Tolerogenic peptides

Abstract

The invention relates to a tolerogenic polypeptide capable of binding to an MHC Class II molecule independent of antigen processing, which is derived from Der p 1, and its uses in the treatment or prevention of an allergy. The invention also relates to method of treating or preventing an allergy in a subject in need thereof, comprising administering to the subject a therapeutically affective amount of the tolerogenic polypeptide.

Description

[0001]TOLEROGENIC PEPTIDES TECHNICAL FIELD The present invention relates to tolerogenic polypeptides capable of binding to an MHC Class II molecule independent of antigen processing, which is derived from Der p 1, and their uses in treating or preventing allergy. BACKGROUND House dust mite (HDM) is the most common indoor aero-allergen associated with allergic rhinitis (AR) and asthma, affecting 1-2% of the global population1. It is linked to industrialisation, and is rising rapidly with a significant economic impact.Sensitisation to HDM strain Dermatophagoides pteronyssinus is high in the West3,4 andis found to be a significant risk factor for asthma in children sensitised before 5 yearsof age 5,6.Allergen-Specific Immunotherapy (AIT) (7,8) has long been used to desensitise patientssuffering from allergies, including HDM (9). In AIT, increasing doses of allergenextract, administered over time via subcutaneous or sublingual routes (11-15), modifyboth innate and adaptive immune responses and can lead to induction of allergen tolerance.Both routes can reduce the progression of AR16 and may also prevent asthma onset ingrass pollen and HDM allergic subjects17,18, with improved outcomes in children and in mono-sensitised individuals19. For sustained immune protection, at least three years of AIT is required19-21. Currently, AIT is the only disease-modifying treatment for allergy patients and is now embedded in the treatment of HDM allergy22. However, all routes of AIT administration are associated with local and systemic allergic reactions25,26, with an increased risk of anaphylaxis in some cases. Allergic reactions are directly linked to the administration of whole allergen (or allergen extracts) and their recognition by allergen-specific IgE bound to mast cells. The risk of adverse events together with long courses of treatment impacts on both patient uptake and compliance of AIT27. There is currently a need for new or improved methods and compositions to induce tolerance to HDM and therefore treat or prevent allergy to HDM. SUMMARY OF INVENTIONIn a first aspect, there is provided a tolerogenic polypeptide capable of binding to anMHC Class II molecule independent of antigen processing, which is derived from Der p 1. The polypeptide may be about 6-30 amino acids in length. The polypeptide may comprise or consist of, or may be derived from, any polypeptide of Figure 3C, or a sequence with about 90%, 95%, or 99% identity thereto.The polypeptide may comprise or consist of an amino acid sequence of IGIKDLD (SEQID NO: 1), or a sequence with about 90%, 95%, or 99% identity thereto. The polypeptide may comprise or consist of an amino acid sequence of RRKAVIIGIKDLDAFRKRR (SEQ ID NO: 2) or RRKIAVIIGIKDLDAFRKRR (SEQ ID NO: 3) or a sequence with about 90%, 95%, or 99% identity thereto. In another aspect, there is provided a nucleic acid encoding one or more polypeptide of the invention. In another aspect, there is provided a vector comprising a nucleic acid disclosed herein. The vector may be an expression vector. The vector may be a plasmid. The vector may be a viral vector, such as a retroviral vector, lentiviral vector or adenoviral vector. Thevector may comprise a nucleic acid disclosed herein. Such nucleic acids may be used todeliver one or more polypeptide of the invention. In another aspect, there is provided a pharmaceutical composition comprising one or more tolerogenic polypeptide, nucleic acid or vector of the invention. In another aspect, there is provided a means for binding a Der p 1-specific MHC Class II molecule. The means may bind the Der p 1-specific MHC Class II molecule independently of antigen processing. The means may be one or more polypeptide of the invention. In another aspect, there is provided a tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of the invention for use in medicine. In another aspect, there is provided a tolerogenic polypeptide, nucleic acid, vector,pharmaceutical composition or means of the invention for use in the treatment orprevention of an allergy.In another aspect, there is provided one or more tolerogenic polypeptide, nucleic acid,vector, pharmaceutical composition or means of the invention for use in inducing tolerogenesis to an allergen in a subject in need thereof.A method of inducing tolerance to an allergen in a subject in need thereof, comprisingadministering to the subject a therapeutically affective amount of one or more tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of the invention.In another aspect, there is provided a method of treating or preventing an allergy in asubject in need thereof, comprising administering to the subject a therapeutically affective amount of one or more tolerogenic polypeptide, nucleic acid, vector,pharmaceutical composition or means of the invention.In another aspect, there is provided the use of one or more tolerogenic polypeptide,nucleic acid, vector, pharmaceutical composition or means of the invention in themanufacture of a medicament for treating or preventing an allergy.In any aspect, the allergy may be one or more of HDM allergy, allergic rhinitis orasthma. The allergy may be caused by exposure to HDM, and in particular to Der p 1 exposure. The allergen may be HDM, in particular Der p 1 derived from HDM. DETAILED DESCRIPTION The term “tolerogenic” as used herein means capable of inducing tolerance to a particular antigen. Immune tolerance refers to a range of host processes that prevent potentially harmful immune responses and results in a state of unresponsiveness of the immune system to substances or tissue(s) that have the capacity to elicit an immune response. Immune tolerance is a highly regulated process that enables the discrimination of self from non-self, suppression of allergic responses and prevention of reactive immune responses towards foetal antigens by the maternal immune system. Tolerance may result from or be characterised by the induction of anergy in at least a portion of CD4+ T-cells of a population. In order to activate a T-cell, a peptide must associate with a professional APC capable of delivering two signals to T-cells. The first signal is delivered by the MHC-peptide complex on the cell surface of the APC and is received by the T-cell via the T-cell receptor. The second signal is delivered by costimulatory molecules on the surface of the APC, such as CD80 and CD86, and received by CD28 on the surface of the T-cell. It is thought that when a T-cell receives the first signal in the absence of the second signal, it is not activated and becomes anergic. Anergic T-cells are refractory to subsequent antigenic challenge, and may be capable of suppressing other immune responses. Anergic T-cells are thought to be involved in mediating T-cell tolerance. Peptides that are capable of binding class II MHC molecules without antigen processing will be able to bind MHC molecules on immature APCs. Thus, they are likely to be presented to T-cells without co-stimulation, leading to T-cell anergy and tolerance. Peptides which require processing before they can be presented in conjunction with MHC molecules do not induce tolerance because they have to be processed by mature APCs. Mature APCs (e.g. macrophages, B cells and dendritic cells) are capable of antigen processing, but also of delivering both first and second signals to a T-cell, resulting in T-cell activation. It is envisaged that the peptides of the correct size and conformation capable of binding an MHC molecule independent of antigen processing, such as those described herein, are likely to bind MHC molecules on immature APCs and induce T-cell anergy. The term “polypeptide” as used herein may refer to a fragment of wild-type or reference protein sequence, such as a fragment up to about 40 amino acids in length. Polypeptidesof the invention may comprise a fragment of wild-type or reference protein sequencewhich is at least 7 amino acids in length. Polypeptides of the invention may compriseat least a minimal epitope. The minimal epitope may be a polypeptide derivable fromDer p 1 which is capable of binding to the peptide-binding groove of an MHC class I orII molecule and being recognised by a T-cell. “polypeptide” as used herein may also encompass variants of the fragment(s) of theamino acid sequence, wherein the variant may comprise one or more amino acidmodifications. The variant may comprise at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 modifications. The variant may comprise at least 2, 3, 4 or 5 modifications. The term “polypeptide” as used herein encompasses those obtained from polypeptides derived from naturally occurring proteins or synthetically synthesised polypeptides. A tolerogenic polypeptide of the invention may comprise 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 any of SEQ ID Nos: 1-3. An amino acid modification may refer to a substitution of a wild-type or reference amino acid with another. Such substitutions may be conservative in that they swap a wild-type residue for another with the same or similar structural, chemical and / or physio-chemical properties. "Conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example, nonpolar (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; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Naturally occurring polypeptides or proteins comprise amino acids in the L- configuration. In some instances, an amino acid modification may comprise a substitution with an equivalent amino acid in the D-configuration or a conservative amino acid substitution with a D-amino acid. In other instances, an amino acid modification may comprise substitution with an unusual amino acid provided that the tolerogenic peptide retains its ability to bind an MHC class II molecule independent of antigen processing, such as citrulline, hydroxy proline, beta-alanine, ornithine, norleucine, 3-nitrotyrosine, pyroglutamic acid, nitroarginine, for example. Alternatively, the peptide sequence may comprise modified amino acids, such as homo- amino acids, beta-homo-amino acids, N-methyl amino acids and / or alpha-methyl amino acids. Substitutions may also be ‘non-conservative’ in that a wild-type residue is substituted for an amino acid of a different class, for example an amino acid which is structurally dissimilar, chemically different and / or physio-chemically different or dissimilar. Optionally, or in addition, the peptides of the invention may be modified by the skilled person using methods known in the art to alter one of more pharmacokinetic properties of the tolerogenic peptide. For example, the tolerogenic peptide may be modified in order to increase its the solubility. Hydrophilic lysine or arginine residues may be subsequently added to a peptide to increase the core epitope solubility. Other modifications may include addition or substitution of a wild-type or reference residue with one or more amino acids selected from lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine and / or glutamine. The modified tolerogenic peptide with improved solubility may have a grand average of hydropathy (GRAVY) score lower than 0. A subject to be administered a tolerogenic peptide as described herein may be a humanor non-human mammal. The subject may be known to be predisposed and / or susceptibleto the allergy to be treated. A pharmaceutical composition of the invention may further comprise one or more carriers or excipients. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. The compositions can be in any suitable form, for example tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. Such compositions may be prepared by any known method, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate for oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. Optionally, the suspension may also contain stabilizers. Liposomes and / or nanoparticles can also be used to encapsulate the agent for delivery into the cell. A pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulation may be used simultaneously to achieve systemic administration. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof. Generally, these agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.), although other forms of administration (e.g., oral, mucosal, etc.) can be also used. Preferably, these agents are formulated for administration by intravenous injection. Accordingly, agents of the invention are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like. Pharmaceutical compositions can also be formulated so as to provide quick, sustained or delayed release of their active ingredients after administration to the patient by employing procedures known in the art. The physical and chemical characteristics of the compositions of the invention may be modified or optimized according to the skill in the art, depending on the mode of administration and the particular disease or disorder to be treated. The compositions may be provided in unit dosage form, a sealed container, or as part of a kit, which may include instructions for use and / or a plurality of unit dosage forms. A variety of administration routes for the polypeptides or pharmaceutical compositions of the invention are available. The particular mode selected will depend upon the particular polypeptide or composition selected, the severity of the medical disorder being treated and dosage required for therapeutic efficacy. The methods of this invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include, but are not limited to, oral, buccal, sublingual, inhalation, mucosal, rectal, intranasal, topical, ocular, periocular, intraocular, transdermal, subcutaneous, intra-arterial, intravenous, intramuscular, parenteral, or infusion methodologies. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. An effective amount of a tolerogenic peptide refers to an amount of the peptide that is sufficient to induce immune tolerance to an epitope and / or antigen of interest. Typically, the tolerogenic peptide is able to reduce T-cell activation by at least 20%, such as 30%, 50%, 60% 70%, 80%, or more. The skilled person understands there are various techniques which can be used to test this. Alternatively, a reduction in cytokine induction may be correlated with a reduction in T-cell activation. A pharmaceutical composition of the invention may further comprise one or moreadditional component or therapeutic known to be useful in treating or preventing theallergy, and / or useful for inducing tolerogenesis. As used herein, the term “peptide” and “polypeptide” can be used interchangeably. The principle underlying antigen-specific immunotherapy is that exposure to an antigen in an environment that is tolerogenic (rather than inflammatory) will educate T-cells todownregulate (rather than activate) subsequent T-cell responses to that antigen. The downregulated T-cell response includes the secretion of anti-inflammatory cytokinessuch as IL-2, which can act locally to downregulate other T-cells in the vicinity - aconcept recognised as bystander suppression. The inventors have developed novel tolerogenic peptides derived from Der p 1 that bind major histocompatibility complex (MHC) class II molecules independent of antigen processing and induce T-cell mediated immune tolerance. Importantly, these tolerogenic peptides 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 the peptide binding groove of an MHC molecule without further processing, or binds in an inappropriate conformation, then it will not be tolerogenic in vivo. As not all T cell epitopes induce tolerance to self-antigens, it is critical to identify specific T-cell epitopes that mimic the conformation of a naturally processed antigen capable of being presented on an MHC class II molecule for downregulation of reactive CD4+ T-cells.To achieve this, the inventors have built on previous findings that drawbacks of AITusing whole / extracts of allergen can be averted by substituting with peptide fragments of the allergen. Presentation of dominant T cell epitope peptides to allergen-specific CD4+ T cells have been found to circumvent IgE cross-linking on basophils, reducing the chance of an immediate (type-1) hypersensitivity reaction and avoids the risk of anaphylaxis. Peptide AIT with cat dander, birch and grass pollen allergens has shown fewer side effects and a good safety profile. Therapeutic benefits included a rapid reduction of sIgE: sIgG4 ratio (grass pollen), long-term benefits at 2 years (cat dander), whilst allergen-specific T cells responses skewed from pathogenic Th2 to more protective tolerogenic responses dominated by regulatory T cells (Tr1 or Foxp3+ Treg cells). It was previously shown that short peptide epitopes have tolerogenic properties when designed to mimic the naturally processed antigen acting as antigen processingindependent epitopes and are also highly soluble. Pan- HLA-DR epitopes displaypromiscuity, binding to a wide range of HLA-DR molecules. Thus, selection of one pan- HLA-DR antigen-specific peptide can target patients with a wide range of HLA haplotypes. Additional T cell epitope peptide(s) from the same or other antigens within the disease-inducing allergen can also be identified to create a universal peptide cocktail for treatment. This approach has been successfully employed in peptide immunotherapeutic clinical trials in patients with multiple sclerosis and Graves’ disease. The inventors developed on finding pan-HLA-DR tolerogenic peptides within Der p 1, a major antigen of HDM. Following an in silico search, Der p 1 dominant T cell epitope peptides were identified by the inventors in HDM sensitised patients with allergic rhinitis and / or asthma with different HLA haplotypes. Analogues of these peptides were designed and validated as antigen processing independent epitopes. One antigenprocessing independent epitope in particular was found to induce tolerance against Derp 1 antigen in HLA-DR4 transgenic mice in vivo and is a suitable candidate for testing in AIT for HDM. The invention therefore presents a new and clinically important way to induce tolerance and prevent allergy to HDM. The skilled person will appreciate that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention. BRIEF DESCRIPTION OF THE DRAWINGSFigure 1 – In silico prediction of Der p 1 T cell epitopes Pan-DR binding predictionsof T cell epitopes within the Der p 1 aa sequence to HLA-DR was determined by either ProPred (a) or NetMHCII-2.3 (b) programmes. Core sequences of peptides with the highest pan-DR affinity, identified by both prediction programmes, were selected from each of 5 different regions of Der p 1. Addition of flanking amino acids based on the native sequence of the Der p 1 protein were added to the core sequence to generate 5 peptides of 29-30 amino acid length (Peptide A-E) and synthesised by F-moc chemistry. Figure 2. Serological and T-cell responses to HDM, Der p 1 and Der p 1-derivedpeptide epitopes. A, the level of total serum IgE was compared with HDM and Der p1- sIgE in HDM allergic (n=25), non-HDM allergic (n=10) and healthy control (n=10)subjects (KUA / L). B, corresponding T-cell proliferative responses to Der p 1, Der p 1- derived synthetic peptides A-E and PPD was measured by the incorporation of [3H]- thymidine in PBMC cultures on day 4, 6 and 8. Proliferation was expressed as stimulation index (SI). Missing values due to low cell yield are denoted by (X). C-I, expression of T cell proliferation as transformed stimulation index (SIlog10, mean+ / - SD), in HDM-positive (HDM-sensitised) PBMC compared with HDM-negative PBMC (combined non-HDM sensitised and healthy control groups) on each day of the assay. Values above the dotted horizontal line on each graph denote positive T cell responses to antigens Der p 1 (C), PPD (D) or Der p 1-derived peptide epitopes (E-I). Statistical analysis was as described in Methods and S1, using Satterthwaite's method for t-tests, with significance at *p<0.05, **p<0.01, ***p<0.001.Figure 3. Minimal T cell epitope of Peptide D. A-C, For fine-mapping of T cellepitope(s), splenocytes from HLA-DR4 mice immunised with Der p 1 (A) or PBS in CFA (B), were re-challenged in vitro with peptide D, analogues D1-D6 (15mer peptides, overlapping by 3aa), Der p 1 or PPD, for 72h. T cell stimulation was measured by IFN-gamma secretion (pg / ml, ELISA). C, defining the minimal T cell epitope of peptide D;summary of T cell IFN-gamma secretion (+ / -) in response to truncated analogues (overlapping by 1aa) of D1 and D2. Boundaries of the minimal T cell epitope sequence are in red font. D, proliferative response of human HLA-DR4 PBMC (HLA DRB1*04:01 DRB1*14:54) to analogue D121B, containing the minimal T-cell epitope. Proliferation was measured by the cell incorporation of [3H]-thymidine after 7 days of culture with antigens D121B, D, C, Der p 1 (all at 20ug / ml) or PPD (300I.U / ml) and expressed as stimulation index (SI); values above the dotted horizontal line denote a positive T cell response to test or control antigens. Figure 4. Validation of D peptide analogues as antigen processing independentepitopes. A, in vitro presentation of antigen (D111B, D121B, D or Der p 1) by fixedHLA-DR4 mouse splenocytes (direct binding, no processing, left bars) or unfixed splenocytes (right bars) following co-culture with Der p 1-specific T cell hybridoma cells. Activation was evaluated by IL-2 secretion (pg / ml, ELISA) after 24h. Control wells contained splenocytes, hybridoma, or no antigen (medium). B, direct binding of D121B to MHC Class II on CD11c+ cells in vivo. Two hours post-injection (s.c) ofHLA-DR4 mice with a single dose of peptide D121B (test, left bar) or PBS (control,right bar), splenocyte CD11c+ cells were isolated and co-cultured with Der p 1 specificT cell hybridoma cells in vitro. T-cell hybridoma cells secreted IL-2 (pg / ml) in response to in vivo peptide pre-loading of CD11c+. Graphs show mean of triplicate wells ± SEM, representative of 2 experiments. Figure 5. Induction of tolerance with peptide D121B in HLA-DR4 mice HLA-DR4 mice received escalating s.c. doses of either PBS containing 0.1ug, 1ug, 10ug and 3 x 100 ug D121B peptide (test, right circles) or PBS alone (control, left circles) every 3rd or 4th day. Three days after the last dose both test and control mice were immunised s.c. with 100ug of peptide D in CFA. Ten days later, splenocytes were harvested and restimulated in vitro for 72h with either D111B, D121B, D, B (1ug / ml), Der p 1 (10ug / ml) or no antigen. Responses were evaluated by IFN-gamma in culturesupernatant (dpg / ml: IFN-gamma pg / ml antigen containing culture – IFN-gamma pg / mlculture without antigen, ELISA). Graph shows the mean value ± SD for 10 samples per treatment group collected from three experiments. Data was analysed with an unpaired t-test with Welch’s correction, *p<0.05, **p<0.005, ***p<0.0005, GraphPad Prism 9.0.Figure 6 - Fine mapping of T cell epitopes within Peptide B. To define potential Tcell epitopes residing within peptide B, analogues B1-B6 were synthesised (15-16mer peptides, overlapping by 3aa, (A)) and used to re-challenge splenocytes isolated from HLA-DR4 mice immunised with Der p 1 (A) or PBS in CFA (B). T cell responses induced by B analogues in the in vitro assay were compared with stimulation by peptide B, Der p 1 or PPD antigen controls, as measured by IFN-gamma secretion (pg / ml) after 72h in culture.Figure 7 - Ex vivo binding of Der p 1 antigens to CD11c+ cells following pre-loadingin vivo. HLA-DR4 mice received either a single dose (s.c) of peptide D121B (test, orange bars) or PBS (control, blue bars). Two hours post injection, splenocyte CD11c+cells were isolated and co-cultured with antigen (peptide D, D111B, D121B or Der p1at 100ug / ml or 10ug / ml) in the presence of Der p 1 specific T cell hybridoma cells for 48h in vitro. The T cell hybridoma was activated to secrete IL-2 in all cultures, whether pre-loaded with Der p 1 antigens or PBS. The graph is representative of 2 experiments. MATERIALS AND METHODS Study subjects Consenting subjects (age ≥18<80 years) were recruited from University Hospital Birmingham NHS FT (details, S1, Table S1). Selection was based on clinical history, lack of immunomodulatory therapies or corticosteroid treatment (preceding ≤6 weeks) and skin prick test (SPT) to a standard aero-allergen panel including D pteronyssinus (HDM). A HDM positive SPT was confirmed by HDM-specific (s)IgE in serum in addition to total serum IgE and Der p 1-sIgE (Immunocap250, S1). Subjects were stratified into 3 groups: HDM-sensitised (HDM SPT and HDM-sIgE positive with allergic rhinitis and / or asthma, n=25), atopic controls (sensitised to one or more aero- allergens but not to HDM, n=10) and healthy non-atopic controls (negative SPTs to HDM and all aero-allergens, n=10). Peripheral blood mononuclear cells (PBMC) from each subject were isolated and cryopreserved, as previously described. The study was approved by North West - Greater Manchester Central Research Ethics Committee (REC 18 / NW0726, Protocol number RG_18-207). Antigens Binding predictions of peptides from the Der p 1 sequence to HLA-DR were conducted in silico using ProPred and NetMHCII-2.3 programmes. Peptides with the highest pan- DR affinity and their related analogues were synthesised by Fmoc chemistry and where required, solubility was optimised by substitution or addition of N-terminal and C- terminal amino acid (aa) tags (S2.A). Der p 1 protein was purchased from Citeq Biologics BV. Mice HLA-DR4 transgenic mice, expressing human HLA-DRA*0101, HLA-DRB1*0401 and murine CD3-human CD446were bred and housed under specific pathogen free conditions in the Biomedical Services Unit, University of Birmingham, in accordance with the local ethical review panel and UK Home Office regulations. Mice (8-16 weeks) were age and sex matched for all experiments. Human PBMC assay Thawed PBMC were re-suspended and cultured with Der p 1 antigen, peptide (A, B, C, D, E or analogues of B or D), Mycobacterium tuberculosis purified protein derivative (PPD, positive control) or no antigen (negative control). T-cell proliferative responses were evaluated on days 4-8 by measuring incorporation of 3[H]-thymidine, expressed as Stimulation Index (SI) (S2.D.1) Mouse immunisation and in vitro recall assay Mice were immunised with antigen Der p 1 (test) or PBS (control) in Complete Freund’s adjuvant (CFA), injected subcutaneously (s.c) in the lower dorsal region (S2.C). After ten days, mice were sacrificed and splenocyte T cell responses were evaluated by the secretion of interferon gamma (IFN-gamma) into culture medium after 72h (ELISA, S2.D.2). Peptide antigen processing independent epitopeantigen processing independentepitope validationPresentation of antigen (Der p 1 or peptides) by fixed (test) or unfixed (control) HLA- DR4 mouse splenocytes was determined following activation of a Der p 1-specific T- cell hybridoma (S2.B), and subsequent IL-2 secretion (ELISA, S2.D.3) in vitro. Presentation by fixed APC indicates that the peptide does not require further processing. Direct binding of peptide D121B to MHC Class II on mouse HLA-DR4 splenic dendritic cells (DC) in vivo was determined 2h post injection (s.c.): isolated CD11c+ DC were isolated and incubated with a Der p 1-specific T-cell hybridoma and activation assessed by IL-2 secretion (ELISA, S2.D.4). Induction of tolerance in mice HLA-DR4 transgenic mice received escalating doses (0.1ug, 1ug, 10ug, 3 x 100ug) of peptide D121B (test) or PBS (control), by s.c. injection (scruff of the neck) every 3rd or 4th day. Three days after the final dose, test and control mice were immunised with 100ug of peptide D in CFA (s.c. lower dorsal area, S2.C). After 10 days, splenocytes were harvested and restimulated in vitro by culturing with various antigens for 3 days (S2.D.2). T cell stimulation was assessed by IFN-gamma secretion (ELISA). Statistical analysis PBMC responses to allergens in HDM sensitised and HDM non-sensitised (combined non-HDM sensitised atopic and healthy non-atopic controls) were compared in a cross- sectional study design devised in R, only main effects were considered in this exploratory work (details in S2.E). Significance of t-tests (Satterthwaite) was set at *p<0.05, **p<0.01, ***p<0.001. For mouse tolerance experiments, data was analysed with an unpaired Student’s t-test with Welch’s correction using GraphPad Prism 9.0. Significance levels were set at *p<0.05, **p<0.005, ***p<0.0005. S1: Study subjects and inclusion criteria Subjects (age ≥18<80 years) were identified from clinical and laboratory databases and allergy / asthma clinics in University Hospital Birmingham (UHB) NHS Foundation Trust, UK. Healthy non-atopic controls were identified via local advertisement at UHB. Subjects were excluded from the study if they were on immunomodulatory therapies (eg. omalizumab, anti-TNF etc) or received regular / recent oral corticosteroid treatment (preceding ≤6 weeks), had a significant psychiatric condition, were smokers or unable to give informed consent. Initial subject selection was based on clinical history and responses to a skin prick test (SPT) to a standard aero allergen panel of D pteronyssinus, D farina, grass pollen, tree pollen (silver birch), cat, dog and aspergillus (ALK Abello). An allergen response with a wheal diameter ≥5mm greater than negative control was deemed positive. An HDM SPT response was confirmed by the presence of HDM-specific (s) IgE in serum (, Phadia, ImmunoCap250). In addition, serum levels of Der p 1-sIgE and total IgE were evaluated (ImmunoCap 250). Based on these analyses, subjects were stratified into 3 groups:• HDM-sensitised (HDM SPT and HDM-sIgE positive with allergic rhinitis and / orasthma, n=25)• non-HDM sensitised (negative HDM SPT but positive to ≥1 aero-allergen(atopic positive control), n=10)• healthy non-atopic negative controls (negative SPTs to HDM and all aero-allergens, n=10). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll® paque PLUS) from a citrated peripheral blood sample collected from each subject and cryopreserved in liquid nitrogen, as previously described42. All subjects provided written informed consent. The study was approved by the NorthWest - Greater Manchester Central Research Ethics Committee (REC reference18 / NW0726, Protocol number RG_18-207). S2.A: Reconstitution of antigens Lyophilized synthetic peptides were reconstituted in dimethyl sulfoxide, stored at - 80oC, then thawed and diluted in phosphate buffered saline (PBS) as indicated. Der p1 protein was reconstituted in PBS and diluted as indicated. S2.B: Generation of Der p 1 T cells hybridomas T-cell hybridomas were generated as described1. Briefly, splenocytes isolated from HLA-DR4 mice immunized with Der p 1 (50ug / mouse) were re-stimulated ex vivo with Der p 1 protein at 50ug / ml in supplemented X-VIVO 15 medium for 5 days. Following fusion with thymoma BW5147 cells, Der p 1-specific T cell hybridoma clones were selected with hypoxanthine-aminopterin-thymidine medium (added after 24h). To testfor Der p 1 specificity, expanded hybridoma clones were co-cultured with DR4-expressing Priess cells (HLA DRB1: 04 and HLA DRB4: 101, EACC #8605211) and Der p 1 antigen for 24h; activation of T cell clones was detected by IL-2 secretion in culture supernatant (ELISA, BioLegend). S2.C: Mouse immunisation Mice were immunised with an emulsion of either Der p 1 (50ug), peptide B (100ug) or peptide D (100ug) in 100ul Complete Freund’s adjuvant (CFA) supplemented with 400ug heat-killed Mycobacterium tuberculosis H37RA, injected subcutaneously (s.c.) at two sites in the lower dorsal region (50ul / site). Control mice were immunised with PBS alone in CFA with H37RA. Ten days after immunisation, mice were sacrificed and spleens collected. S2.D: In vitro assay conditions 1. Human PBMC assay Medium: Thawed PBMC were cultured in RPMI 1640 medium supplemented with 20mM HEPES, 100U / ml Penicillin, 100ug / ml streptomycin and 10% heat-inactivated autologous plasma40, cultured in a 24 well plate at 1.2 X 106cells / well at 37oC / 5%CO2. Antigens:• Test - Der p 1, peptide A, B, C, D or E, peptide analogues of B or D at 20ug / ml• Positive control - Mycobacterium tuberculosis purified protein derivative (300I.U / ml, PPD, Bovigam, Thermo Fisher (positive control)• Negative control - no antigen (medium alone)• Assay readout: T-cell proliferation, kinetic assay, monitored in duplicatealiquots of cell suspensions on days 4, 6 and 8 by measuring cell incorporation of 3[H]- thymidine, as previously described2. Proliferation was considered positive when cell incorporated cpm >1000 and Stimulation Index (SI) >3 (SI: cpm with antigen / cpm no antigen).• Assay readout to assess response to analogue D121B: essentially as above,except T-cell proliferation was assessed in triplicate aliquots of cell suspensions from triplicate culture wells / antigen on day 7. 2. In vitro recall assay following mouse immunisation Medium: Disaggregated splenocytes were seeded in triplicate in X-VIVO 15 supplemented with 100U / ml Penicillin, 100ug / ml streptomycin, and 0.05uM 2- mercaptoethanol at 0.5 X 106cells in 96-well flat-bottomed tissue culture plates at 37oC / 5%CO2 for 72h Recall Antigens:• Test - Der p 1 (10-50ug / ml) or peptides (1-100ug / ml)• Positive control - Mycobacterium tuberculosis purified protein derivative (300I.U / ml, PPD, Bovigam, Thermo Fisher (positive control)• Negative control - no antigen (medium alone)Assay readout: T cell responses were evaluated by the secretion of interferon gamma (IFN-gamma, pg / ml) into culture medium (enzyme-linked immunosorbent assay (ELISA), BioLegend).3. Peptide antigen processing independent epitope validation: presentation of allergenby fixed APC Splenocytes from HLA-DR4 mice were fixed with 0.5% paraformaldehyde in PBS for 5 min at 20oC, quenched with 0.4M glycine in PBS for 5 min and washed with cold PBS. Medium: Resuspended cells were cultured in RPMI 1640 medium supplemented with 20mM HEPES, 100U / ml Penicillin, 100ug / ml streptomycin, 0.05uM 2-mercaptoethanol and 5% foetal calf serum cultured in a 96 well plate. Cells:• • Fixed (test) or unfixed (control) splenocytes at 2x105 cells / well together with• • Der p 1-specific T cell hybridoma cells at 1x105cells / wellAntigens: Der p 1 at 50ug / ml or peptides at 100ug / ml Assay readout: T-cell hybridoma activation was determined by IL-2 secretion after 24 hours of incubation with splenocytes and antigen at 37oC / 5%CO2(ELISA, BioLegend).4. Peptide antigen processing independent epitope validation: direct binding of peptideto MHC Class II on CD11c+ cells in vivo Direct binding of a potential peptide tolerogen to MHC Class II on APC was tested in vivo. HLA-DR4 mice received either a single 100ug dose of peptide D121B (test) or PBS alone (control), injected s.c. in the scruff of the neck. After 2h, spleens were harvested and CD11c+ dendritic cells (DC) isolated (microbeads, Miltenyi Biotec) Medium: Resuspended cells were cultured in RPMI 1640 medium supplemented with 20mM HEPES, 100U / ml Penicillin, 100ug / ml streptomycin, 0.05uM 2-mercaptoethanol and 5% foetal calf serum cultured in a 96 well plate. Cells:• • CD11c+ DCs at 0.5x105 CD11c+ cells / well together with• • Der p 1-specific T cell hybridoma cells at 1x105cells / wellControl cultures: either Der p 1, peptide D or analogues D111B, D121B (100ug / ml) antigens Assay readout: T-cell hybridoma activation was determined by IL-2 secretion after 48hours of incubation with CD11c+ DCs, + / - antigen at 37oC / 5%CO2 (ELISA,BioLegend). S2.E: Statistical analysis The sample size calculation (95% power, 5% significance) for this study was based on a previous study3and exceeded the minimum suggested by Lancaster4(n=30). To compare PBMC responses to allergens, subjects were divided into HDM-positive (HDM-allergic) and HDM-negative individuals (combined groups non-HDM allergic and healthy controls) in a cross-sectional study design, devised in R5. The experiment was conducted on a convenience sample, to undertake exploratory analysis. The variables were proliferation (SI), exposure to allergen (either HDM, Der p 1, peptide A,B, C, D or E) and length of exposure (day of sampling). A logarithmic transformationof SI was performed to improve variance stability (SI log10). A two-level regression was undertaken of log (SI) regressed on exposure group (HDM-positive or HDM-negative) and day of experiment (4, 6, or 8), with observations clustered within subjects. Only main effects were considered in this exploratory work. Due to a low PBMC yield from peripheral blood from one HDM-negative subject, insufficient cells were available for sampling on 3 different days of culture; PBMC cultures were omitted for day 4. Missing measurements were not associated with exposure to allergen and so assumed to be missing at random and therefore, not a source of bias in the multilevel analysis. For the analysis, each antigen was considered separately. Satterthwaite's method was used for t-tests, with significance set at *p<0.05, **p<0.01, ***p<0.001. For mouse tolerance experiments, data was analysed with an unpaired Student’s t-test with Welch’s correction using GraphPad Prism 9.0. Significance levels were set at *p<0.05, **p<0.005, ***p<0.0005. Table S2. Statistical analysis of PBMC responses to Der p 1 peptide allergens in HDM-positive and HDM-negative subjects SIlog10HDM+ vs SI HDM- (day 4,6,8)mean / antigen / day Antigen t tests (Satterthwaite) t-value p day HDM-positiveHDM-negative p Mean (SD) Mean (SD) Der p 1 1.427 0.1607 43.03 (3.43) 3.56 (5.54) 0.698 6 7.69 (13.06) 2.80 (3.19) 0.1108 8.12 (13.31) 2.99 (3.78) 0.103Peptide0.583 0.5627 41.59 (0.63) 2.91 (5.31) 0.225 A 6 3.65 (6.78) 1.84 (1.39) 0.248 8 2.19 (2.70) 1.78 (1.62) 0.554 Peptide1.994 0.0525 43.81 (4.28) 3.28 (3.47) 0.661 B 6 9.84 (11.54) 3.60 (2.92) 0.023* 8 7.12 (6.38) 4.31 (4.93) 0.112 Peptide0.401 0.6904 41.96 (1.28) 2.58 (3.57) 0.425 C 6 3.78 (7.01) 1.98 (1.83) 0.272 8 2.41 (3.16) 2.72 (4.24) 0.784 Peptide 2.633 4 8.28 (9.49) 5.01 (7.72) 0.227 D 0.0117* 6 15.08 (17.80) 2.81 (1.69) 0.004** 8 24.12 (44.19) 5.67 (6.80) 0.072 Peptide0.404 0.6881 41.76 (1.04) 2.34 (3.25) 0.406 E 6 3.44 (8.52) 2.03 (3.39) 0.489 8 2.12 (2.97) 1.48 (1.06) 0.365 PPD 0.981 0.3319 433.84 (67.51) 27.63 (43.14) 0.728 6 72.51 (112.98) 42.11 (78.79) 0.313 8 47.97 (97.83) 29.36 (53.14) 0.449 For Satterthwaite t-tests, significance was set at *p<0.05, **p<0.01, ***p<0.001 EXAMPLESExample 1 – Selection of tolerogenic T cell epitopesT cell epitopes were predicted according to MHC class II binding affinity. The highest affinity pan-HLA-DR peptides predicted by both ProPred and NetMHCII programmes were selected based on the 9-mer or 15-mer core sequences (Fig.1). Test peptides were designed to span the predicted core epitope by adding flanking amino acids from theDer p 1 protein. Five peptides of 29-30 amino acid (aa) length were identified aspotential T cell epitopes (Pep A to E, Fig.1).Example 2 - Serum IgE analysis of subjectsMost subjects showed concordance between HDM sensitisation as detected by the SPT and serum sIgE. All subjects within the HDM-allergic group were positive for HDM- sIgE (Fig.2A), this group displayed the highest level of both HDM-specific and total serum IgE (Fig.2A). Individuals in the non-HDM sensitised atopic positive control group had no (9 / 10) or very low levels of HDM-specific IgE (0.58 kUA / L) with none in the healthy control group (0 / 10). Most HDM-allergic subjects had sIgE antibodies specific for Der p 1, a HDM constituent protein. (23 / 25). In contrast, no Der p 1-sIgE was found in either non-HDM sensitised atopic positive control or healthy non-atopic controls.Example 3 - Human T cell responses to Der p 1 and Der p 1-derived epitopesScreening of PBMC proliferation in the presence of Der p 1 antigen or in silico selected and designed pan-HLA-DR test peptides A to E (Fig.1) revealed variation in peak day T-cell responses (day 4, 6 or 8, Fig.2B), as previously observed in this kinetic in vitro assay. T cell responses to peptides B and D were elevated and more frequent (15 / 25 and 17 / 25 respectively) in the HDM-sensitised group compared with the non-HDM- sensitised groups (Fig.2 B). Some subjects demonstrated a T-cell response to Der p 1 antigen, in all 3 study groups (Fig.2B), despite lacking a Der p 1 sIgE. For the two HDM sensitised individuals with negative Der p 1 sIgE, T cells were activated with peptides B or D (SI 3-15) and strongly with Der p 1 in one subject (SI>16) (Fig.2B). Most individuals demonstrated a clear response to the positive control antigen PPD; non-responders were characterised by either weak T cell responses to all other test antigens (no response or SI >7) or had not been inoculated with BCG (PPD SI>3 on any assay day) (Fig.2 B). Non-PPD responders were distributed evenly between HDM sensitised and non-HDM sensitised groups. Initial observations indicated no distinct differences between T cell responses to Der p 1 or Der p 1-derived test peptides in the non-HDM sensitised or healthy control groups; therefore, these were combined into one group for statistical analysis, described henceforth as the HDM-negative group. Variation between the HDM-positive and the HDM-negative group was significant for Peptide D at the 5% level (*p0.0117, t2.633, Fig.2(H)). Furthermore, there was a trend towards significance between test and control responses to Peptide B (p0.0525, t1.994, Fig.2(F)). Little evidence of differences appeared between groups responding to other antigens (Fig.2C-E,G,I), since the reported t values were small in absolute value (Table S2). Preliminary analysis of HLA DR type indicates that indeed, both peptides D and B are pan-DR binding, with no over-representation of any particular allele. The response to peptide D was clear and there was a trend towards a response to peptide B; this warranted further study of both peptides in an HLA-DR transgenic mouse model.Example 4 - Minimal T cell epitope of Peptide DTo define T cell epitopes residing within peptide B or D, a panel of overlapping 15-mer peptides shifting in sequence by 3 aa was used to fine map these regions using splenocytes from HLA-DR4 transgenic mice. Splenocytes from mice immunised with Der p 1 responded to Der p 1, peptide D and two of the N-terminal 15-mer peptides from peptide D (D1 and D2) in an in vitro recall assay (Fig. 3(A)). No recall responses were observed with these antigens following immunisation with CFA alone (Fig. 3B). This confirms that T cell responses to peptide 15mers D1 (aa 1-15) and D2 (aa 4-19) were specific when presented to HLA-DR4. Furthermore, it suggests that the minimal T cell epitope in the N-terminal region resides within aa sequence 4-15 of peptide D. To identify the minimal T cell epitope within peptide D, T cell responses to a set of overlapping (1aa) and truncated peptide analogues of D1 and D2 were evaluated in splenocyte assays. The core sequence was defined as a 7mer, IGIKDLD, with 2 analogues D111B and D121B with their solubility optimised by addition of hydrophilic amino acids (Fig.3C). Analogue D121B induced a proliferative response in PBMC from a Der p 1, HLA DR4 (HLA DRB1*04:01 DRB1*14:54) positive individual (Fig. 3D) indicating that it is a relevant epitope in humans. Furthermore, the PBMC also responded to peptide C, which has an overlapping sequence with D121B (Fig.1) Despite positive responses to peptide B, none of the shorter overlapping 15-mer B peptides could activate splenocytes in the in vitro recall assay (Fig. 6). Thus, only analogues D1 and D2 could be assessed for peptide immunotherapy in the HLA-DR4 mouse model.Example 5 - Validation of D121B as an antigen processing independent epitope inHLA-DR4 transgenic mice The potential of peptides D111B and D121B as tolerogens was evaluated by theirantigen processing independent epitope characteristics. Antigen processing independentepitopes do not require processing, thus can be presented effectively to T cells by fixed APC. Presentation of peptide D, Der p 1, D111B and D121B by live naïve HLA-DR4 mouse splenocytes (APCs) resulted in IL-2 secretion by a Der p 1 hybridoma, but none in control cultures (Fig. 4A, unfixed APC). However, upon fixation of APC, neither the 30-mer peptide D nor the intact antigen Der p 1 could activate the hybridoma, whereas analogues D111B and D121B both elicited an IL-2 response (Fig. 4(A), fixed APC). This provides evidence that epitopes D111B and D121B bind directly to MHC II without further antigen processing and therefore function as antigen processing independent epitopes. To induce tolerance, previous studies have shown that an antigen processingindependent epitope must be soluble and bind directly to HLA-DR on steady stateCD11c+ DC in lymphoid organs. Following a single s.c injection of peptide D121B in an HLA-DR4 mouse, a Der p 1-specific T cell hybridoma was activated ex vivo, showing that peptide D121B had indeed bound to splenocyte CD11c+ cells in vivo (Fig.4B). No response was seen with CD11c+ DCs isolated from PBS treated mice (Fig.4B); however, cells maintained their ability to present Der p 1 and peptides when antigens were added ex vivo (Fig. 7). Both the in vitro fixed APC assay and the in vivo CD11c+ binding experiments validate peptide D121B as an antigen processing independent epitope, thus a suitable candidate tolerogen.Example 6 - Peptide D121B induces tolerance in HLA-DR4 miceAdministration of repeated doses of a soluble antigen processing independent epitope, by dose escalation, induces tolerance associated with T cell anergy and Treg cell generation. Dose escalation with D121B prior to immunisation with peptide D in HLA- DR4 transgenic mice resulted in a suppressed immune response in splenocytes re-stimulated in vitro with Der p 1 or peptide D analogues as compared with PBS controls(Fig. 5). As expected, no response was observed in D121B or PBS treated splenocytes re-stimulated with peptide B. Treatment with the antigen processing independentepitope D121B led to reduced IFN-gamma secretion to the homologous peptide D121B(p0.0032), overlapping peptide D111B (p0.0036), peptide D (p0.0011) and Der p 1 (p0.0022). This provides evidence that the pan-DR binding antigen processingindependent epitope D121B is a good tolerogen to mediate antigen-specificimmunotherapy in individuals with HDM-induced allergic rhinitis / asthma.Example 7 – peptides of the invention are hydrophilic and highly soluble.The peptides of the invention were developed and selected to be hydrophilic and highly soluble, which is a requirement for inducing tolerance successfully (Shepard et al, 2021, Font. Immunol., 12:654201). Peptides of the invention were demonstrated to have negative GRAVY scores, which demonstrate that they are hydrophilic and therefore water soluble. In contrast, other peptides purported to be useful in inducing tolerance to HDM are less hydrophilic and less water soluble, which would translate to a less effective tolerogen.Table 3 – Peptide GRAVY scoresPeptide Sequence Grand HydrophobicityScore D1.1 AVIIGIKDLDAFRHY 0.51D1.2 IAVIIGIKDLDAFRH 0.89D111B RRKIAVIIGIKDLDAFRKRR -0.46D121B RRKAVIIGIKDLDAFRKRR -0.72SEQ 1 ALAQTHSAIAVIIGIKDLDA 0.965SEQ 2 VIIGIKDLDAFRHYDGRTII 0.285SEQ 3 REALAQTHSAIAVIIGIKDLDA 0.513Summary The inventors identified pan-HLA-DR T cell epitopes of Der p 1 (Figure 1). There are multiple elements determining the immunodominance of T cell epitopes from a given antigen in humans. These include HLA class II binding affinity, the ability of human APC to reveal the epitope during antigen processing and finally the presence of T cells carrying a TCR complimentary to the conformation created by binding of the peptide epitope to a specific MHC class II molecule. Having identified epitopes by two independent algorithms, the inventors extended the predicted epitopes to create long (~30mer) peptides to optimise the generation of naturally processed epitopes from the longer peptide. It is important to design pan-HLA-DR binding epitopes since, unlike in many autoimmune diseases, MHC is less closely associated with allergic asthma. The inventors compared immune responses to Der p 1 and the five predicted pan-DR binding epitopes (Figure 2). This revealed a correlation between recognition of Der p 1 and epitopes B and D with a significant increase in the response to D among subjects sensitised to house dust mite and a trend in response to B. Some individuals responded to peptides B and D without recognition of Der p 1. One explanation would be that the peptide sequences are shared with other Der p antigens; however, there is no homology between peptide D and Der p 2, Der p 5, Der p 7 or Der p 23. Alternatively, it is possible that the APC found in the PBMC cultures are not optimal for processing of epitopes B and D from the intact antigen. The most likely explanation, however, is that the concentration of Der p 1 protein in the cultures was too low to elicit a response in these individuals. Immunisation of HLA-DR4 transgenic mice with Der p 1 led to a strong immune response to both peptide D and the intact Der p 1 protein itself (Figure 3). These mice do not express endogenous mouse class II MHC proteins. This proves that peptide D contains a naturally processed epitope from Der p 1 identity of which was further mapped to the N-terminus of the peptide. It was not possible to identify an epitope from peptide B. This is most probably because the epitope created by processing of the whole peptide B cannot be recreated by any of the shorter peptide sequences. Evidence of such dominant cryptic epitopes has been reported in other antigens. The dominant epitope from peptide D in HLA-DR4 transgenic mice mapped to residues IGIKDLD which is predicted to be a pan-HLA-DR binding epitope (Figure 4). In addition to HLA-DR binding predicted for DRB1*0301, *0404, *0701 and *1401, the NetMHCII pan algorithm shows that D121B is predicted to bind specific HLA-DQ molecules (DQA10102-DQB10501; DQA10102-DQB10604; DQA10103-DQB10501; DQA10103-DQB10603) further broadening its specificity and likely impact on tolerance induction. Importantly, the IGIKDLD sequence is found in both peptides C and D which would explain why there is recognition of both peptides in some individuals (Figure 2). The inventors’ previous work defines rules governing the design of tolerogenic peptide epitopes. The first rule is that the peptide must mimic the naturally processed T cell epitope to induce tolerance in those T cells specific for Der p 1. This is confirmed by demonstrating that the peptide functions as an antigen processing independent T cell epitope (Figure 4). Secondly, it has been shown that tolerogenic T cell epitopes selectively bind to unstable MHC II molecules on steady-state dendritic cells. These cells are known to induce tolerance primarily because they express low levels of costimulatory molecules. It has previously been shown that tolerogenic peptides must be highly soluble to reach steady-state dendritic cells in lymphoid organs. Peptide D121B was, therefore, designed to function as an antigen processing independent epitope, was modified to optimise solubility and hence shown to bind directly to CD11c, steady-state dendritic cells following subcutaneous injection. Finally, proof that D121B functions as a tolerogenic peptide was provided by tolerance experiments in HLA-DR4 transgenic mice. These experiments showed that D121B suppressed the immune response to both peptide D and Der p 1 providing proof that this antigen processingindependent epitope was capable of tolerising T cells specific for the naturallyprocessed epitope.Here it is shown that an antigen processing independent epitope from Der p 1 can beused to suppress the immune response to the antigen in a relevant HLA-DR transgenic mouse. Importantly, the peptide antigen processing independent epitopes are hydrophilic and highly soluble, which will further provide benefits in the induction of tolerance. Antigen processing independent epitopes have a good safety profile in the four clinical trials conducted to date in autoimmune diseases. 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Claims

CLAIMS 1. A tolerogenic polypeptide capable of binding to an MHC Class II molecule independent of antigen processing, which is derived from Der p 1.

2. The tolerogenic polypeptide of claim 1, wherein the polypeptide is about 6-30 amino acids in length.

3. The tolerogenic polypeptide of claim 1 or claim 2, wherein the polypeptide comprises or consists of, or is derived from, any polypeptide of Figure 3C, or a sequence with about 90%, 95%, or 99% identity thereto.

4. The tolerogenic polypeptide of any of claims 1-3, wherein the polypeptide comprises or consists of an amino acid sequence of IGIKDLD (SEQ ID NO: 1), or a sequence with about 90%, 95%, or 99% identity thereto.

5. The tolerogenic polypeptide of any of claims 1-4, wherein the polypeptide comprises or consists of an amino acid sequence of RRKAVIIGIKDLDAFRKRR (SEQ ID NO: 2) or RRKIAVIIGIKDLDAFRKRR (SEQ ID NO: 3) or a sequence with about 90%, 95%, or 99% identity thereto.

6. A nucleic acid encoding one or more polypeptide of any of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. The nucleic acid of claim 7, wherein the vector is an expression vector, a plasmid, or viral vector.

9. A pharmaceutical composition comprising one or more tolerogenic polypeptide, nucleic acid or vector of any of claims 1-8.

10. A means for binding a Der p 1-specific MHC Class II molecule, optionally wherein the means binds the Der p 1-specific MHC Class II molecule independently of antigen processing.

11. The means of claim 10, wherein the means comprises or consists of one or more tolerogenic polypeptide of any of claims 1-5.

12. The tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of any of claims 1-11, for use in medicine.

13. The tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of any of claims 1-11, for use in the treatment or prevention of an allergy.

14. The tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of any of claims 1-11, for use in inducing tolerogenesis to an allergen in a subject in need thereof.

15. A method of inducing tolerance to an allergen in a subject in need thereof, comprising administering to the subject a therapeutically affective amount of one or more tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of any of claims 1-11.

16. A method of treating or preventing an allergy in a subject in need thereof, comprising administering to the subject a therapeutically affective amount of one or more tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of any of claims 1-11.

17. The use of one or more tolerogenic polypeptide, nucleic acid, vector, pharmaceutical composition or means of any of claims 1-11, in the manufacture of a medicament for treating or preventing an allergy.

18. The use or method of any of claims 12-17, wherein the allergy is one or more of HDM allergy, allergic rhinitis or asthma, and / or the allergen is HDM, such as Der p 1 derived from HDM.

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