with or without addition of exogenous antigen in vitro. +Cells were co-cultured with 1x10 5 GAD65 P10-specific hybridomas for 48 hours, and the T cell hybridoma response was measured by secreted IL-2.

[0064] Tolerance induction and immunization exposure HLA-DR4 transgenic mice were subcutaneously injected with GAD65 P10 Sol peptide every 3 - 4 days in a dose escalation of 0.1 μg, 1 μg, 10 μg, 100 μg, 100 μg. On day 21, all mice were exposed to 100 μg of GAD65 P10 30-mer in CFA. Ten days after exposure, spleen cells were isolated and used for the FAIM assay.

Example

[0065] Identification of tolerogenic antigen processing-independent T cell epitopes (apitopes) Tolerogenic apitopes: a ntigen p rocessing i ndependent T -cell epit opes(Antigen processing-independent T cell epitope)) The workflow for designing and validating starts with the identification of peptides (pan-DR binding factors) predicted to bind to diverse HLA-DR molecules using a combination of publicly available MHC binding algorithms (Figure 1A). In silico analysis identified peptides of 9 or 15 amino acids within GAD65 and extended these to ~30-mer peptides (Figure 1B) to facilitate antigen processing and presentation of endogenously processed epitopes (Anderton et al., 2002). To test whether the designed 30-mer peptides could be correctly processed and preferentially induce an immune response in T1D patients, the inventors screened for GAD65 30-mer reactivity in isolated peripheral blood mononuclear cells (PBMCs) from 20 non-diabetic healthy controls and 40 adults with T1D. The HLA-DRB1*04:01 and / or HLA-DRB1*03:01 alleles are strongly associated with T1D disease (Pociot and McDermott 2002, Noble and Valdes 2011), and 45% (18 / 40) of the T1D patients tested carried at least one HLA-DRB1*04:01 allele and 35% (14 / 40) had at least one HLA-DRB1*03:01 allele (Figure 1C). In the initial peptide screening, to provide the most appropriate HLA comparator, healthy control PBMCs were HLA genotyped to ensure that a high proportion of the healthy cohort carried the HLA-DRB1*04:01 and / or HLA-DRB1*03:01 alleles (Figure 1C). 3 On day 7 of the H-thymidine incorporation assay, 20% (4 / 20) of healthy controls showed a positive response to the GAD65 P10 30-mer compared to 58% (23 / 40) of T1D patients (Figure 1C). This demonstrated a significantly higher response in T1D patients (Fisher's exact probability test; p = 0.0069). Furthermore, the mean magnitude of the proliferative response was also significantly increased in T1D patients (Figure 1D; Mann-Whitney test, p = 0.0146)

[0066] Since an increased reactivity to the P10 30-mer peptide of GAD65 was demonstrated in T1D patients, the next step was to evaluate peptide immunogenicity in appropriate human HLA-DR transgenic mice, as illustrated in Figure 1A, and to generate peptide-specific hybridomas using responsive HLA-DR transgenic mice. HLA-DR4 transgenic mice expressing the HLA-DRB1*04:01 allele responded well to immunization with the GAD65 P10 30-mer (data not shown) and were used to generate P10 30-mer-specific T cell hybridomas (Figure 2A). Generation of hybridomas that react to both the GAD65 protein and the P10 30-mer peptide means that these hybridomas are able to recognize the naturally processed peptides presented by APCs. This is a property that has been shown to be very important for the success of peptide immunotherapy design (Anderton et al 2002). Initial screening of hybridoma cultures with HLA-DRB1*04:01-expressing APCs showed that hybridoma clone 6 reacted to both the P10 30-mer and the full-length GAD65 protein (Figure 2B), and this clone was then subcloned to single cells by limiting dilution (Figure 2C).

[0067] Enhanced solubility of tolerogenic peptides Peptide solubility is an important property required for tolerogenic epitope design (Shepard et al., 2021), and thus the inventors next sought to elucidate the P10 30-mer minimal core epitope and test analogs modified to optimize solubility. This was achieved by sequentially removing amino acids from the N and C termini of the 15-mer following identification of the reactive 15-mer within the 30-mer to identify the minimal "core" amino acids critical for MHC class II binding and hybridoma TCR stimulation (Figures 3A-D). Applying this methodology, the inventors identified the P10.5 15-mer (Figure 3B) and the minimal core epitope (P10.5.C; SEQ ID NO: 20, Figures 3D-E) within the P10 30-mer. Versions designed based on the core minimal epitope included retention of non-essential residues and / or addition of multiple lysine residues to both the N and C termini (Figure 3E). Both the P10.5.C.6K (SEQ ID NO: 22) and P10.5.C+1.6K (SEQ ID NO: 23) versions had improved GRAVY scores of -2.1 and -1.84, respectively, and both peptides were highly soluble in PBS. P10.5.C.6K contains only the essential core epitope with additional lysine residues, but when it again includes a single non-essential alanine at the C terminus (P10.5.C+1.6K), the strength of the single clone hybridoma response increased 10-fold (Figure 3F).

[0068] Binding of tolerogenic peptides to MHC class II and APCs The next step was to determine whether GAD65 P10.5.C+1.6K (P10Sol; SEQ ID NO: 23) was able to: (i) bind to MHC class II molecules without antigen processing, and (ii) steady state CD11c +It was to test whether it could bind to APCs. These characteristics are excellent indicators of whether a peptide can induce tolerance and be characterized as an epitope (Shepard et al., 2021). Formaldehyde fixation of splenocytes inhibits antigen processing. Therefore, to test whether GAD65 P10Sol can directly bind to cell surface MHC class II without antigen processing, the GAD65 P10-specific hybridoma SC3 was co-cultured with fixed or non-fixed splenocytes from HLA-DR4 transgenic mice. The response to the full-length GAD65 protein requires antigen processing from non-fixed splenocytes, whereas both the GAD65 P10 30-mer and GAD65 P10Sol (SEQ ID NO: 23) induced a strong IL-2 response from hybridoma SC3 when co-cultured with either fixed or non-fixed splenocytes (Figure 3G). This demonstrated that both peptides can directly bind to MHC class II independent of antigen processing. P10Sol is in steady-state CD11c + To test whether P10Sol can bind to APCs in vivo, 80 μg of P10Sol or PBS was injected into HLA-DR4 transgenic mice, and CD11c + splenocytes were isolated 1 hour later and co-cultured with the GAD65 P10 hybridomas SC3 or SC22. CD11c isolated from mice injected with the P10Sol peptide + cells activated and induced a strong IL-2 response in P10-specific hybridomas without the exogenous addition of peptides / proteins to the in vitro cultures, indicating rapid and efficient in vivo presentation of the soluble P10Sol peptide (Figure 3H). However, for CD11c + cells from PBS-injected mice, the P10-specific hybridoma IL-2 response was only observed when P10Sol peptide or GAD65 protein was exogenously added to the in vitro cultures (Figure 3H). Collectively, this indicates that P10Sol is in steady-state CD11c in a manner independent of antigen processing +It was demonstrated to be able to bind to APC, which is a characteristic of the epitope, and thus, it was demonstrated that P10Sol is a suitable candidate for tolerance induction studies.

[0069] Tolerance induction The soluble candidate peptide GAD65 P10Sol (SEQ ID NO: 23) was used as a representative peptide and tested for tolerance induction in a suitable HLA-DR transgenic mouse model (Figure 1A). HLA-DR4 transgenic mice were treated with either a dose escalation of GAD65 P10Sol or a PBS control for 17 / 18 days and then exposed to P10 30-mer in a strong adjuvant (Figure 4A). Ten days after exposure, spleens were harvested and an in vitro fluorescence dye dilution activation-induced marker (FAIM: F fluorescent dye dilution A Activation I Induced M marker) assay was set up (Figure 4A). The FAIM assay involves a primary flow cytometry readout with fluorescent dye-labeled CD4 + cell enrichment cultures to evaluate CD4 T cell fluorescent dye dilution and cell surface activation marker expression, and optionally 3 readouts paired with 3H-thymidine incorporation and secreted cytokine measurements. Here, the FAIM assay was validated by performing a parallel 3 3H-thymidine incorporation assay. In-house testing of the FAIM assay to detect the induction of CD4 T cell tolerance with dose escalations of B10PL mice and myelin basic protein-derived tolerogenic peptides (Burton et al., 2014) was performed for cytokines, 3Flow cytometry readings of the FAIM assay, including the use of H-thymidine and CD71 as a novel activation-induced marker, demonstrated the ability to successfully distinguish the induction of tolerance (data not shown). This novel activation-induced marker, CD71, is the transferrin receptor involved in iron uptake and has an expression pattern strongly associated with Ki67 and cell proliferation (Lastovicka et al., 2009; Motamedi et al., 2016). Flow cytometry analysis from day 7 of in vitro stimulation using titrations of GAD65 P10 30mer and P10Sol (gating strategies for PBS-treated and P10Sol-treated mice are shown in Figures 4B - C) revealed that the total CTV dilution proliferation (CTV (mid) ) CD4 T cells were significantly decreased in P10Sol-treated mice (Figure 5A; Sidak's multiple comparison test). Furthermore, in P10Sol-treated mice, antigen-reactive CTV (mid) CD4 T cells co-expressing the combination of activation markers CD25 / CD71 or CD25 / OX40 were either almost absent or significantly decreased (Figures 5B - C; Sidak's multiple comparison test). This decreased response measured by flow cytometry readings of the FAIM assay was supported by paired 3 H-thymidine incorporation readings, which showed a statistically non-significant but strong trend towards a decrease in the overall proliferation response in cultured cells from P10Sol-treated mice restimulated with P10Sol or P10 30mer in vitro (Figure 5D). The inventors tetramerized the P10Sol peptide - DRB1*04:01 MHCII monomer, P10Sol(DRB1*04:01)-PE, for validation (Figure 6A) and used this to evaluate whether the proliferating CD4 T cells expressing activation markers were antigen-specific. Tetramer staining was included in samples stimulated with 10 μg / mL of P10Sol or P10 30mer, which showed selective binding to antigen-reactive proliferating CD4 T cells (Figures 6B - C). Antigen-specific tetramer + FAIM +The number of cells was highly enriched in PBS-treated mice and almost absent in mice treated with increasing doses of P10Sol (Figure 6D). Furthermore, the combination of CD25 / CD71 on reactive CD4 T cells identified a significant increase in the frequency of tetramer + cells (Figure 6E). This also held true when analyzing the enrichment of co-expression of activation markers within total tetramer + CD4 T cells (Figure 6F). P10Sol was able to directly bind to MHCII without antigen processing and demonstrated the ability to induce tolerance in HLA-DR4 transgenic mice.

[0070] Since the properties of P10Sol and the response to it in T cell hybridomas and HLA-DR4 transgenic mice were demonstrated, the next important step was to evaluate whether T1D patients could also respond to P10Sol. The inventors used the FAIM assay on PBMCs from a cohort of 44 adult T1D patients to measure FAIM + (CD25 + CD71 + CTV (mid) CD4 + T cells) and 3 the H-thymidine proliferation response as a pair (Figure 7A). The FAIM + analysis highlighted that 89% (34 / 38) of T1D patients could respond to the GAD65 P10 30-mer, and 38% (13 / 34) of these also responded to P10Sol (Figure 7A). Importantly, the FAIM + flow cytometry readings detected 16 additional positive responses (10.2% of the total results), which were below the positive threshold of the paired 3 H-thymidine proliferation readings, whereas 3 results detected by H-thymidine but not accompanied by FAIM + were only one (0.6% of the total results) (Figure 7A - B). This demonstrated that P10Sol is a natural epitope recognized by the TCR of individuals with type 1 diabetes.

[0071] Conclusion The present inventors 3 used a combination of methods based on H-thymidine incorporation and activation-induced markers to identify and validate novel peptide epitopes capable of inducing immune tolerance to self-antigens. Using P10Sol as a representative peptide, increasing doses were able to inhibit CD4 + T cell-specific proliferation, as measured by flow cytometry, which was supported by paired 3 H-thymidine incorporation readings. Furthermore, the number of CD4 + OX40 + and CD25 + CD71 + co-expressing CD4 + T cells decreased in mice treated with increasing doses of P10Sol. Using the P10Sol peptide-MHC class II tetramer, a higher frequency of antigen-specific proliferating CD4 T cells was identified only when using a novel combination of activation markers CD25 and CD71, as compared to the use of CD25 and OX40 alone, thus demonstrating a novel combination of CD25 and CD71. The present inventors then demonstrated that P10Sol identified by the described method was able to induce a CD4 T cell response in PBMCs from T1D patients, that this was a disease-significant epitope, warranting further clinical development.

[0072] References Anderton, S., Viner, N., Matharu, P. et al. Influence of a dominant cryptic epitope on autoimmune T cell tolerance. Nat Immunol 3, 175-181 (2002). https: / / doi.org / 10.1038 / ni756 Atkinson et al., 2014. Lancet. 2014 Jan 4;383(9911):69-82. doi:10.1016 / S0140-6736(13)60591-7. Babon et al., 2016. Nat Med 2016 Dec;22(12):1482-1487. doi: 10.1038 / nm.4203. Epub 2016 Oct 31 Burton et al., 2014. Nat Commun. 2014 Sep 3;5:4741. doi:10.1038 / ncomms5741 Feutren et al., 1986. Lancet. 1986 Jul 19;2(8499):119-24. doi:10.1016 / s0140-6736(86)91943-4. Fugger et al., 1994. Proc Natl Acad Sci USA. 1994 Jun 21;91(13):6151-5. doi:10.1073 / pnas.91.13.6151 Herold et al., 2019. N Engl J Med. 2019 Aug 15;381(7):603-613. doi:10.1056 / NEJMoa1902226. Jensen et al., 2018. Immunology. 2018 Jul;154(3):394-406. Doi: 10.1111 / imm.12889. Epub 2018 Feb 6 Kroll et al., 2013. J Clin Virol. 2013 Jun; 57(2): 115-119. Last’ovicka et al., 2009. Cell Immunol. 2009;256(1-2):79-85. doi:10.1016 / j.cellimm.2009.01.007 Mazza et al., 2002. Clin Exp Immunol. 2002 Jun;128(3):538-47. doi: 10.1046 / j.1365-2249.2002.01831.x Motamedi et al., 2016. J Immunol Methods. 2016 Oct;437:43-52. doi:10.1016 / j.jim.2016.08.002 Mullard 2022. Nature Reviews Drug Discovery. Doi 10.1038 / D41573-022-00198-9 Naruse et al., 1997. Tissue Antigens. 1997 Feb;49(2):152-9. doi: 10.1111 / j.1399-0039.1997.tb02729.x Noble and Valdes 2011. Curr Diab Rep. 2011 Dec;11(6):533-42. doi:10.1007 / s11892-011-0223-x Pociot and McDermott 2002. Genes Immun. 2002 Aug;3(5):235-49. Doi: 10.1038 / sj.gene.6363875. Peterson and Haskins 1996. Diabetes. Mar;45(3):328-36. doi:10.2337 / diab.45.3.328 Shepard et al., 2021. Front Immunol. 2021 Apr 14;12:654201. doi:10.3389 / fimmu.2021.654201 Singh and Raghava 2001. Bioinformatics. 2001 Dec;17(12):1236-7. Doi: 10.1093 / bioinformatics / 17.12.1236 Wraith, D. Antigen-specific immunotherapy. Nature 530, 422-423 (2016). https: / / doi.org / 10.1038 / nature17300

Claims

1. An antigenic peptide capable of binding to MHC class II molecules without depending on antigen processing, for use in the treatment of type 1 diabetes, said antigenic peptide being derived from a protein expressed by pancreatic cells.

2. The antigenic peptide for use according to claim 1, wherein said peptide derived from a protein expressed by pancreatic cells comprises a fragment of glutamic acid decarboxylase 65 (GAD65) or a variant thereof.

3. The antigenic peptide for use according to claim 2, wherein said fragment of GAD65 comprises 5 to 40 amino acids in length.

4. The antigenic peptide for use according to claim 2 or 3, wherein said fragment of GAD65 comprises 5 to 20 amino acids in length.

5. The antigenic peptide for use according to any one of claims 1 to 4, wherein said peptide is selected from SEQ ID NOs: 20 to 23.

6. The antigenic peptide for use according to any one of claims 1 to 5, wherein said peptide is SEQ ID NO:

23.

7. The antigenic peptide for use according to any one of claims 1 to 4, wherein said peptide comprises the amino acid sequence DAAWGGGLLMSRKHKWKLSGVERANSVTWN, IFSPGGAISNMYAMMIARFKMFPEVKEKGMA, DLERRILEAKQKGFVPFLVSATAGTTVYGA, or a fragment thereof.

8. The antigenic peptide for use according to any one of claims 2 to 7, wherein said fragment comprises 5 to 20 amino acids in length.

9. The antigenic peptide for use according to any one of claims 2 to 7, wherein said fragment comprises 5 to 15 amino acids in length.

10. Said peptide is: (i) one or more amino acid substitutions (including conservative substitutions), (ii) one or more amino acid deletions, (iii) one or more amino acid additions, and / or (iv) one or more sequence inversions The antigenic peptide for use according to any one of claims 1 to 9, comprising one or more amino acid modifications selected from the above.

11. The antigenic peptide for use according to any preceding claim, wherein said peptide is selected from SEQ ID NOs: 1 to 65.

12. The antigenic peptide for use according to any preceding claim, wherein said peptide is selected from SEQ ID NOs: 1 to 23.

13. A tolerogenic peptide for use according to any preceding claim, wherein the peptide further comprises one or more amino acid modifications that increase the solubility of the peptide.

14. The tolerogenic peptide for use according to claim 13, wherein the one or more amino acid modifications comprise addition and / or substitution of one or more amino acids selected from lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine and / or glutamine of the wild-type or reference residue.

15. The tolerogenic peptide for use according to claim 13, wherein the one or more amino acid modifications comprise addition of 1 to 4 lysine residues to the N-terminus and / or C-terminus of the peptide.

16. The tolerogenic peptide for use according to claim 15, wherein the peptide is selected from KKKWKLSGVERKKK, KKKWKLSGVERAKKK, KKKRISNMYAMMIARRKKK, KKKKISNMYAMMIARKKKK and / or KKERRILEAKQKGFVPKK.

17. A tolerogenic peptide for use according to any preceding claim, wherein the peptide is used in combination with one or more other treatment(s) for type 1 diabetes.

18. A pharmaceutical composition comprising an effective amount of one or more tolerogenic peptides selected from SEQ ID NOs: 1 to 65; optionally, the peptide further comprises one or more amino acid modifications that increase the solubility of the peptide.

19. The pharmaceutical composition according to claim 18, wherein the peptide is formulated with one or more pharmaceutically acceptable excipients, stabilizers, wetting agents, emulsifiers, salts, buffers and / or adjuvants.

20. The pharmaceutical composition according to claim 18 or 19, wherein the tolerogenic peptide is combined with one or more active ingredient(s) and / or treatment(s) for type 1 diabetes.

21. The tolerogenic peptide, which is capable of binding to MHC class II molecules without depending on antigen processing, and is derived from pancreatic cells or a synthetic variant thereof.

22. The tolerogenic peptide according to claim 21, wherein the peptide derived from pancreatic cells comprises a fragment of glutamic acid decarboxylase 65 (GAD65).

23. The tolerogenic peptide according to claim 21 or 22, wherein the fragment of GAD65 comprises 5 to 40 amino acids in length.

24. The tolerogenic peptide according to any one of claims 21 to 23, wherein the fragment of GAD65 comprises 5 to 20 amino acids.

25. The tolerogenic peptide according to any one of claims 21 to 24, wherein the peptide comprises the amino acid sequence DAAWGGGLLMSRKHKWKLSGVERANSVTWN, IFSPGGAISNMYAMMIARFKMFPEVKEKGMA, DLERRILEAKQKGFVPFLVSATAGTTVYGA, or a fragment thereof.

26. The tolerogenic peptide according to claim 25, wherein the fragment comprises 5 to 20 amino acids.

27. The tolerogenic peptide according to claim 25, wherein the fragment comprises 5 to 15 amino acids.

28. The tolerogenic peptide according to any one of claims 21 to 27, wherein the fragment comprises the sequence WKLSGVER, ISNMYAMMIA or ERRILEAKQKGFVP.

29. The peptide is: (i) one or more amino acid substitutions (including conservative substitutions), (ii) one or more amino acid deletions, (iii) one or more amino acid additions, and / or (iv) one or more sequence inversions The tolerogenic peptide according to any one of claims 21 to 28, comprising one or more amino acid modifications selected from the above.

30. The tolerogenic peptide according to any one of claims 21 to 29, wherein the peptide is selected from SEQ ID NOs: 1 to 65.

31. The tolerogenic peptide according to any one of claims 21 to 30, wherein the peptide further comprises one or more amino acid modifications that increase the solubility of the peptide.

32. The tolerogenic peptide according to claim 31, wherein the one or more amino acid modifications comprise addition and / or substitution of one or more amino acids selected from lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine and / or glutamine of the wild-type or reference residue.

33. The tolerogenic peptide according to claim 31, wherein the one or more amino acid modifications comprise addition of one to four lysine residues to the N-terminus and / or C-terminus of the peptide.

34. The tolerogenic peptide according to claim 33, wherein the peptide is selected from KKKWKLSGVERKKK, KKKWKLSGVERAKKK, KKKRISNMYAMMIARRKKK, KKKKISNMYAMMIARKKKK and / or KKERRILEAKQKGFVPKK.

35. A pharmaceutical composition comprising an effective amount of one or more tolerogenic peptides according to any one of claims 21 to 34.

36. (i) For use as a medicament, (ii) in medicine, and / or (iii) in therapy A tolerogenic peptide according to any preceding claim.

37. The tolerogenic peptide according to any one of claims 21 to 36, wherein the peptide further comprises a label with one or more moieties selected from a radionuclide, a peptide tag, a luminescent molecule, a fluorescent molecule, a quencher molecule, a pH-sensitive molecule, an oxygen-sensitive molecule, or a combination thereof.

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