Celiac disease epitopes

Abstract

The invention provides a peptide comprising an epitope, e.g. a T cell epitope, comprising the amino acid sequence PYPQQQQPY, or an epitope, e.g. a T cell epitope, comprising the amino acid sequence PYPQQQQPY in which one or more Q residues are replaced by E residues, the peptide being 50 amino acids or less in length. Also provided are conjugates or complexes comprising said peptides and MHC molecules, and various therapeutic and diagnostic uses of said peptides and complexes.

Description

[Technical field]

[0001] The present invention relates generally to the field of epitopes, in particular T-cell epitopes. The present invention further relates to peptides comprising said epitopes and to products relating to or containing said epitopes or peptides. Such products are of therapeutic and diagnostic use, in particular in the treatment or diagnosis of celiac disease (CeD). [Background technology]

[0002] Celiac disease (CeD) is an immune-mediated disorder involving an immune response, including an abnormal intestinal T cell response, to dietary gluten proteins, particularly from cereals such as wheat (gliadins and glutenins), barley (hordeins), and rye (secalins).

[0003] CeD has a strong genetic basis (genetic predisposition) and the disease is characterized by gluten-reactive CD4 T cells that recognize deamidated gluten peptides in the context of HLA-DQ2 (especially HLA-DQ2.5 but also HLA-DQ2.2) or HLA-DQ8 molecules (Non-patent Document 1).

[0004] Thus, the disease has a strong HLA association, with approximately 90% of patients expressing HLA-DQ2.5 (DQA1*05-DQB1*02), and most of the remaining patients expressing HLA-DQ8 (DQA1*03-DQB1*03:02) or HLA-DQ2.2 (DQA1*02:01-DQB1*02).Gluten proteins are resistant to proteolysis due to their high proline content, resulting in long immunogenic peptide fragments remaining in the intestine. Currently, T cell responses to wheat gluten are thought to be dominated by reactivity to two epitopes of α-gliadin, DQ2.5-glia-α1a (PFPQPELPY (SEQ ID NO:4)) and DQ2.5-glia-α2 (PQPELPYPQ (SEQ ID NO:5)), and two epitopes of ω-gliadin, DQ2.5-glia-ω1 (PFPQPEQPF (SEQ ID NO:6)) and DQ2.5-glia-ω2 (PQPEQPFPW (SEQ ID NO:7)), that can be found within the proteolysis-resistant α-gliadin 33-mer peptide. Importantly, the immunogenicity of gluten peptides is greatly enhanced by post-translational modification by the transglutaminase 2 (TG2) enzyme, which converts specific glutamine residues (Q) to glutamic acid (E) by deamidation. Introduction of negatively charged anchor residues would make the peptide more suitable for HLA-DQ2.5 binding, presumably by increasing the stability of the pMHC (peptide-major histocompatibility complex).

[0005] Other features of CeD are the autoantigen transglutaminase 2 (TG2) and antibodies against gluten peptides (Non-Patent Document 2, Non-Patent Document 3). Both gluten peptide-reactive B cells and TG2-reactive B cells are thought to participate in an amplification loop for pathogenic T cell responses by acting as antigen-presenting cells for gluten-reactive (anti-gluten) CD4+ T cells. Thus, both B cell (antibody) and T cell responses are involved in CeD (Non-Patent Document 4, Non-Patent Document 5).

[0006] The immune response in CeD is classically observed against proline- and glutamine-rich gluten proteins from wheat, barley, and rye, although some patients also show sensitivity to oat (avenin). CeD primarily affects the small intestine, and classical symptoms include gastrointestinal problems such as chronic diarrhea, abdominal bloating and pain, malabsorption, and loss of appetite. However, diagnosis is often difficult because these symptoms are common to other diseases and conditions. Patients may have severe symptoms and undergo testing for a long period of time before a diagnosis of CeD is made.

[0007] The only established treatment for CeD is lifelong adherence to a gluten-free diet, which generally results in recovery of the intestinal mucosa, improves symptoms, and reduces the risk of developing complications. However, such diets are very difficult to manage and adhere to, in addition to making the re-establishment of homeostasis uncertain (Non-Patent Document 6). Thus, relapse and recurrence are very common in CeD patients.

[0008] In addition, CeD is difficult to diagnose: although diagnostic assays based on the detection of TG2 antibodies or anti-gluten peptide antibodies are highly disease-specific, these assays have problems that prevent their routine use (e.g. low sensitivity or procedural complexity), which means that many patients with CeD cannot be diagnosed using current methods.

[0009] The incidence of CeD varies from as low as 1 in 300 to as high as 1 in 40 in different parts of the world, averaging 1 in 100 to 1 in 170. However, it is estimated that 80% of cases remain undiagnosed. Both children and adults can be affected by CeD.

[0010] Improved treatment and diagnostic methods for CeD are therefore highly desirable. Ideally, they would allow early diagnosis in asymptomatic people, for example by screening.

[0011] To this end, over the past few years, research has been carried out to identify and characterize gluten-derived T cell epitopes (gluten-derived peptides) that may act to trigger abnormal T cell responses, i.e., that may have a role in CeD. CD4+ T cells from CeD patients, but not from healthy subjects, recognize gluten peptides when presented by disease-associated HLA-DQ molecules (such as DQ2.5, DQ2.2, and DQ8 discussed above). In addition, it is often observed that gluten-reactive T cells from CeD patients become much better at recognizing antigenic peptides when certain glutamine (Q) residues in the peptides are converted to glutamic acid residues (E) by the enzyme TG2. These converted peptides are also called deamidated or deamidated gluten peptides. CD4+ T cells that recognize gluten peptides / epitopes presented by disease-associated HLA-DQ molecules are thought to be drivers of the disease.

[0012] Many different HLA-DQ-restricted T cell epitopes derived from gliadin (α-, γ-, and ω-gliadins), glutenins (both high and low molecular weight glutenins), hordeins, secalins, and avenins have been identified (Non-Patent Document 7). These T cell epitopes have a core region of nine amino acids (9-mers), but most CD4+ T cells recognize peptides longer than nine amino acids due to the inclusion of N- and C-terminal flanking residues. In addition, gluten-specific T cell responses generally depend on or are strongly promoted by deamidation of gluten. Although many different T cell epitopes have been identified, it is necessary to identify others to improve therapeutic and diagnostic options for CeD patients. This is especially because it is widely recognized that the pool of CeD-active gluten epitopes recognized by CD4+ T cells is far from complete, since the epitopes recognized by many T cells are unknown (Non-Patent Document 8). [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Lindfors et al., 2019, Nature Reviews Disease Primers, 5(3) [Non-Patent Document 2] Osman et al., Clin. Exp. Immunol., 2000;121(2):248-254 [Non-Patent Document 3] Dorum et al., 2016, Scientific Reports (Sci.Rep.) 6, 25565 [Non-Patent Document 4] Solid, 2002, above [Non-Patent Document 5] Stamnaes and Solid, Semin. Immuno. 2015;27(5):343-352 [Non-Patent Document 6] Stamnes et al., Adv Sci (Weinheim), Vol. 8(4), 2021 [Non-Patent Document 7] Solid et al., 2020, Immunogenetics 72, 85-88 [Non-Patent Document 8] Sherf et al., March 17, 2020, Frontiers in Nutrition (Front.Nutr.) [Non-Patent Document 9] Walkowiak et al., Nature, 2020, 588(7837):277 [Non-Patent Document 10] Raki et al., Gastroenterology, 2017, 153(3):787 Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention is based on the identification of a new T cell epitope associated with CeD. Surprisingly, this epitope has not been found in classical hexaploid bread wheat (Triticum aestivum) before, but in diploid wild type wheat (Tritcum urartu). In particular, the size and complexity of the wheat genome, as well as the lack of genome assembly data for multiple wheat accessions, may be responsible for the concealment of CeD-related epitopes, including the new epitopes disclosed herein, in hexaploid bread wheat (Non-Patent Document 9). This likely underlies the predominant lack of known epitope reactivity in CeD (Non-Patent Document 10). These epitopes therefore provide exciting new opportunities for the treatment and diagnosis of CeD.

[0015] This epitope was not identified using conventional techniques, but rather by the surprising discovery that an antibody selected for its ability to interact with the immunodominant α-gliadin epitope DQ2.5-glia-α1a displayed several properties that, upon further investigation, suggested that the antibody may also recognize additional T cell epitopes in CeD patients. Extensive database analysis combined with in vitro testing demonstrated that the antibody is capable of recognizing a novel T cell epitope associated with CeD. This T cell epitope has a 9-mer core sequence (non-deamidated sequence) PYPQQQQPY (SEQ ID NO:8). Its deamidated form is also provided.

[0016] Thus, in one embodiment, the invention provides a peptide, e.g., an isolated peptide, comprising an epitope comprising (or consisting of) the amino acid sequence PYPQQQQPY (SEQ ID NO:8), or an epitope comprising (or consisting of) the amino acid sequence PYPQQQQPY (SEQ ID NO:8) in which one or more Q residues are replaced by E residues.

[0017] Alternatively, the invention provides a peptide, e.g., an isolated peptide, that comprises the amino acid sequence PYPQQQQPY (SEQ ID NO:8), or that comprises the amino acid sequence PYPQQQQPY (SEQ ID NO:8) with one or more Q residues substituted with an E residue. [Brief description of the drawings]

[0018] [Figure 1] A. Binding properties of HLA-DQ2.5:DQ2.5-glia-α1a specific antibody 107. Eight different HLA-DQ2.5:gluten peptide complexes and HLA-DQ2.5:CLIP2 were used in ELISA for specificity analysis (n=2). mAb 2.12.E11 specific for the β chain of HLA-DQ2 was included to check for pMHC capture levels. Error bars indicate mean ± SD of duplicates. B. Plasma cells (PC) and B cells of intestinal biopsies present DQ2.5-glia-α1a peptide. Detection of DQ2.5-glia-α1a presentation in PC and B cells in single cell suspensions prepared from intestinal biopsies from either untreated celiac disease (UCD) or treated celiac disease (TCD) patients or healthy controls was performed. Mouse IgG2b mAb107 was used for detection and the percentage of positive cells was determined relative to the use of secondary antibody alone. Control patients were stratified in CD19+ PCs. Each symbol corresponds to one individual. [Figure 2-1] A. Biophysical characterization of leads. SPR was used to rank Fab fragments based on off-rate binding to HLA-DQ2.5:DQ2.5-glia-α1a. Individual clone IDs are indicated. [Figure 2-2]B. Biophysical characterization of the leads. Fab was reformatted to full length hIgG1 and analyzed by ELISA against a panel of related soluble peptide:HLA-DQ2.5 complexes. Error bars represent mean ± SD of duplicates. C. Biophysical characterization of the leads. Sequence comparison of the 9-mer core epitope of each peptide used in B. [Diagram 3] Figure 1: Assessment of mAb4.7C binding to pMHC on cells. A. Mouse A20 B cells engineered to express HLA-DQ2.5 with covalently linked peptides as indicated were stained with 5 μg / ml of antibody. B. Raji B cells were loaded in vitro with 50 μM gluten peptides as noted and stained with 5 μg / ml of antibody (12-mer α1a peptide: QLQPFPQPELPY (SEQ ID NO:53); CLIP2 peptide (MATPLLMQALPMGAL (SEQ ID NO:54)): 33-mer: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO:55)). [Figure 4-1] A. Ability of mAb4.7C to inhibit T cell activation. Activation of gliadin-specific SKW3 T cells. Raji B cells were loaded with serial dilutions of peptide and co-cultured with engineered gliadin-specific SKW3 T cells. T cell activation was measured as CD69+CD19- cells by flow cytometry. Error bars represent mean ± SD of duplicates (n=2). [Figure 4-2] B. Ability of mAb4.7C to inhibit T cell activation. 1 μM mAb4.7C or 0.1 μM pan-HLA antibodies were added to Raji B cells prior to incubation with T cells at a peptide dose that resulted in 60% of maximal T cell activation as measured by CD69 upregulation. T cell activation was calculated relative to peptide-specific T cell activation without the presence of antibody. [Diagram 5]Figure 1: pHLA-specific antibodies detect the presentation of gluten peptides in cells derived from small intestinal biopsies of CeD patients. Single cell suspensions were prepared from untreated HLA-DQ2.5+ CeD patients (n=8) (A) or controls with normal intestinal histology (n=3) (B). Cells were gated as live large lymphocytes CD3-CD11c-CD14-CD38+CD27+CD19+CD45+PC (A). Bound mIgG2b antibodies were detected by Alexa-546-conjugated secondary antibodies, and the frequency of positive cells was calculated based on gates set according to the staining of an isotype control antibody (Isotype). The mean percentage of each group is shown as a horizontal line, and the dotted line represents the mean background staining of the isotype control. Each CeD patient is represented by a unique color, and the changes in biopsy histology according to the modified Marsh score are shown. [Figure 6] Figure 1. Identification of Triticum urartu peptides homologous to DQ2.5-glia-α1a from Triticum aestivum. The peptide of the ancestral wheat species Triticum urartu, annotated as Uniprot entry A0A0E3SZN6_TRIUA, was identified by searching for DQ2.5-glia-α1a homologous peptides with no proline but preferably glycine at p10 against Triticum taxa using the ScanProsite search engine (https: / / prosite.expasy.org / scanprosite / ). Positions p6 and p10 are boxed for clarity (A). The complete amino acid sequence of Uniprot entry A0A0E3SZN6_TRIUA is shown (B). The hypothetical nonameric core of the DQ2.5-glia-α1a homologous peptide is highlighted in bold and underlined. [Figure 7]Figure 1: Evaluation of antibody binding to the A0A0E3SZN6_TRIUA epitope candidates. The A0A0E3SZN6_TRIUA peptides were aligned to the homologous DQ2.5-glia-α1a and DQ2.5-glia-α2 epitopes for comparison and the corresponding Qln (Q) to Glu (E) exchanges at p4 and p6 positions. Known P positions are indicated, and in particular the DQ2.5-glia-α1a epitope contains an artificial p10 glycine stretch (A). The indicated peptides (50 μM) were pulsed onto human DQ2.5+Raji cells as described and antibody binding was examined in a FACS (B). [Figure 8-1] A. Evaluation of antibody binding to physiologically relevant forms of the potential A0A0E3SZN6_TRIUA epitope. The T. Urartu A0A0E3SZN6_TRIUA sequence was in silico digested with trypsin and chymotrypsin with the relevant digestion fragments underlined. B. Evaluation of antibody binding to physiologically relevant forms of the potential A0A0E3SZN6_TRIUA epitope. The T. aestivum Q9M4L6_WHEAT sequence was in silico digested with trypsin and chymotrypsin with the relevant digestion fragments underlined. [Figure 8-2] C. Evaluation of antibody binding to physiologically relevant forms of the A0A0E3SZN6_TRIUA epitope candidates. For comparison, the relevant sequences were aligned to the homologous DQ2.5-glia-α1a and DQ2.5-glia-α2 epitopes and the Qln (Q) to Glu (E) exchange at the p6 position. The known P position is indicated. D. Evaluation of antibody binding to physiologically relevant forms of the A0A0E3SZN6_TRIUA epitope candidates. The indicated peptides were pulsed (50 μM) into human DQ2.5+Raji cells as described and antibody binding was examined in a FACS. [Figure 9-1]A. Evaluation of in vitro chymotrypsin proteolysis of A0A0E3SZN6_TRIUA protein. T. aestivum Q9M4L6_WHEAT (A) protein was produced in E. coli and affinity purified, followed by in vitro chymotrypsin digestion and mass spectrometry of the resulting peptide segments (D). Panel A: SDS-PAGE and Western blot analysis of Q9M4L6_WHEAT. Lane M1: Protein marker, GenScript, Cat. No. M00516, Lane M2: Protein marker, GenScript, Cat. No. M00521, Lane 1: BSA (2.00 μg), Lane 2: Q9M4L6_WHEAT (reduced condition, 2.00 μg), Lane 3: Q9M4L6_WHEAT (non-reduced condition, 2.00 μg), Lane 4: Q9M4L6_WHEAT (reduced condition), Lane 5: Q9M4L6_WHEAT (non-reduced condition), Primary antibody: Mouse anti-His mAb (GenScript, Cat. No. A00186). B. Evaluation of in vitro chymotrypsin proteolysis of A0A0E3SZN6_TRIUA protein. T. urartu A0A0E3SZN6_TRIUA (B) protein was produced in E. coli and affinity purified, followed by in vitro chymotrypsin digestion and mass spectrometry analysis of the resulting peptide segments (E). Panel B: SDS-PAGE and Western blot analysis of A0A0E3SZN6_TRIUA. Lane M1: Protein marker, GenScript, Cat. No. M00516, Lane M2: Protein marker, GenScript, Cat. No. M00521, Lane 1: BSA (2.00 μg), Lane 2: A0A0E3SZN6_TRIUA (reducing conditions, 2.00 μg), Lane 3: A0A0E3SZN6_TRIUA (reducing conditions), Primary antibody: Mouse anti-His mAb (GenScript, Cat. No. A00186). [Figure 9-2]Figure 1. Evaluation of in vitro chymotrypsin proteolysis of C.A0A0E3SZN6_TRIUA protein. T. aestivum Q9FUW7_WHEAT (C) protein was produced in E. coli and affinity purified, followed by in vitro chymotrypsin digestion and mass spectrometry analysis of the resulting peptide segments (F). Panel C: SDS-PAGE and Western blot analysis of Q9FUW7_WHEAT. Lane M1: Protein Marker, GenScript, Cat. No. M00516, Lane M2: Protein Marker, GenScript, Cat. No. M00521, Lane 1: BSA (2.00 μg), Lane 2: Q9FUW7_WHEAT (reducing conditions, 2.00 μg), Lane 3: Q9FUW7_WHEAT (reducing conditions), Primary antibody: Mouse anti-His mAb (GenScript, Cat. No. A00186). D. Evaluation of in vitro chymotrypsin proteolysis of A0A0E3SZN6_TRIUA protein. T. aestivum Q9M4L6_WHEAT(A) protein was produced in E. coli and affinity purified, followed by in vitro chymotrypsin digestion and mass spectrometry analysis of the resulting peptide segments (D). The predicted 9-mer core regions of relevant epitopes are listed. [Figure 9-3] E. Evaluation of in vitro chymotrypsin proteolysis of A0A0E3SZN6_TRIUA protein. T. Urartu A0A0E3SZN6_TRIUA (B) protein was produced in E. coli and affinity purified, followed by in vitro chymotrypsin digestion and mass spectrometry of the resulting peptide segments (E). The predicted 9-mer core regions of relevant epitopes are listed. F. Evaluation of in vitro chymotrypsin proteolysis of A0A0E3SZN6_TRIUA protein. T. aestivum Q9FUW7_WHEAT (C) protein was produced in E. coli and affinity purified, followed by in vitro chymotrypsin digestion and mass spectrometry of the resulting peptide segments (F). The predicted 9-mer core regions of relevant epitopes are listed. [Figure 10-1] A. Evaluation of CeD-derived gluten peptide-specific B cell receptor (BCR) binding to A0A0E3SZN6_TRIUA epitope candidates. Shows the common epitope of CeD prototype gluten-specific BCR 1E01 / 1E03 compared with the proven T. aestivum omega Q9FUW7_WHEAT and T. urartu A0A0E3SZN6_TRIUA epitope candidate sequences. B. Evaluation of CeD-derived gluten peptide-specific B cell receptor (BCR) binding to A0A0E3SZN6_TRIUA epitope candidates. Figure 1 shows the common epitope of the CeD prototype gluten-specific BCR 1E01 / 1E03 compared with the proven T. aestivum omega Q9FUW7_WHEAT and T. urartu A0A0E3SZN6_TRIUA epitope candidate sequences. C. Evaluation of binding of CeD-derived gluten peptide-specific B cell receptors (BCRs) to the A0A0E3SZN6_TRIUA epitope candidates. The 1E01 / 1E03 BCRs were tested for binding to immobilized peptides in ELISA in the form of reassembled soluble IgG as described. [Figure 10-2] D. Evaluation of CeD-derived gluten peptide-specific B cell receptor (BCR) binding to the A0A0E3SZN6_TRIUA epitope candidates. EC50 determination for comparison of 1E03 BCR binding to reactive PC2 and A0A0E3SZN6 peptides in a peptide catcher ELISA. [Figure 11-1]A. Production of recombinant soluble pHLA produced in insect cells. Soluble recombinant versions of HLA-DQ2.5 ectodomain with gliadin peptide covalently linked using a 15aa synthetic linker (GAGSLVPRGSGGGGS (SEQ ID NO:56)) were generated in Sf9 insect cells by GenScript and biotinylated essentially as described (Karsten et al., J Immunol, 2001, 167:4861). Integrity was assessed by evaluating equal amounts of protein by SDS PAGE and Western blot. For each version, the following gliadin peptide was used: (A) QLQPFPQPELPY (SEQ ID NO:53). Panel A: SDS-PAGE and Western blot analysis of DQB1*0201:DQ25_glia_a1a_DQA1*0501. Lane M1: Protein marker, GenScript, Cat. No. M00516, Lane M2: Protein marker, GenScript, Cat. No. M00521, Lane 1: BSA (2.00 μg), Lane 2: DQB1*0201:DQ25_glia_a1a_DQA1*0501 (reduced condition, 2.00 μg), Lane 3: DQB1*0201:DQ25_glia_a1a_DQA1*0501 (reduced condition), Primary antibody: Mouse anti-His mAb (GenScript, Cat. No. A00186), Primary antibody: Mouse anti-FLAG mAb (GenScript, Cat. No. A00187), Primary antibody: Streptavidin-HRP (GenScript, Cat. No. M00091). [Figure 11-2]B. Production of recombinant soluble pHLA produced in insect cells. Soluble recombinant versions of HLA-DQ2.5 ectodomain with gliadin peptide covalently linked using a 15aa synthetic linker (GAGSLVPRGSGGGGS (SEQ ID NO:56)) were generated in Sf9 insect cells by GenScript and biotinylated essentially as described (Karsten et al., J Immunol, 2001, 167:4861). Integrity was assessed by evaluating equal amounts of protein by SDS PAGE and Western blot. For each version, the following gliadin peptide was used: (B) QPEQPYPQQEQPY (SEQ ID NO:31). Panel B: SDS-PAGE and Western blot analysis of DQB1*0201:DQ25_glia_a1a_mutant_DQA1*0501. Lane M1: Protein marker, GenScript, Cat. No. M00516, Lane M2: Protein marker, GenScript, Cat. No. M00521, Lane 1: BSA (2.00 μg), Lane 2: DQB1*0201:DQ25_glia_a1a_mutant_DQA1*0501 (reducing condition, 2.00 μg), Lane 3: DQB1*0201:DQ25_glia_a1a_mutant_DQA1*0501 (reducing condition), Primary antibody: Mouse anti-His mAb (GenScript, Cat. No. A00186), Primary antibody: Mouse anti-FLAG mAb (GenScript, Cat. No. A00187), Primary antibody: Streptavidin-HRP (GenScript, Cat. No. M00091). [Figure 11-3]C. Production of recombinant soluble pHLA produced in insect cells. Soluble recombinant versions of HLA-DQ2.5 ectodomain with gliadin peptide covalently linked using a 15aa synthetic linker (GAGSLVPRGSGGGGS (SEQ ID NO:56)) were generated in Sf9 insect cells by GenScript and biotinylated essentially as described (Karsten et al., J Immunol, 2001, 167:4861). Integrity was assessed by evaluating equal amounts of protein by SDS PAGE and Western blot. For each version, the following gliadin peptide was used: (C) PQPELPYPQPE (SEQ ID NO:57). Panel C: SDS-PAGE and Western blot analysis of DQB1*0201:DQ25_glia_a2_DQA1*0501. Lane M1: Protein marker, GenScript, Cat. No. M00516, Lane M2: Protein marker, GenScript, Cat. No. M00521, Lane 1: BSA (2.00 μg), Lane 2: DQB1*0201:DQ25_glia_a2_DQA1*0501 (reduced condition, 2.00 μg), Lane 3: DQB1*0201:DQ25_glia_a2_DQA1*0501 (reduced condition), Primary antibody: Mouse anti-His mAb (GenScript, Cat. No. A00186), Primary antibody: Mouse anti-FLAG mAb (GenScript, Cat. No. A00187), Primary antibody: Streptavidin-HRP (GenScript, Cat. No. M00091). D. Production of recombinant soluble pHLA produced in insect cells. A deamidated version of the T. urartu A0A0E3SZN6_TRIUA identified sequence (A0A0E3SZN6_p-2E_p6E_medium) containing both BCR and T cell epitope candidate sequences is reannotated here as DQ2.5-glia-NTP-001. [Figure 12-1]A. Evaluation of antibody binding to recombinant soluble pHLA (rs-pHLA) produced in insect cells. Biotinylated soluble recombinant versions of HLA-DQ2.5 with the indicated peptides were immobilized on neuravidin-coated wells to evaluate reactivity with the indicated antibodies (mAbs) in ELISA. Detection of rs-pHLA using conformation-specific pan-HLA-DQ (mAb SPVL3) and pan-HLA-DR (mAb L243). B. Evaluation of antibody binding to recombinant soluble pHLA (rs-pHLA) produced in insect cells. Biotinylated soluble recombinant versions of HLA-DQ2.5 with the indicated peptides were immobilized on neuravidin-coated wells to evaluate reactivity with the indicated antibodies (mAbs) in ELISA. Detection of rs-pHLA by use of conformation-specific pan-HLA-DQ (mAb SPVL3) and pan-HLA-DR (mAb L243) is shown. [Figure 12-2] C. Evaluation of antibody binding to recombinant soluble pHLA (rs-pHLA) produced in insect cells. Biotinylated soluble recombinant versions of HLA-DQ2.5 with the indicated peptides were immobilized on neuravidin-coated wells to assess reactivity with the indicated antibodies (mAbs) in ELISA. Detection of rs-pHLA using conformation-specific pan-HLA-DQ (mAb SPVL3) and pan-HLA-DR (mAb L243) is shown. D. Evaluation of antibody binding to recombinant soluble pHLA (rs-pHLA) produced in insect cells. Biotinylated soluble recombinant versions of HLA-DQ2.5 with the indicated peptides were immobilized on neuravidin-coated wells to assess reactivity with the indicated antibodies (mAbs) in ELISA. Detection of individual rs-pHLA using TCR-like mAbs 107, 4.7C, and 3.C11, respectively, is shown. [Figure 12-3]E. Evaluation of antibody binding to recombinant soluble pHLA (rs-pHLA) produced in insect cells. Biotinylated soluble recombinant versions of HLA-DQ2.5 with the indicated peptides were immobilized on neuravidin-coated wells to evaluate reactivity with the indicated antibodies (mAbs) in ELISA. Detection of individual rs-pHLA using TCR-like mAbs 107, 4.7C, and 3.C11, respectively. F. Evaluation of antibody binding to recombinant soluble pHLA (rs-pHLA) produced in insect cells. Biotinylated soluble recombinant versions of HLA-DQ2.5 with the indicated peptides were immobilized on neuravidin-coated wells to evaluate reactivity with the indicated antibodies (mAbs) in ELISA. Detection of individual rs-pHLA using TCR-like mAbs 107, 4.7C, and 3.C11, respectively. [Figure 13]Figure 1: Assessment of T cell activation using TCR-reconstituted SKW3 T cells from CeD patients. (A) Raji B cells were loaded with serial dilutions of DQ2.5-glia-α1a peptide (N-QLQPFPQPELPY-C (SEQ ID NO:53)) and co-cultured with engineered gliadin-specific SKW3 T cells. T cell activation was measured as CD69+CD19- cells in flow cytometry. (B) Raji B cells were loaded with 10 μM of the indicated peptides and co-cultured with engineered gliadin-specific SKW3 T cells. T cell activation was measured as CD69+CD19- cells in flow cytometry. The following peptides were used: DQ2.5-Glia-α2(N-PQPELPYPQPE-C(SEQ ID NO:57)), A0A0E3SZN6_p4Q_p6Q_medium(N-QPQQPYPQQQQPY-C(SEQ ID NO:57)), respectively NO:20)), A0A0E3SZN6_p4Q_p6E_medium(N-QPQQPYPQQEQPY-C(SEQ ID NO:28)), A0A0E3SZN6_p4E_p6E_medium(N-QPQQPYPEQEQPY-C(SEQ ID NO:58)), A0A0E3SZN6_p6E_long(N-QPQQPYPQQEQPYGTSL-C(SEQ ID NO:30)). Phorbol myristate acetate (PMA) was used as a positive control to assess peptide-independent maximal T cell activation, and a baseline threshold for non-activated T cells was established by co-culturing SKW3 and Raji cells, respectively, in the absence of peptide. [Figure 14]Figure 1. Intracellular IFNγ flow assessment of CD4 T cell peptide stimulation capacity using PBMCs. Cryopreserved HLA-DQ2.5 typed PBMCs from confirmed healthy controls (HC) and CeD patients were purchased (HemaCare-Cellero) and used for autologous in vitro T cell peptide stimulation assays followed by intracellular IFNγ staining in flow. (A) PBMCs from CeD donor 595 were used to set an intracellular IFNγ baseline detection gate for CD3 / CD4 T cells using anti-IFNγ-PE. (B and C) Bar graphs representing intracellular IFNγ detection in the CD4+ / IFNγ+ (B) and CD4- / IFNγ+ (C) gates shown in A. PBMCs from CeD donors 595 and HC575 were stimulated with 20 μM DQ2.5-glial 33-mer (N-LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF-C (SEQ ID NO:59)) and A0A0E3SZN6_p6E_long (N-QPQQPYPQQEQPYGTSL-C (SEQ ID NO:30)) for 48 hours prior to intracellular T cell IFNγ detection. [Figure 15] Figure 13: Intracellular IFN-γ flow assessment of CD4 T cell peptide stimulation capacity using PBMCs. Cryopreserved HLA-DQ2.5 typed PBMCs from confirmed healthy controls (HC) and CeD patients were purchased (Hemacare-Cellero) and used for intracellular IFN-γ staining in flow following autologous in vitro T cell peptide stimulation assay. As in Figure 14, anti-IFN-γ-PE was used to set the intracellular IFN-γ baseline detection gate for CD3 / CD4 T cells. PBMCs from CeD donors (585, 595, and 600) and HCs (557 and 558) were stimulated with 20 μM DQ2.5-glial 33-mer (N-LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF-C (SEQ ID NO: 59)) and A0A0E3SZN6_p6E_long (N-QPQQPYPQQEQPYGTSL-C (SEQ ID NO: 30)) for 48 h prior to intracellular T cell IFN-γ detection (A and B). Values ​​are presented as mean and ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Thus, the peptides of the present invention comprise the above 9 amino acid sequences (9-mers), which may be considered as core sequences or core epitope sequences. However, when such core sequences are associated with MHC molecules, i.e., when peptide-MHC (p-MHC) complexes are involved, the flanking residues at the N-terminus and / or C-terminus of the 9-mer are also typically important for the interaction. Thus, peptides with longer sequences are also contemplated, such as peptides with the above 9-mers and additional amino acids at the N-terminus and / or C-terminus. Any suitable number of additional flanking amino acid residues may be included. For example, such additional residues may be included when the peptide is capable of associating with, forming a complex with, or binding to an MHC molecule, such as, for example, HLA-DQ2.5 or HLA-DQ2.2. Preferred examples include sequences with up to 4 amino acid residues, e.g., 1, 2, 3, or 4, at the N-terminus and / or C-terminus of the above 9-mer. Other preferred examples include sequences in which position 9 of the 9-mer as well as positions 8 and 9 are terminated by Y or PY residues, respectively, i.e., sequences without any flanking residues at the C-terminus. Some exemplary sequences are shown in Figure 9E.

[0020] The two residues immediately adjacent (flanking) the 9-mer, i.e. positions -1 or 10 (when the nine amino acids of the 9-mer are referred to as positions 1 to 9 from the N-terminus to the C-terminus), may be particularly important for the interaction of the peptide with the MHC molecule, such as for example the interaction of the peptide with the peptide binding groove of the MHC molecule. Thus, preferred peptides of the invention may contain a Q residue at position -1 and / or a G residue at position 10, e.g. the amino acid sequence QPYPQQ Q QPY (SEQ ID NO: 9), PYPQQ Q QPYG (SEQ ID NO: 10) or QPYPQQ QQPYG (SEQ ID NO:11), or a deamidated version thereof. In such deamidated versions or forms of the peptide, any one or more Q residues may be replaced with an E residue, with preferred and convenient positions for deamidation (or positioning of the E residue) being underlined.

[0021] Other preferred peptides of the invention may include a 9-mer of N-terminal QQ and / or a 9-mer of C-terminal GT. For example, such peptides may have the amino acid sequence Q QPYPQQ Q QPY (SEQ ID NO: 12), PYPQQ Q QPYGT (SEQ ID NO:13), or Q QPYPQQ Q QPYGT (SEQ ID NO:14), or a deamidated version thereof. Q QPYPQQ Q QPY (SEQ ID NO: 12), or Q QPYPQQ Q Peptides comprising QPYG (SEQ ID NO:15), or a deamidated version thereof, are preferred. In such deamidated versions or forms of the peptide, any one or more Q residues may be replaced with an E residue, with preferred and convenient positions for deamidation (or positioning of the E residue) being underlined. In exemplary peptides, one or both of these positions may be deamidated (or otherwise provided) to replace the Q residue with an E residue.

[0022] Other preferred peptides of the invention may include a 9-mer of N-terminal PQQ and / or a 9-mer of C-terminal GTS. For example, such peptides may include the amino acid sequence P Q QPYPQQ Q QPY (SEQ ID NO: 16), PYPQQ Q QPYGTS (SEQ ID NO: 17) or P Q QPYPQQ QQPYGTS (SEQ ID NO:18), or a deamidated version thereof. Q QPYPQQ Q QPY (SEQ ID NO: 16) or P Q QPYPQQ Q Peptides comprising QPYG (SEQ ID NO:19), or deamidated versions thereof, are preferred. In such deamidated versions or forms of the peptide, any one or more Q residues may be replaced with an E residue, with preferred and convenient positions for deamidation (or positioning of the E residue) being underlined. In exemplary peptides, one or both of these positions may be deamidated (or otherwise provided) to replace the Q residue with an E residue.

[0023] Other preferred peptides of the invention may comprise a 9-mer of QPQQ at the N-terminus and / or a 9-mer of GTSL at the C-terminus. For example, such peptides may have the amino acid sequence QP Q QPYPQQ Q QPY (SEQ ID NO: 20), PYPQQ Q QPYGTSL (SEQ ID NO: 21) or QP Q QPYPQQ Q QPYGTSL (SEQ ID NO:22), or a deamidated version thereof. Q QPYPQQ Q QPY(SEQ ID NO:20), QP Q QPYPQQ Q Peptides comprising QPYG (SEQ ID NO:23), or deamidated versions thereof, are preferred. In such deamidated versions or forms of the peptide, any one or more Q residues may be replaced with an E residue, with preferred and convenient positions for deamidation (or positioning of the E residue) being underlined. In exemplary peptides, one or both of these positions may be deamidated (or otherwise provided) to replace the Q residue with an E residue.

[0024] Combinations of the above examples of 1, 2, 3 or 4 flanking residues are also provided, so for example one flanking residue at the N-terminus or C-terminus can be combined with 2, 3 or 4 of the above flanking residues at the other end, or two flanking residues at the N-terminus or C-terminus can be combined with 1, 3 or 4 of the above flanking residues at the other end, or three flanking residues at the N-terminus or C-terminus can be combined with 1, 2 or 4 of the above flanking residues at the other end, or four flanking residues at the N-terminus or C-terminus can be combined with 1, 2 or 3 of the above flanking residues at the other end.

[0025] Other exemplary peptides of the invention include additional amino acids only at the N-terminus of the 9-mer. The peptides of the present invention are generally at most 50 amino acids in length (or less than 50 amino acids in length). Thus, preferred peptides are at most 50, 45, 40, 38, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 amino acids in length (or less). In this embodiment of the present invention, the minimum length of the peptide is 9 amino acids, so as to include the core 9-mer epitope sequence, such as the core T-cell epitope sequence outlined above, or its deamidated form. Thus, preferred peptides are at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. For example, from 9 to 50, 45, 40, 35, 30, or 25 amino acids in length, such as, for example, from 10, 11, 12, 13, 14, 15, 16, or 17 to 50, 45, 40, 35, 30, 25, or 20 amino acids in length. Other preferred peptides are up to 15 amino acids in length, such as, for example, from 9 to 15 amino acids in length, such as, for example, from 13 amino acids in length. Other preferred peptides are up to 20 amino acids in length, such as, for example, from 9 to 20 amino acids in length, such as, for example, from 17 amino acids in length. Other preferred peptides are up to 25 or 30 amino acids in length, such as, for example, from 9 to 25 or 9 to 30 amino acids in length, such as, for example, from 13 or 17 amino acids in length. Other preferred peptides are up to 40 amino acids in length, such as, for example, from 9 to 40 or 9 to 38 amino acids in length, such as, for example, as shown in Figure 9E, which also shows the sequences of some exemplary peptides of the invention. Deamidated (or E residue-containing) versions thereof may be preferred.

[0026] Thus, embodiments of the present invention provide a peptide, e.g., an isolated peptide, comprising an epitope comprising (or consisting of) the amino acid sequence PYPQQQQPY (SEQ ID NO:8), or an epitope comprising (or consisting of) the amino acid sequence PYPQQQQPY (SEQ ID NO:8) with one or more Q residues substituted with E residues, which peptide is 50 amino acids or less in length, and has the preferred lengths and preferred exemplary sequences described above and elsewhere herein.

[0027] It will be appreciated that the peptide or isolated peptide according to the present invention does not include the full-length omega gliadin protein from Triticum urartu (i.e. wild-type or naturally occurring omega gliadin Triticum urartu protein) in which the peptides and epitopes of the present invention can be found, or any other full-length (wild-type or naturally occurring) gliadin protein, or any other full-length (wild-type or naturally occurring) protein of the omega gliadin family, or any other full-length (wild-type or naturally occurring) protein. Thus, the peptide or isolated peptide according to the present invention does not include the full-length SEQ ID NO:1 (corresponding to Uniprot database entry number A0A0E3SZN6 (https: / / www.uniprot.org / uniprotA0A0E3SZN6) or Uniparc accession number: UPI000618D06A).

[0028] Since the peptides or isolated peptides of the invention are proposed to be related to CeD, they may be found in or derived from gluten or gliadin proteins, and thus such sequences will generally be naturally occurring sequences, such as fragments of naturally occurring sequences or deamidated versions thereof.

[0029] Thus, the peptides or isolated peptides of the invention correspond (or essentially correspond) to, for example, a region or fragment (or epitope) of a gluten or gliadin protein, such as, for example, a region or fragment (or epitope) of an entire or full-length gluten or gliadin protein, such as, for example, a full-length (wild-type or naturally occurring) omega gliadin Triticum urartu protein (e.g., as described elsewhere herein) or an equivalent or similar sequence in an alternative gluten or gliadin protein. Some peptides of the invention are considered to be naturally occurring (i.e., have their own naturally occurring counterpart or are naturally occurring, e.g., are not naturally occurring, e.g., are not naturally occurring, e.g., are not naturally occurring, e.g., are not naturally occurring fragments). Thus, some peptides of the invention (or complexes or conjugates comprising said peptides) may be considered to be artificial peptides, or synthetic peptides, or man-made peptides, or non-natural peptides.

[0030] "Corresponding" in this context means that the amino acid sequence of the isolated peptide (SEQ ID NO:) matches the amino acid sequence of the equivalent region or epitope of the wild-type (or naturally occurring) omega gliadin Triticum urartu protein (SEQ ID NO:1). "Essentially corresponding" means that the amino acid sequence of the peptide (SEQ ID NO:) is identifiable as being based on (or derived from, or a modified form of) the sequence of the equivalent region or epitope of the wild-type (or naturally occurring) omega gliadin Triticum urartu protein (SEQ ID NO:1). For example, a peptide having a sequence that "essentially corresponds" to an equivalent region or epitope of a wild-type (or naturally occurring) omega gliadin Triticum urartu protein typically has one or more (e.g., 1, 2, 3, 4, or 5, preferably 1, 2, or 3) amino acid substitutions, additions, or deletions compared to a peptide that corresponds (i.e., exactly corresponds) to the sequence of the equivalent region or epitope of a wild-type (or naturally occurring) omega gliadin Triticum urartu protein. Thus, a peptide having a sequence that "essentially corresponds" to an equivalent region or epitope of a wild-type (or naturally occurring) omega gliadin Triticum urartu protein may be considered a "substantially homologous" peptide sequence as defined elsewhere herein.

[0031] Preferred peptides of the invention comprise the amino acid sequence QPQQPYPQQQQPY (SEQ ID NO:20) (13-mer), PQQPYPQQQQPYGT (SEQ ID NO:24) (14-mer), or QPQQPYPQQQQPYGTSL (SEQ ID NO:22) (17-mer), or said sequences in which one or more of the Q residues are replaced by an E residue.

[0032] As mentioned elsewhere herein, peptides or epitopes related to CeD generally contain one or more residues where Q has been changed to an E residue. Such E-containing sequences are preferred in some embodiments, and are also referred to herein as deamidated peptides, deamidated versions, or deamidated forms, or other similar or equivalent terms. Thus, peptide sequences in which one or more or all Q residues are replaced by E residues (or where an E residue is present in place of a Q residue) are contemplated by the present invention. Thus, with reference to the 9-mer core sequence described herein (PYPQQQQPY (SEQ ID NO:8)), one, two, three, or four Q residues at (or corresponding to) positions 4, 5, 6, or 7 of the 9-mer may be replaced by E residues. However, with reference to the 9-mer core sequence, a preferred peptide of the present invention is one in which the Q residue at position 6 is replaced by an E residue. Other peptides of the present invention are those in which the Q residue at position 4 is replaced by an E residue. Other peptides of the present invention are those in which the Q residue at position 6 and the Q residue at position 4 are replaced by E residues. When this 9-mer is present within a longer peptide, the appropriate location of the E residue will be adjusted according to where in the sequence the 9-mer is present, In other words, these positions (and other amino acid positions described herein, except where otherwise indicated) are defined relative to the 9-mer core sequence, and the longer peptide sequence will have E residues at positions corresponding to those noted above in the 9-mer (e.g., position 6 and / or position 4, or positions corresponding to position 6 and / or position 4).

[0033] Thus, preferred peptides of the invention comprise the sequence PYPQQEQPY (SEQ ID NO:25), PYPEQEQPY (SEQ ID NO:26) or PYPEQQQPY (SEQ ID NO:27).

[0034] As described elsewhere herein, in CeD patients, the transglutaminase 2 (TG2) enzyme is believed to be responsible for the physiological deamidation of gluten-derived sequences observed during the progression of CeD. Thus, peptides comprising sequences deamidated by TG2 enzyme, or comprising sequences obtainable by TG2 deamidation, or comprising sequences corresponding to sequences deamidated by TG2 enzyme are preferred. These sequences thus represent exemplary preferred deamidated peptides of the present invention. TG2 enzyme can use the QXP residue as a substrate and convert this sequence to EXP by a deamidation reaction (Solid, 2002, Nature Reviews Immunology 2:647-655). Thus, the Q residue at position 6 in the above-mentioned non-deamidated (healthy or wild-type) 9-mer PYPQQQQPY (SEQ ID NO:8) is the residue most likely to be deamidated in CeD patients. Thus, peptides comprising the sequence PYPQQEQPY (SEQ ID NO:25) are particularly preferred. A specific example of such a peptide is QP Q QPYPQQEQPY (SEQ ID NO: 28), P Q QPYPQQEQPYGT (SEQ ID NO:29), or QP Q The peptide contains QPYPQQEQPYGTSL (SEQ ID NO:30).

[0035] These longer peptides, QP Q QPYPQQEQPY (SEQ ID NO: 28), P Q QPYPQQEQPYGT (SEQ ID NO:29), or QP Q In QPYPQQEQPYGTSL (SEQ ID NO:30), and some of the other longer peptides of the invention, there is an additional classical substrate for the TG2 enzyme (the QXP sequence / motif), i.e., where the sequence QQP can be deamidated to EQP. This motif is shown in the sequence above, with the Q underlined, which can be deamidated to an E residue. Again, peptides with an E residue at this position are preferred in some embodiments.

[0036] In other embodiments, a peptide of the invention comprises the sequence QPQQPYPQQQQPY (SEQ ID NO:20), in which the Q at residue 3 is replaced with an E residue, and / or the Q at residue 10 (e.g., corresponding to position 6 in the core 9-mer of an epitope of the invention, such as a T-cell epitope) is replaced with an E residue. Optionally, or additionally, the Q at residue 8 (e.g., corresponding to position 4 in the core 9-mer of an epitope of the invention, such as a T-cell epitope) is replaced with an E residue.

[0037] Thus, in some embodiments, a peptide of the invention comprises the sequence PQQPYPQQQQPYGT (SEQ ID NO:24), in which the Q at residue 2 is replaced with an E residue, and / or the Q at residue 9 (e.g., corresponding to position 6 in the core 9-mer of an epitope of the invention, such as a T-cell epitope) is replaced with an E residue. Optionally, or additionally, the Q at residue 7 (e.g., corresponding to position 4 in the core 9-mer of an epitope of the invention, such as a T-cell epitope) is replaced with an E residue.

[0038] In other embodiments, a peptide of the invention comprises the sequence QPQQPYPQQQQPYGTSL (SEQ ID NO:22), in which the Q at residue 3 is replaced with an E residue, and / or the Q at residue 10 (e.g., corresponding to position 6 in the core 9-mer of an epitope of the invention, such as a T-cell epitope) is replaced with an E residue. Optionally, or additionally, the Q at residue 8 (e.g., corresponding to position 4 in the core 9-mer of an epitope of the invention, such as a T-cell epitope) is replaced with an E residue.

[0039] A preferred example of such an embodiment is a peptide comprising the sequence: QP Q QPYPQQ E QPY (SEQ ID NO: 28), QP E QPYPQQ E QPY (SEQ ID NO: 31), QP E QPYPQQQ QPY (SEQ ID NO: 32), QP Q QPYPQQ E QPYGTSL (SEQ ID NO: 30), QP E QPYPQQ E QPYGTSL (SEQ ID NO: 33), QP E QPYPQQ Q QPYGTSL (SEQ ID NO: 34), P Q QPYPQQ E QPYGT (SEQ ID NO: 29), P E QPYPQQ E QPYGT (SEQ ID NO:35), or P E QPYPQQ Q QPYGT (SEQ ID NO:36).

[0040] The second underlined residue in these peptides corresponds to position 6 in the core 9-mer of an epitope of the invention, eg, a T cell epitope 9-mer. Other specific examples may include the above peptides which include an additional E residue at a position corresponding to position 4 in a core 9-mer of an epitope of the invention, e.g., a T-cell epitope 9-mer, or may include the above peptides where there is only one E residue at a position corresponding to position 4 in a core 9-mer of an epitope of the invention, e.g., a T-cell epitope 9-mer (in other words, both underlined residues in the nine peptides listed above are Q residues and the position corresponding to position 4 in a core 9-mer of an epitope of the invention, e.g., a T-cell epitope 9-mer is an E residue).

[0041] As mentioned elsewhere herein, CeD is believed to involve both B cell and T cell responses. Herein, peptides are described that include the core 9-mer of the newly identified epitopes, e.g., T cell epitopes, of the invention. However, some of the peptides described above and elsewhere herein also contain a second epitope, QPQQPYP (SEQ ID NO:37), which may act as, e.g., B cell epitopes. Peptides that include or further include such epitopes, or deamidated versions of such epitopes (or versions in which E residues are present in place of Q residues), e.g., one, two, or three of the Q residues at positions 1, 3, or 4 (or positions corresponding to these positions) of the B cell epitope, are replaced by E residues.

[0042] The Q residue at position 3 of the sequence QPQQPYP (SEQ ID NO:37) of this epitope, e.g., B-cell epitope, is most likely a substrate for the TG2 enzyme (it is in the motif QQP, which is the classical QXP motif for the TG2 enzyme, and should be converted to EQP). Thus, because of the proposed role of TG2 in the progression of CeD, a particularly preferred second epitope or B-cell epitope sequence found in the peptides of the invention comprises the sequence QPEQPYP (SEQ ID NO:38).

[0043] Thus, an alternative embodiment of the present invention provides an epitope, e.g. a B-cell epitope, having the sequence QPQQPYP (SEQ ID NO:37) or a deamidated version thereof as described above (or a version in which an E residue is present instead of the Q residue), in particular QPEQPYP (SEQ ID NO:38). A peptide sequence comprising the sequence QPQQPYP (SEQ ID NO:37) of this epitope, e.g. a B-cell epitope, or a deamidated version thereof as described above, in particular QPEQPYP (SEQ ID NO:38), provides a further embodiment.

[0044] For example, as defined herein, some peptides of the present invention comprise both a first or core 9-mer-containing epitope, e.g., a T cell epitope, and a second epitope, e.g., a B cell epitope. Preferred such peptides have a first epitope, e.g., a T cell epitope, and a second epitope, e.g., a B cell epitope, overlapping each other. In other words, in such embodiments, at least one of the amino acid residues of the first or T cell epitope also forms part of the second or B cell epitope, and vice versa. For example, in a particularly preferred peptide of the present invention, the PYP residues at the end of the second (or B cell) epitope also form part of the first (or T cell) epitope, e.g., providing the first three amino acids of the first (or T cell) epitope. A preferred example of such an embodiment is a peptide comprising the following sequence:

[0045] QP Q QPYPQQ E QPY (SEQ ID NO: 28), QP E QPYPQQ E QPY (SEQ ID NO: 31), QP E QPYPQQ Q QPY (SEQ ID NO: 32), QP Q QPYPQQ E QPYGTSL (SEQ ID NO: 30), QP E QPYPQQ E QPYGTSL(SEQ ID NO:33), or QP E QPYPQQ Q QPYGTSL (SEQ ID NO: 34).

[0046] A particularly preferred peptide according to this embodiment has the sequence QP E QPYPQQ E QPY (SEQ ID NO:31) or QP E QPYPQQ EQPYGTSL (SEQ ID NO:33). Another example is the sequence QP E QPYPQQ E QPYG (SEQ ID NO:39), or versions thereof having a Q residue in place of one or both E residues.

[0047] When dietary gluten is ingested, it is degraded by proteases in the intestinal space, e.g., in the lumen of the small intestine, to form partially digested gluten peptides or proteolysis-resistant peptides. Deamidation of these peptides by TG2 may then occur, e.g., in the lamina propria of the small intestine, as part of the mechanism of CeD progression. Recognition of such deamidated peptides by T cells and B cells is believed to be involved in CeD. Thus, in some embodiments of the invention, preferred peptides contain, comprise, consist of, or correspond to such naturally occurring (native) peptides, e.g., naturally occurring partially digested gluten peptides or naturally occurring proteolysis-resistant peptides (or deamidated versions thereof, in particular naturally occurring deamidated versions thereof). Trypsin digestion analysis of the peptides of the invention has shown that the peptide sequences QPQQPYPQQQQPY (SEQ ID NO:20) and / or QPQQPYPQQQQPYGTSL (SEQ ID NO:22), or naturally occurring variants thereof, such as substantially homologous sequences, e.g., having Y or PY as the final (or C-terminal) amino acid of the substantially homologous sequence (as shown at the end of a first sequence), or having L or SL as the final (or C-terminal) amino acid of the substantially homologous sequence (as shown at the end of a second sequence), are most likely to naturally occur (or be generated) in the small intestine. Thus, in some embodiments, these sequences, or sequences comprising (or consisting of) these sequences, or sequences generated (e.g., in vitro) or predicted (e.g., in silico) by trypsin digestion, e.g., trypsin and / or chymotrypsin digestion, or naturally occurring variants thereof (or deamidated versions thereof as described elsewhere herein) are preferred. Exemplary peptide sequences are shown in Figure 9E, and peptides having or comprising (or consisting of) these sequences are exemplary sequences, particularly peptides that end (or terminate) in residues PY or Y, such as those described in Figure 9E or elsewhere herein.In particular, deamidated (or E residue-containing) versions of such peptides are provided, preferably in which the E residue is present at a position described elsewhere herein, such as one or more of positions 6, -2, and 4, e.g., positions 6 and / or -2, relative to the 9-mer core sequence shown in Figure 9E.

[0048] The preferred peptide or epitope of the present invention is an isolated peptide or epitope.The preferred epitope of the present invention is a T cell epitope or a B cell epitope, particularly a T cell epitope.Therefore, the preferred peptide of the present invention comprises, contains, consists of, or corresponds to the sequence of a T cell epitope or a B cell epitope.Other preferred peptides of the present invention comprise, contain, consist of, or correspond to the sequence containing a T cell epitope and a B cell epitope.

[0049] The term "T cell epitope" as used herein refers to an amino acid sequence capable of binding to, associating with, forming a complex with, or being presented in the antigen peptide groove of an appropriate MHC / HLA molecule, here HLA-DQ2.5 or HLA-DQ2.2 molecule. In addition, such T cell epitopes, when associated with said HLA molecule, can be recognized or bound by a T cell receptor to activate T cells or promote T cell reactivity, e.g., stimulate T cell proliferation. These T cells are sometimes referred to herein as gluten-specific (or gluten-reactive) T cells or gluten-specific (or gluten-reactive) CD4+ T cells. The typical length of the core region of a T cell epitope is 9 amino acids.

[0050] The term "B cell epitope" as used herein refers to an amino acid sequence that can be recognized or bound by an antibody molecule (or B cell receptor). Such antibody molecules can be present on B cells (as B cell receptors) or in soluble form. The length of the peptide sequence recognized or bound by an antibody, e.g., the length of a B cell epitope, is highly variable. As mentioned above, the particular B cell epitope described herein contains 7 amino acids, but B cell epitopes can be shorter or longer.

[0051] The peptides or epitopes of the invention, such as, for example, T cell epitopes, may be classified as gliadin-omega (ω) peptides or epitopes. The peptides or epitopes of the invention, such as, for example, T cell epitopes, are preferably DQ2.5-restricted. In other words, they are peptides or epitopes that can bind to, associate with, or be presented by, the MHC class II / HLA molecule HLA-DQ2.5. Alternatively or additionally, the peptides or epitopes of the invention, such as, for example, T cell epitopes, may be DQ2.2-restricted. In other words, they are peptides or epitopes that can bind to, associate with, or be presented by, the MHC class II / HLA molecule HLA-DQ2.2. Thus, the peptides or epitopes of the invention may form a complex (pMHC complex) with the MHC class II / HLA molecule HLA-DQ2.5 or HLA-DQ2.2, preferably HLA-DQ2.5.

[0052] HLA-DQ2.5 (encoded by DQA1*05 and DQB1*02) is a specific type of MHC class II molecule that has a strong association with CeD. HLA-DQ2.2 (encoded by DQA1*02:01-DQB1*02) is another specific type of MHC class II molecule that has an association with CeD.

[0053] HLA-DQ2.5 is an α-chain (typically 1 Domain and α 2domain, typically encoded by DQA1*05), and a β chain (typically 1 Domains and Beta 2 domain and typically encoded by DQB1*02. The amino acid sequences of the α and β chains of HLA-DQ2.5 are set forth herein (the α chain sequence is set forth in SEQ ID NO:2 and the β chain sequence is set forth in SEQ ID NO:3).

[0054] HLA-DQ2.5 (or HLA-DQ2.2) may present gliadin peptides or epitopes, such as, for example, α-gliadin or ω-gliadin peptides or epitopes, to T cells (e.g., CD4+ T cells). Preferred peptides or epitopes presented by HLA-DQ2.5 (or HLA-DQ2.2) are the peptides or epitopes of the invention described herein, such as T cell epitopes. The amino acid sequences of various deamidated DQ2.5 epitopes and peptides of the invention are provided elsewhere herein. These deamidated forms of the DQ2.5 epitopes and peptides of the invention may be considered CeD-associated forms of the DQ2.5 epitopes and peptides, which are generally preferred.

[0055] Although longer peptides of the invention, such as peptides such as QPQQPYPQQQQPY (SEQ ID NO:20), PQQPYPQQQQPYGT (SEQ ID NO:24), or QPQQPYPQQQQPYGTSL (SEQ ID NO:22) (or deamidated versions thereof as described herein), may associate with (or bind to) HLA-DQ2.5 (or HLA-DQ2.2), the binding groove (or binding pocket or peptide groove, or antigen peptide groove) of an HLA-DQ2.5 (or HLA-DQ2.2) molecule (i.e., an MHC molecule) can only present or accommodate a single 9-mer epitope at a given time (e.g., the DQ2.5-core epitope PYPQQQQPY (SEQ ID NO:8), or a deamidated equivalent sequence as described herein). Which epitopes are presented by HLA-DQ2.5 (or HLA-DQ2.2) is determined by the "register" (or position) at which the longer peptide is bound (associated) with HLA-DQ2.5 (or HLA-DQ2.2).

[0056] A non-disease associated form (or "native" form or non-deamidated form or "healthy" form) of an epitope or peptide, such as a DQ2.5 epitope or peptide of the present invention comprises the sequence PYPQQQQPY (SEQ ID NO:8).

[0057] A preferred disease associated form (deamidated form or CeD associated form or pathogenic form) of an epitope or peptide, such as a DQ2.5 epitope or peptide of the invention comprises the sequence PYPQQEQPY (SEQ ID NO:25).

[0058] In addition, the non-disease associated form of the epitope may be present on a longer peptide, e.g., a longer peptide that is proteolysis resistant, which longer peptide comprises (or consists of) the sequence QPQQPYPQQQQPY (SEQ ID NO:20) or QPQQPYPQQQQPYGTSL (SEQ ID NO:22), or a naturally occurring variant thereof.

[0059] Similarly, a disease-associated form of an epitope may also be present on a longer peptide, such as a longer peptide that is proteolysis-resistant, which may comprise (or consist of) a deamidated form of the sequence QPQQPYPQQQQPY (SEQ ID NO:20) or QPQQPYPQQQQPYGTSL (SEQ ID NO:22), or a naturally occurring variant thereof, such as:

[0060] QP Q QPYPQQ E QPY (SEQ ID NO: 28), QP E QPYPQQ E QPY (SEQ ID NO: 31), QP E QPYPQQ Q QPY (SEQ ID NO: 32), QP Q QPYPQQ E QPYGTSL (SEQ ID NO: 30), QP E QPYPQQ E QPYGTSL (SEQ ID NO: 33), QP E QPYPQQ Q QPYGTSL (SEQ ID NO: 34), or any other E residue-containing peptide described elsewhere herein, or a naturally occurring variant of any of the above.

[0061] Also provided are peptides or epitopes comprising sequences substantially homologous to any of the sequences provided herein.

[0062] In the context of the peptide sequences of the present invention, a sequence that is "substantially homologous" to a given amino acid sequence may be a sequence that has or includes a sequence that contains 1, 2, 3, 4, 5, or 6 (preferably 1, 2, or 3) amino acid substitutions, deletions, or additions when compared to the given amino acid sequence, or a sequence that has at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the given amino acid sequence, or a sequence that has at least 6 consecutive amino acids of the given amino acid sequence.

[0063] In some preferred embodiments, an amino acid sequence that is "substantially homologous" to a peptide of the invention is a sequence that has or contains a sequence that has one, two, or three amino acid substitutions or additions or deletions (preferably one or two, more preferably one) when compared to the amino acid sequence of a given peptide.

[0064] An amino acid sequence "substantially homologous" to a peptide of the invention includes a sequence comprising (or consisting of) at least 6 consecutive amino acids of the isolated peptide (or a sequence comprising or consisting of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 15, at least 20, at least 25, or at least 30 consecutive amino acids of the isolated peptide). The typical length of the core region of a T cell epitope presented by an appropriate HLA molecule and recognized or bound by a T cell receptor is 9 amino acids. The typical length of a peptide / protein sequence recognized or bound by an antibody, such as the typical length of a B cell epitope, will be 6 or 7 amino acids or more. As described elsewhere herein, peptides comprising T cell and / or B cell epitopes are preferred, such as peptides comprising the sequences PYPQQQQPY (SEQ ID NO:8) and / or QPQQPYP (SEQ ID NO:37), or deamidated versions thereof (these sequences may overlap when both are present). Thus, other exemplary peptides of the invention include sequences that are substantially homologous to sequences that include the T cell and / or B cell epitopes described herein.

[0065] Alternative or substantially homologous peptides of the invention include sequences that are longer than 9 amino acids and include the 9-mer core sequence PYPQQQQPY (SEQ ID NO:8). In other words, preferred substantially homologous peptides have a peptide sequence as defined herein, but with amino acid variations located outside the 9-mer core sequence, such as, for example, amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 changes, preferably 1 or 2, more preferably 1) as described herein. Suitable lengths for such peptides are described elsewhere herein.

[0066] The changes in the amino acid sequence may be of conservative or non-conservative amino acids. Preferably, the changes are amino acid substitutions. Preferably, the changes are conservative amino acid substitutions.

[0067] As used herein, "conservative amino acid substitution" refers to an amino acid residue that is replaced by another amino acid residue that has a similar side chain.Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0068] The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid sequences of the invention that perform substantially the same function in substantially the same manner as the amino acid sequences of the invention. For example, any substantially homologous peptide encompassed by the invention should typically retain the ability to act as a T cell epitope and / or a B cell epitope, as appropriate. In the case of T cell epitopes, such substantially homologous peptides retain the ability to bind or associate with, for example, MHC class II / HLA molecules, particularly HLA-DQ2, such as HLA-DQ2.5 (or HLA-DQ2.2).

[0069] Alternatively or additionally (preferably additionally), such substantially homologous peptides have the ability to bind to, interact with or be recognised by a T cell receptor (conveniently a TCR that binds to or recognises the unmodified or unmutated sequence) and / or the ability to activate or stimulate a T cell response, such as, for example, T cell proliferation or inflammatory cytokine production.

[0070] In the case of B cell epitopes, such substantially homologous peptides retain the ability to be bound, for example, by anti-gluten or anti-gliadin (e.g., anti-T. Urartu omega gliadin) antibodies or B cell receptors, particularly antibodies (or BCRs) that can bind to or recognize the peptides of the invention (e.g., unmodified or unmutated peptides).

[0071] Alternatively, or in addition, such substantially homologous peptides retain the ability to act as peptides or epitopes against which (or against) omega gliadin, such as the omega gliadin of T. Urartu, can be generated (or produced).

[0072] The above-mentioned functional properties of the substantially homologous peptides of the invention can be conveniently evaluated by comparison with the unmodified or wild-type or parent peptide sequence of the invention. Typically, such functional properties of the substantially homologous peptides will be observed to at least the same amount, level, or extent as (or when compared to) that observed with the unmodified, wild-type, or parent peptide sequence of the invention.

[0073] Methods for carrying out the above-described manipulations of amino acids (eg, to generate "substantially homologous" sequences) are well known to those of skill in the art. Preferably, such peptides or substantially homologous peptides of the invention retain a Q or, preferably, an E residue at the position corresponding to position 6 of the core 9-mer described herein.

[0074] Other preferred peptides of the invention or substantially homologous peptides include a G residue at position 10, or at a position corresponding to position 10, with respect to the core 9-mer described herein. Exemplary such sequences would include a G residue at position 10, or GT residues at positions 10 and 11, respectively, or G and L residues at positions 10 and 13, respectively, or GTSL residues at positions 10, 11, 12, and 13, respectively (or corresponding positions).

[0075] Other preferred peptides or substantially homologous peptides of the invention include naturally occurring peptides (or sequences substantially homologous thereto). In particular, preferred peptides of the invention are those corresponding to peptides found in the small intestine, such as naturally occurring proteolytic fragments (or proteolytic resistant peptides) obtained or obtainable by the action of proteases in the gastrointestinal tract. To confirm the sequence of the preferred naturally occurring peptides (or substantially homologous peptides), the action of such proteases can be mimicked in vitro, such as by carrying out tryptic digestion. Such studies show that preferred peptides or substantially homologous peptides of the invention have Y or PY as the final (or C-terminal) amino acid. Other preferred peptides or substantially homologous peptides of the invention have L or SL as the final (or C-terminal) amino acid. The Y residue can be found, for example, at position 9 (or a position corresponding to position 9) of the 9-mer core described herein. The L residue can be found, for example, at position 13 (or a position corresponding to position 13) with respect to the 9-mer core described herein. Thus, peptides having these residues at these positions are preferred, while other positions may have variant amino acid sequences, for example other positions may contain any amino acid.More preferred peptides also contain the 9-mer core.

[0076] Other preferred peptides of the invention or substantially homologous peptides have Q or E residues at positions corresponding to positions 4, 5, 6, and 7 of the 9-mer core, and preferably have a Y residue at position 9 (or the corresponding residue). As described elsewhere herein, E residues at positions 4 or 6, preferably 6, in such peptides are preferred, with exemplary positions 4-7 (or the corresponding residues) in this case being QQEQ or EQEQ.

[0077] Other preferred peptides of the invention or substantially homologous peptides comprise a Q or, preferably, an E residue at a position corresponding to position -2 of the first or T cell epitope, or at a position corresponding to position 3 of the second or B cell epitope. In a preferred embodiment, this Q or E residue is the same residue in both the first (T cell) and second (B cell) epitopes.

[0078] Other preferred peptides of the invention or substantially homologous peptides comprise a Y residue at a position corresponding to position 2 of the first or T cell epitope, or at a position corresponding to position 6 of the second or B cell epitope. In a preferred embodiment, the Y residue is the same residue in both the first (T cell) and second (B cell) epitope.

[0079] Other preferred peptides of the invention or substantially homologous peptides may have aspartic acid (D) residues in place of glutamic acid (E) residues at one or more positions. For the avoidance of doubt, reference herein to a deamidated version or form of a peptide etc of the invention includes such a form of the peptide produced by any process, i.e. it does not refer solely to an E residue produced at a particular position by deamidation of a Q residue (although the peptide may be so produced, if appropriate), but refers to a peptide or amino acid sequence in which an E residue is present in place of a Q residue at one or more positions, by any suitable means, for example synthetic or recombinant production of a peptide having those E residues. Thus, as used herein most broadly, reference to a deamidated version or form of a peptide refers to a sequence having one or more E residues present in the place of, i.e. in place of, one or more Q residues present in the original peptide sequence.

[0080] Homology (e.g., sequence identity) may be assessed by any convenient method, but to determine the degree of homology (e.g., identity) between sequences, computer programs that perform multiple alignment of sequences, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994), are useful. If desired, the Clustal W algorithm can be used in conjunction with the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and a gap extension penalty of 0.1 to obtain the highest order match between two sequences where at least 50% of the total length of one of the sequences is included in the alignment. Another method that can be used to align sequences is the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981) to obtain the highest order match between two sequences and to determine the number of identical amino acids between the two sequences.Other methods for calculating the percentage of identity between two amino acid sequences are generally recognized in the art and include, for example, those described in Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) and in "Computational Molecular Biology", Lesk, ed. Oxford University Press, New York, 1988, "Biocomputing: Informatics and Genomics Projects".

[0081] Typically, a computer program will be used for such calculations. Programs for comparing and aligning pairs of sequences, such as ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990), and gapped BLAST (Altschul et al., Nucleic Acids Research, 1999, 113-110, 1999). Res. 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res. 12:387, 1984), are also useful for this purpose. In addition, the European Bioinformatics institute's Dali server provides structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998).

[0082] To provide a reference point, sequences according to the invention having at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology, sequence identity, etc. may be determined using the ALIGN program with default parameters (such as that available at the GENESTREAM network server on the internet, IGH, Montpellier, France).

[0083] In some embodiments, the present invention provides peptides, e.g., isolated peptides, that comprise (or consist of) extended or truncated versions of the sequences of (or sequences substantially homologous to) the peptides, e.g., isolated peptides, disclosed herein.

[0084] The peptides of the invention may comprise (or consist of) extended versions of the peptide sequences disclosed herein or of amino acid sequences substantially homologous to the peptide sequences disclosed herein, for example, one or more additional amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids, or 1-5 or 1-10 or 1-20 amino acids) may be present at one or both termini of the peptide sequence (or a sequence substantially homologous thereto).

[0085] Nucleic acid molecules comprising (or consisting of) a nucleotide sequence encoding a peptide or epitope or conjugate or complex of the invention as defined herein, or nucleic acid molecules substantially homologous thereto, form further aspects of the invention. Vectors or expression vectors comprising the nucleic acid molecules of the invention, or cells, e.g. host cells, comprising said expression vectors or nucleic acid molecules, form further aspects. Said vectors or cells may be used as alternatives to the nucleic acid molecules in the various aspects of the invention described herein, e.g. in therapeutic or diagnostic uses and methods.

[0086] The term "substantially homologous" as used herein in reference to nucleic acid sequences includes sequences having at least 65%, 70%, or 75%, preferably at least 80%, and even more preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the starting nucleic acid sequence.

[0087] The term "nucleic acid sequence" or "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences that include non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. The nucleic acid molecule may be double-stranded or single-stranded. The nucleic acid molecule may be wholly or partially synthetic or recombinant.

[0088] One or more nucleic acid molecules may be used to encode the desired sequences described here or elsewhere herein.

[0089] In an embodiment of the invention, a peptide or protein consisting of or consisting essentially of the amino acid sequence of a peptide or epitope or conjugate or complex as described herein, or a sequence substantially homologous thereto, or a nucleic acid molecule encoding said peptide or epitope or conjugate or complex, or a nucleic acid molecule substantially homologous thereto, form a further aspect of the invention.

[0090] A further aspect of the present invention provides a conjugate (or complex) comprising a peptide or epitope of the present invention. Typically, the conjugate (or complex) is configured to present the peptide or epitope to a T cell, such as a CD4+ T cell. Typically, the conjugate (or complex) is configured to present the peptide or epitope to a T cell receptor. The conjugate (or complex) may comprise at least one peptide or epitope of the present invention as defined elsewhere herein, coupled (e.g., linked or connected or joined or conjugated or bonded) or otherwise associated with a second component or another entity.

[0091] Exemplary conjugates (or complexes) may include peptides or epitopes of the invention bound, e.g., physically bound, to MHC molecules, typically MHC class II molecules (HLA molecules), to form peptide-MHC (p-MHC) conjugates / complexes. Such binding may be achieved by methods known and reported in the art. For example, peptides or epitopes of the invention may be bound to the α or β chain (or fragments or variants thereof, e.g., functional fragments or variants, e.g., truncated fragments or variants) of MHC or MHC class II molecules, e.g., via peptide or other linker or linkage means (e.g., chemical linkers or linkages). Thus, such linkers or linkages are typically artificial, synthetic, or non-naturally occurring linkers or linkages. Peptide linkers with suitable sequences, e.g., non-natural or artificial sequences, may preferably be conveniently provided as gene fusions. Alternatively, application of click chemistry, e.g., sortase A, may be used to provide such linkages.

[0092] Such binding or binding means can typically and advantageously be carried out via permanent, covalent or irreversible linkage.Similarly, said binding can be via association, such as physical association, where for example a peptide or epitope of the invention can be bound or associated with another entity or second component, such as for example an MHC molecule, without being joined by a physical linkage.Thus, the free or isolated peptide of the invention can be conveniently loaded or bound to an MHC molecule.

[0093] Exemplary functional fragments or variants of MHC or MHC class II molecules are those that retain the ability to bind or otherwise associate with a peptide or epitope of the invention. For example, several engineered HLA variants, such as truncation variants, have been described in the art, any of which may be used.

[0094] As elsewhere herein, the preferred MHC class II molecules for use in such conjugates (or complexes) are HLA-DQ2 molecules (or chains or fragments thereof), in particular HLA-DQ2.5 or HLA-DQ2.2 molecules (or chains or fragments thereof). Such molecules thus comprise MHC or HLA molecules, such as HLA-DQ2.5 molecules, which present (or are "loaded") with peptides or epitopes, such as gliadin epitopes, such as ω-gliadin epitopes, of the invention. In other words, such conjugates may be HLA-DQ2.5-peptide or HLA-DQ2.2-peptide conjugates / complexes (pMHC), in which the peptides or epitopes of the invention are presented (or housed) in the antigen-binding groove. Such pMHC complexes containing class II MHC molecules, such as HLA-DQ2.5-peptide or HLA-DQ2.2-peptide complexes, may also be referred to as pMHCII. Such pMHC complexes may be produced in soluble form, e.g. as isolated or recombinant molecules (e.g. recombinant soluble pMHC or pHLA molecules), may be associated with or loaded on or in another entity or carrier or formulation, may be coated or attached to a solid support, e.g. a nanoparticle or other solid support as described elsewhere herein, may be expressed in a cell, e.g. by recombinant expression, may be associated with another particle, carrier, or formulation, such as a lipid-based particle, carrier, or formulation, e.g. a liposome or micelle, etc. Such pMHC-containing entities (including multimers as described below and elsewhere herein) may be used therapeutically, e.g. for induction of T cell anergy, or may be used in diagnostic applications or detection methods described elsewhere herein, e.g. for detection (e.g. staining) of T cells, or for example for activation of T cells.

[0095] As mentioned above, the conjugate or complex of the present invention may contain two or more peptides or epitopes of the present invention bound to a second entity, such as an MHC molecule. Thus, conjugates or complexes are provided that include peptide-MHC (pMHC) multimers, such as having two, three, or four or more peptide-MHC complexes (pMHC). Particularly preferred multimeric conjugates are in the form of dimers or tetramers, such as molecules in which two or four peptide-MHC conjugates / complexes, respectively, are provided together in a single complex or molecule. Any suitable structure or design may be used for these conjugates with two or more peptide-MHC complexes, and suitable formats have been reported in the art (e.g., dimers are described in Clemente-Casares et al., 2002, Nature Immunology 3, 383-391). Additionally, the tetramer may take any of the forms described in the art, such as, for example, by conjugating each pMHC complex to a biotin-containing moiety and using biotin-streptavidin interactions to join four separate pMHC complexes together to form a tetramer (see, for example, Quarsten et al., J. Immunol., 2001, 167(9), 4861-8). In such multimeric conjugates, it is preferred that each pMHC arm of the construct, such as each individual pMHC complex, retains the ability to bind to a T cell receptor, e.g., on the surface of a CD4+ T cell.

[0096] Other conjugates may comprise the peptide or epitope of the present invention and a peptide carrier, where the peptide or epitope is bound to the peptide carrier. The peptide carrier typically enhances or can be selected to enhance immunogenicity. Thus, such conjugates are particularly useful when it is desired to induce an antigen response against the peptide or epitope. Thus, such conjugates are suitable for generating or producing antibodies against the peptide or epitope of the present invention. As described elsewhere herein, such antibodies that can bind or specifically bind to the peptide or epitope of the present invention provide further embodiments of the present invention.

[0097] Also provided is one or more nucleic acid molecules encoding such conjugates or complexes.

[0098] As indicated above, in some embodiments, the peptide (or conjugate or complex, such as pMHC) of the invention may be present on (i.e. attached or bound to) a solid support (e.g. a particle or planar support, such as a bead or microbead or nanoparticle or plate or microtiter plate). Thus, in one aspect, the invention provides a solid support to which the peptide or conjugate or complex of the invention is attached (directly or indirectly). Typically, multiple copies will be attached. Such solid supports are particularly suitable for or employed in in vitro diagnostic applications.

[0099] As noted above and described elsewhere herein, in some embodiments the peptides of the invention (or conjugates or complexes, such as pMHC) may be present in association with other entities, formulations or carriers, such as lipid-based formulations or carriers, such as liposomes or micelles. Thus, carriers or formulations, such as lipid-based carriers or formulations, comprising the peptides, conjugates or complexes of the invention form a further aspect.

[0100] Further aspects provide antigen presenting cells (APCs) or other cells that contain, present or are loaded with the peptides or epitopes or conjugates or complexes of the invention. In such embodiments, the peptides or epitopes of the invention are typically located on or associated with the cell surface. Any suitable method of preparing such cells may be used. Thus, cells may be exogenously loaded with the peptides or epitopes of the invention when the cells naturally express or contain MHC class II molecules on their surface that can bind to the peptides or epitopes. Alternatively, cells may be engineered to express the peptides or epitopes of the invention, such as by recombinant means. For example, cells may be transfected with constructs that result in the expression of appropriate pMHC (e.g., pMHC conjugates or complexes of the invention) on their surface, such as such cells may contain nucleic acid molecules of the invention. As described elsewhere herein, such cells may be used therapeutically, such as for inducing T cell anergy.

[0101] Any suitable cell type may be used. Preferred APCs may take the form of dendritic cells, macrophages, or B lymphocytes, particularly plasma cells.

[0102] A further aspect of the present invention provides binding proteins that bind or specifically bind to the peptides or epitopes or complexes or conjugates of the present invention. In particular, binding proteins that specifically bind to the peptides or epitopes or complexes or conjugates of the present invention are preferred. Binding proteins that bind or specifically bind to the deamidated (or E residue-containing) peptides or epitopes or complexes or conjugates of the present invention are also provided and are preferred in some embodiments.

[0103] In some embodiments, the binding protein is present on or expressed on a cell (cell surface), which may be a cell, such as a eukaryotic cell, such as a T cell or an NK cell, which contains or expresses the binding protein, such as a CAR T cell or a TCR T cell or a TCR-NK cell or a CAR-NK cell.

[0104] Thus, a further aspect of the invention provides a method for expressing a binding protein of the invention on the surface of a cell, e.g. a eukaryotic cell such as a T cell or a NK cell, said method may conveniently comprise the step of providing a cell with a nucleic acid molecule or construct encoding said binding protein, to allow expression of said binding protein on the surface of the cell to occur.

[0105] Because the binding proteins of the invention may target peptides and epitopes associated with CeD, in some embodiments the binding proteins of the invention may be associated or conjugated (attached) to other entities, such as other useful therapeutic entities or moieties.

[0106] For example, the binding proteins of the invention (e.g., antibodies or T cell receptors) may be conjugated or associated with a toxic or cytotoxic moiety, or some other payload, such as, for example, an inhibitory RNA molecule, such as, for example, an siRNA molecule, for example, in the form of, for example, an antibody drug conjugate (ADC). In some embodiments, the binding proteins of the invention, such as, for example, an antibody molecule, may be internalized or allow internalization into a target cell. Since the binding proteins of the invention may target pMHC complexes on APCs, when the binding proteins are then internalized, this provides a convenient way to ensure that the payload enters the target cell. Thus, depending on the payload selected, APCs expressing pMHC targets (e.g., APCs associated with CeD) may be killed or eliminated (e.g., when the payload is a cytotoxic molecule or an appropriate inhibitory RNA / siRNA molecule) or gene or protein levels may be altered (e.g., when the payload is an inhibitory RNA / siRNA). Thus, such conjugates are used therapeutically as described herein, for example, in the treatment of CeD.

[0107] Alternatively, the binding proteins of the invention (e.g., antibodies, such as TCR-like antibodies, or T cell receptors) may be conjugated or associated with a second binding protein having specificity for another entity, such as an effector cell, which may then be recruited. Suitable effector cells for which the second binding protein has specificity may be any type of cell, such as a T cell or a NK cell. Thus, bispecific molecules comprising the binding proteins of the invention are also provided, such as bispecific antibodies and cell engagers, such as bispecific T cell engager molecules (BiTEs). Suitable second binding proteins, such as antibodies or T cell receptors, can be readily selected depending on the entity or effector cell to be targeted, examples of which are well known and reported in the art. For example, when the target is a T cell or an NK cell, the second binding protein, e.g., an antibody or a T cell receptor, is optionally selected to bind to a T cell surface protein (e.g., CD3 or CD16, e.g., in the form of an anti-CD3 or anti-CD16 unit, e.g., an anti-CD3 or anti-CD16 antibody) or an NK cell surface protein (e.g., NKG2D, e.g., in the form of an anti-NKG2D unit, e.g., an anti-NKG2D antibody).

[0108] In addition, the binding proteins of the invention may be associated or conjugated (attached) to an entity that may be detected or that allows for detection, such as an entity that may be associated with a label or other detectable moiety. Such a labeled or detectable binding protein (e.g., an antibody) may then be used to detect a peptide, epitope, or pMHC complex of the invention, such as to detect an APC that displays a peptide or epitope of the invention in association with an MHC molecule. In addition, the binding proteins of the invention may target a peptide, epitope, or pMHC complex associated with CeD, such as on the surface of an antigen presenting cell, and thus the binding protein, such as an antibody, may be used to block or inhibit the interaction of T cells (e.g., gluten-specific T cells or pathogenic T cells) with pMHC complexes, thereby inhibiting or reducing T cell activation or proliferation.

[0109] Thus, the binding proteins of the invention may also be used therapeutically, for example in treatment methods such as those described elsewhere herein.

[0110] A preferred such binding protein is a T cell receptor (TCR). Said T cell receptor may be present on a cell, such as a cell, e.g. a eukaryotic cell, that contains or expresses said T cell receptor on its surface, e.g. a T cell, or an NK cell or other cell type that expresses a TCR, e.g. by recombinant means, or said T cell receptor may be a soluble T cell receptor (TCR), such as a TCR that is not associated with a cell membrane, e.g. produced by recombinant means. Thus, a further embodiment of the invention provides a cell, e.g. a T cell or an NK cell, that has or expresses a cell surface TCR that can bind or specifically bind to a peptide or epitope or complex or conjugate of the invention. Another embodiment provides a soluble TCR that can bind or specifically bind to a peptide or epitope or complex or conjugate of the invention. In particular, TCRs or T cells that can bind or specifically bind to a pMHC complex or molecule of the invention are preferred. TCRs or T cells that exhibit specific binding are also preferred. Fragments of the TCR are also included which retain functional activity, for example the ability to bind or specifically bind to a pMHC complex or molecule of the invention. TCRs which bind or specifically bind to a deamidated (or E residue containing) peptide or epitope or complex or conjugate of the invention are also provided and are preferred in some embodiments.

[0111] Another preferred binding protein is or comprises an antibody or an antigen-binding domain of an antibody that specifically binds to a peptide or epitope or a complex or conjugate of the invention. An antibody or an antigen-binding domain of an antibody that binds or specifically binds to a pMHC or pHLA complex may be referred to as a TCR-like antibody. Also provided is an antibody or an antigen-binding domain of an antibody that binds (or specifically binds) to a peptide or epitope of the invention, particularly a peptide that comprises a T cell epitope of the invention, such as a peptide that comprises a T cell epitope of the invention having a 9-mer core sequence, but does not comprise a B cell epitope of the invention (e.g., in naked, isolated, or uncomplexed form, as opposed to a complex or conjugate such as pMHC). Also provided is an antibody or an antigen-binding domain of an antibody that binds (or specifically binds) to a complex or conjugate of the invention (e.g., a peptide of the invention in the form of pMHC, as opposed to a peptide of the invention in naked, isolated, or uncomplexed form). In such an embodiment, said peptide of pMHC preferably comprises a T cell epitope of the invention, for example a T cell epitope of the invention having a 9-mer core sequence, but no B cell epitope of the invention.

[0112] Such binding proteins may be present on a cell, such as a cell, e.g. a eukaryotic cell, that contains or expresses said antibody or an antigen-binding domain of an antibody on its surface, such as a T cell, such as in the form of a CAR T cell, or such binding proteins may be soluble or isolated binding proteins, such as an antibody or an antigen-binding domain of an antibody that is not associated with a cell membrane, produced, e.g. by recombinant means.

[0113] Preferably, the binding protein comprising an antigen-binding domain of an antibody is an antibody or an antigen-binding fragment thereof.

[0114] As will be understood by one of skill in the art, immunological binding reagents encompassed by the term "antibody" include or extend to all antibodies and antigen-binding fragments thereof, including whole antibodies, dimeric, trimeric, and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof.

[0115] Thus, the term "antibody" is used to denote any antibody-like molecule having an antigen-binding region comprising one or more CDRs and a framework (FR) region (or one or more VH and / or VL regions), and this term includes, for example, Fab', Fab, F(ab') and / or F(ab') domains. 2 Antibody fragments containing antigen-binding domains such as, single domain antibodies (DAB), TandAb dimers, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabodies; kappa (lambda) bodies (scFv-CL fusions); BiTE (bispecific T cell engagers, scFv-scFv tandems for attracting T cells); DVD-Ig (dual variable domain antibodies, bispecific format); SIP (small immunoprotein, a type of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimers); DART (ds stabilized diabody "dual affinity retargeting" and small antibody mimetics.

[0116] As used herein, the terms "specifically bind" or "specifically recognize" in the context of a peptide or epitope or a complex or conjugate of the invention refer to a peptide or epitope of the invention, such as the sequence PYPQQQQPY (SEQ ID NO: 1), by which is meant a binding protein (e.g., a TCR, an antibody, or an antigen-binding domain of an antibody) that is capable of binding to, e.g., a peptide or epitope comprising HLA-DQ2.5 or a deamidated (or E residue containing) version thereof, or a complex or conjugate of the invention, e.g., a complex or conjugate comprising said peptide or epitope of the invention carried or presented on HLA-DQ2.5 or HLA-DQ2.2, and that does not cross-react (or bind) or does not significantly cross-react (or bind) with other peptides, e.g., other naked, isolated, or unconjugated peptides, or other peptides carried or presented on HLA-DQ2.5 or HLA-DQ2.2 (e.g., other celiac disease associated peptides, or other gliadin or gliadin derived peptides, or variants of gliadin derived peptides, or other gluten derived peptides). Preferably, the binding protein (e.g., a TCR, an antibody, or an antigen-binding domain of an antibody) does not cross-react (or does not bind) or does not significantly cross-react (or does not bind) with DQ2.5:DQ2.5-glia-α1a. Other preferred binding proteins (e.g., a TCR, an antibody, or an antigen-binding domain of an antibody) do not cross-react (or does not bind) or does not significantly cross-react (or does not bind) with peptides that include the B cell epitopes described herein but do not include the core 9-mer T cell epitopes of the invention, such as naked, isolated, or unconjugated peptides that include the B cell epitopes described herein, or peptides that include the B cell epitopes described herein loaded or presented on an MHC molecule (pMHC), such as HLA-DQ2.5 or HLA-DQ2.2, but do not include the core 9-mer T cell epitopes of the invention.

[0117] HLA-DQ2.5:DQ2.5-glia-α1a refers to an HLA-DQ2.5 molecule that presents (or "carries") the DQ2.5-glia-α1a epitope (PFPQPELPY (SEQ ID NO:4)). In other words, HLA-DQ2.5:DQ2.5-glia-α1a refers to an HLA-DQ2.5-peptide complex (pMHC) that presents (or houses) the DQ2.5-glia-α1a epitope in its antigen-binding groove.

[0118] Other preferred binding proteins (e.g., TCRs, antibodies, or antigen-binding domains of antibodies) are specific for the deamidated (or E residue-containing) peptides of the invention. Such binding proteins bind to the deamidated (or E residue-containing) peptides of the invention, either alone (naked or isolated form) or when associated with an MHC molecule, but do not cross-react (or bind) or significantly cross-react (or significantly bind) with the non-deamidated or healthy forms of the peptides of the invention.

[0119] In some embodiments, a binding protein of the invention, particularly an antibody or an antigen-binding domain of an antibody, has a binding affinity of 1 nM or less (e.g., 1 pM to 1 nM, or 10 pM to 1 nM, or 20 pM to 1 nM, or 50 pM to 1 nM, or 100 pM to 1 nM, or 1 pM to 500 pM, or 10 pM to 500 pM, or 20 pM to 500 pM, or 50 pM to 500 pM, or 100 pM to 500 pM, or 1 pM to 100 pM, or 10 pM to 100 pM, or or 20 pM to 100 pM, or 50 pM to 100 pM), preferably 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less (e.g., 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, or 10 pM or less), or 100 pM or less (e.g., 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, or 1 pM or less). D Preferably, the binding affinity and affinity range values ​​mentioned above apply when the antigen binding protein is in scFv format or Fab format or whole antibody format.

[0120] Binding affinity (K D ) can be measured by any suitable technique that will be known to those skilled in the art. A convenient technique would be the use of surface plasmon resonance (SPR), for example BIAcore.

[0121] Thus, the binding proteins of the invention recognize or bind to residues found in a peptide or epitope of the invention, and thus do not bind to a single MHC molecule, e.g., an empty MHC molecule or an MHC molecule not loaded with a peptide, although the binding protein may also contact or interact with some residues outside of the peptide or epitope.

[0122] In some embodiments, the binding protein is not or does not include an antibody or an antigen-binding fragment thereof, hi other embodiments, the binding protein is not or does not include the antigen-binding domain of an antibody.

[0123] In some embodiments, the binding protein is or comprises an antibody or an antigen-binding domain of an antibody, with the proviso that said binding protein does not bind to HLA-DQ2.5:DQ2.5, which presents the alpha 1a gliadin peptide, hi other embodiments, said binding protein does not bind to HLA-DQ2.2, which presents the alpha 1a gliadin peptide.

[0124] In some embodiments, the binding protein is or comprises an antibody or antigen binding protein (or an antigen binding domain of an antibody), with the proviso that said binding protein is not an antibody or antigen binding protein or domain that binds to HLA-DQ2.5:DQ2.5 presenting alpha 1a gliadin peptide, said antibody or antigen binding protein (or antigen binding domain of an antibody) comprising at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), and said antibody or antigen binding protein or domain comprising: a variable heavy (VH) CDR1 comprising the amino acid sequence GDSVSSNSAA (SEQ ID NO:40), or a sequence containing one, two or three amino acid substitutions, additions or deletions thereof; a variable heavy chain (VH) CDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO:41), or a sequence containing one, two or three amino acid substitutions, additions or deletions thereto; Amino acid sequence ARDX 4 X 5 X 6 GWX 9 X 10A variable heavy chain (VH) CDR3 comprising YGMDV (SEQ ID NO:42), X 4 can be any amino acid, preferably S or R (SEQ ID NO:43); X 5 can be any amino acid, preferably S or T (SEQ ID NO:43); X 6 can be any amino acid, preferably S or T (SEQ ID NO:43); X 9 can be any amino acid, preferably H or N or G (SEQ ID NO:43); X 10 may be any amino acid, preferably P or A (SEQ ID NO:43); A variable light (VL) CDR1 comprising the amino acid sequence HDISSY (SEQ ID NO: 44), or a sequence containing one, two or three amino acid substitutions, additions or deletions thereof; A variable light (VL) CDR2 comprising the amino acid sequence AAS (SEQ ID NO:45), or a sequence containing one amino acid substitution, addition, or deletion relative to said sequence; Amino acid sequence QX 2 LNSYPLX 9 X 10 (SEQ ID NO:46), wherein X 2 can be any amino acid, preferably D or Q (SEQ ID NO:47); X 9 may be any amino acid or no amino acid, preferably no amino acid or L (SEQ ID NO:47); X 10 and a variable light chain (VL) CDR3, wherein A may be any amino acid or no amino acid, preferably no amino acid or T (SEQ ID NO:47).

[0125] In other embodiments, antibodies or antigen binding proteins (or antigen binding domains of antibodies) that bind to HLA-DQ2.5:DQ2.5, which presents the α1a gliadin peptide, and that contain one or more (e.g., one, two, three, four, five, or all six) of the above CDRs are also excluded from the scope of the present invention.

[0126] Generally, in some embodiments, antibody molecules having CDR regions as defined above are excluded from the scope of the present invention.

[0127] In some embodiments, the binding protein is or comprises an antibody or antigen binding protein (or an antigen binding domain of an antibody), with the proviso that said binding protein is not an antibody or antigen binding protein (or an antigen binding domain of an antibody) that binds to HLA-DQ2.5:DQ2.5 presenting α1a gliadin peptide, said antibody or antigen binding protein (or antigen binding domain of an antibody) comprises at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), and said antibody or antigen binding protein (or antigen binding domain of an antibody) comprises A variable heavy chain (VH) CDR1 comprising the amino acid sequence GDSVSSNSAA (SEQ ID NO:40), or a sequence containing one, two or three amino acid substitutions, additions or deletions relative to said sequence; a variable heavy chain (VH) CDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO:41), or a sequence containing one, two or three amino acid substitutions, additions or deletions thereto; Amino acid sequence ARDX 4 X 5 X 6 GWX 9 X 10 A variable heavy chain (VH) CDR3 comprising YGMDV (SEQ ID NO:42), X 4 can be any amino acid, preferably S or R (SEQ ID NO:43); X 5 can be any amino acid, preferably S or T (SEQ ID NO:43); X 6 can be any amino acid, preferably S or T (SEQ ID NO:43); X 9 can be any amino acid, preferably H or N or G (SEQ ID NO:43); X 10 can be any amino acid, preferably P or A (SEQ ID NO:43); More preferably, said (VH) CDR3 comprises the amino acid sequence ARDSSSGWHPYGMDV (SEQ ID NO: 48); A variable light chain (VL) CDR1 comprising the amino acid sequence HDISSY (SEQ ID NO:44), or a sequence containing one, two or three amino acid substitutions, additions or deletions thereof; A variable light (VL) CDR2 comprising the amino acid sequence AAS (SEQ ID NO:45), or a sequence containing one amino acid substitution, addition, or deletion relative to said sequence; and a variable light chain (VL) CDR3 comprising the amino acid sequence QDLNSYPL (SEQ ID NO:49), or a sequence containing one, two, or three amino acid substitutions, additions, or deletions relative to said sequence.

[0128] In other embodiments, antibodies or antigen binding proteins (or antigen binding domains of antibodies) that bind to HLA-DQ2.5:DQ2.5, which presents the α1a gliadin peptide, and that contain one or more (e.g., one, two, three, four, five, or all six) of the above CDRs are also excluded from the scope of the present invention.

[0129] Generally, in some embodiments, antibody molecules having CDR regions as defined above are excluded from the scope of the present invention.

[0130] In some embodiments, the binding protein is or comprises an antibody or antigen binding protein (or an antigen binding domain of an antibody), with the proviso that said binding protein is not an antibody or antigen binding protein (or an antigen binding domain of an antibody) that binds to HLA-DQ2.5:DQ2.5 presenting α1a gliadin peptide, said antibody or antigen binding protein (or antigen binding domain of an antibody) comprises at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), and said antibody or antigen binding protein (or antigen binding domain of an antibody) comprises A variable heavy chain (VH) CDR1 comprising the amino acid sequence GDSVSSNSAA (SEQ ID NO:40), or a sequence containing one, two or three amino acid substitutions, additions or deletions relative to said sequence; A VH CDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO:41), or a sequence containing one, two or three amino acid substitutions, additions or deletions thereto; A VH CDR3 comprising the amino acid sequence ARDRTTGWHPYGMDV (SEQ ID NO:50), or a sequence containing one, two or three amino acid substitutions, additions or deletions relative to said sequence; A variable light chain (VL) CDR1 comprising the amino acid sequence HDISSY (SEQ ID NO:44), or a sequence containing one, two or three amino acid substitutions, additions or deletions thereof; A VL CDR2 comprising the amino acid sequence AAS (SEQ ID NO:45), or a sequence containing a substitution, addition, or deletion of one amino acid relative to said sequence; and a VL CDR3 comprising the amino acid sequence QDLNSYPL (SEQ ID NO:49), or a sequence containing one, two, or three amino acid substitutions, additions, or deletions relative to said sequence.

[0131] In other embodiments, antibodies or antigen binding proteins (or antigen binding domains of antibodies) that bind to HLA-DQ2.5:DQ2.5, which presents the α1a gliadin peptide, and that contain one or more (e.g., one, two, three, four, five, or all six) of the above CDRs are also excluded from the scope of the present invention.

[0132] Generally, in some embodiments, antibody molecules having CDR regions as defined above are excluded from the scope of the present invention.

[0133] An antibody having all six of the following CDR sequences:

[0134] [Table 1]

[0135] is also referred to herein as the 107 antibody. Such antibodies (or other binding proteins having all six of these CDR sequences), such as those defined in Table 1, such as antibodies having the VH and VL domains summarized in Table 1, or one or more of the other sequences summarized in Table 1, are excluded from the scope of the present invention.

[0136] In other embodiments, antibodies (or other binding proteins) having a VH and / or VL domain with at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a given amino acid sequence in Table 1 are excluded, or antibodies (or other binding proteins) having a set of six CDR domains with at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a set of six CDRs (combined set) in Table 1, i.e., the set of CDRs as a whole, are excluded.

[0137] In other embodiments, antibodies (or other binding proteins) are provided having a VH and / or VL domain that has less than 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a given amino acid sequence in Table 1, or antibodies (or other binding proteins) are provided having a set of six CDR domains that have less than 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a set of six CDRs (combined set) of Table 1, i.e., the set of CDRs as a whole.

[0138] An antibody having all six of the following CDR sequences:

[0139] [Table 2]

[0140] is also referred to herein as the 4.7C antibody. Such antibodies (or other binding proteins having all six of these CDR sequences), such as those defined in Table 2, such as antibodies having the VH and VL domains summarized in Table 2, or one or more of the other sequences summarized in Table 2, are excluded from the scope of the present invention.

[0141] In other embodiments, antibodies (or other binding proteins) having a VH and / or VL domain with at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a given amino acid sequence in Table 2 are excluded, or antibodies (or other binding proteins) having a set of six CDR domains with at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a set of six CDRs (combined set) in Table 2, i.e., the set of CDRs as a whole, are excluded.

[0142] In other embodiments, antibodies (or other binding proteins) are provided having a VH and / or VL domain that has less than 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a given amino acid sequence in Table 2, or antibodies (or other binding proteins) are provided having a set of six CDR domains that have less than 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a set of six CDRs (combined set) of Table 2, i.e., the set of CDRs as a whole.

[0143] In other embodiments, the antibodies (or other binding proteins) described in WO 2019 / 158602 are excluded.

[0144] In another embodiment, the VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWFNWVRQAPGKGLEWVGRIKTNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGEPLVNHITILDYWGQGTLVTVSS(SEQ ID NO:51), and / or the following VLs: An antibody (or other binding protein) having DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYRTPPLTFGGGTKVEIK (SEQ ID NO:52); Alternatively, the antibody designated herein as 1E03 is excluded from the scope of the present invention.

[0145] In other embodiments, antibodies (or other binding proteins) having VH and / or VL domains with at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a given amino acid sequence are excluded.

[0146] In other embodiments, antibodies (or other binding proteins) are provided that have VH and / or VL domains that have less than 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a given amino acid sequence.

[0147] Preferred exemplary peptides or epitopes or complexes or conjugates of the invention are described elsewhere herein, and these aspects relating to the binding proteins of the invention apply mutatis mutandis. For example, in some embodiments, binding proteins that bind or specifically bind to a deamidated (or E residue-containing) peptide or peptide complex (e.g., pMHC) of the invention are preferred, such as, for example, a binding protein comprising an antibody or an antigen-binding domain of an antibody, or a TCR.

[0148] Also provided is one or more nucleic acid molecules encoding the binding proteins of the invention.

[0149] A further aspect of the invention provides a method of producing (or making or isolating or identifying or generating) a binding protein such as an antibody, a binding protein comprising an antigen-binding domain of an antibody, a TCR (or T cell) of the invention, said method using a peptide or epitope or conjugate or complex (e.g. a pMHC complex) of the invention. Viewed alternatively, the invention provides the use of a peptide or epitope or conjugate or complex (e.g. a pMHC complex) of the invention for the identification (or isolation or generation or generation) of a binding protein such as an antibody, a binding protein comprising an antigen-binding domain of an antibody, a TCR (or T cell) of the invention.

[0150] Thus, a further aspect of the invention provides a method for producing (or making or isolating or identifying or generating) an antibody of the invention, or an antibody capable of binding to a peptide or epitope or conjugate or complex of the invention, comprising the step of immunising a non-human animal (e.g. a rabbit) with a peptide (or epitope or complex or conjugate) of the invention. A preferred method comprises the step of obtaining from said animal an antibody produced (or generated) against a peptide (or epitope or complex or conjugate) of the invention, and optionally a step of purifying the antibody product, and / or formulating the antibody or product (or antigen-binding fragment thereof) into a composition comprising at least one additional component, such as, for example, a carrier or excipient, e.g. a pharma- ceutically acceptable carrier or excipient.

[0151] A further aspect of the invention provides a method of producing (or making or isolating or identifying or generating) an antibody of the invention, or an antibody capable of binding to a peptide or epitope or conjugate or complex of the invention, by using a peptide, epitope, complex or conjugate of the invention in hybridoma technology (e.g., conventional hybridoma technology). Viewed alternatively, the invention provides the use of a peptide, epitope, complex or conjugate of the invention to identify (or isolate or generate or generate) an antibody of the invention, or an antibody capable of binding to a peptide or epitope or conjugate or complex of the invention, using hybridoma technology. In some embodiments, a non-human animal (e.g., a mouse) is immunized with a peptide, epitope, complex, or conjugate of the invention, and spleen cells from the immunized animal (e.g., a mouse) are isolated and fused with myeloma cells (e.g., mouse myeloma cells) lacking HGPRT expression (such myeloma cells cannot grow in HAT-containing medium), and hybrid (i.e., fusion or hybridoma) cells are selected using hypoxanthine, aminopterin, and thymine (HAT)-containing medium. Only fused cells grow in HAT-containing medium.

[0152] A further aspect of the invention provides a method of identifying (or isolating or generating) an antibody of the invention or an antibody capable of binding to a peptide or epitope or conjugate or complex of the invention, which comprises screening an antibody library using a peptide or epitope or conjugate or complex of the invention (e.g. a pMHC complex), such as by using phage display techniques (with a phage display antibody library).

[0153] Alternatively, the present invention provides for the use of the peptides or epitopes or conjugates or complexes of the present invention to identify (or isolate or generate or produce) antibodies capable of binding to the antibodies of the present invention, or peptides or epitopes or conjugates or complexes of the present invention, using phage display techniques (by phage display antibody libraries). Suitable phage display techniques and libraries are well known and standard in the art. For example, in some embodiments, the peptides, epitopes, complexes or conjugates of the present invention (typically immobilized on a solid support such as, for example, a bead or a microbead or a plate or a microtiter plate) are contacted with a phage display library (e.g., a bacteriophage library, typically a filamentous bacteriophage library such as, for example, an M13 or fd phage library) that displays (or displays or expresses) a library of antibodies or antibody fragments, such as, for example, scFv or Fab fragments, on the phage surface. Any suitable phage display antibody library may be used, with which the skilled artisan will be familiar (in addition, there are, for example, commercially available phage display antibody libraries, etc.). The identity of the displayed antibody may then be readily determined by eluting the bound phage and isolating and sequencing the phage's nucleic acid (or at least a portion of the nucleic acid encoding the displayed antibody). In some embodiments, elution of the bound phage is followed by one or more (e.g., 1, 2, 3, 4, or 5 or more) additional rounds of contacting and elution before identifying the displayed antibody of the bound phage. Such additional rounds typically further enrich the library.

[0154] Alternative screening methods for identifying (or isolating or generating) antibodies capable of binding to the antibodies or T cells / TCRs of the invention, or peptides or epitopes or conjugates or complexes of the invention, may include screening for antibodies or T cells / TCRs capable of binding or specifically binding to the peptides or epitopes or conjugates or complexes of the invention (e.g., pMHC complexes) using the peptides or epitopes or conjugates or complexes of the invention (e.g., pMHC complexes) against other suitable sources of antibodies or T cells / TCRs, such as suitable antibody or T cell-containing samples from a subject, particularly from a human subject, such as a CeD subject. Suitable exemplary samples are described elsewhere herein. Alternatively, sequence-based screening methods using NGS and bioinformatics may be used to identify such targets (see, for example, Nannini et al., 2021, MAbs.13(1):1864084).

[0155] A preferred conjugate or complex for use in such methods is a pMHC complex or conjugate. Preferred antibodies or T cells / TCRs of the invention produced by such methods are described elsewhere herein, such as antibodies or T cells / TCRs that specifically bind to a peptide or epitope or conjugate or complex (e.g. a pMHC complex) of the invention.

[0156] A further embodiment provides a method of detecting a peptide or epitope or conjugate or complex (e.g. a pMHC complex) of the invention using a binding protein, e.g. an antibody, or a protein comprising an antigen binding domain of an antibody, or a T cell / TCR, such as a binding protein of the invention, which recognizes or specifically recognizes said peptide or epitope or conjugate or complex (e.g. a pMHC complex), comprising, for example, contacting said binding protein with a sample likely to contain said peptide, epitope, conjugate or complex under conditions effective to allow formation of a complex between the binding protein and the peptide, epitope, conjugate or complex, and detecting the complex so formed.

[0157] In another aspect, the invention provides compositions comprising a peptide (or conjugate or complex) of the invention, or a nucleic acid molecule (or molecules) encoding such a peptide or conjugate or complex, or a cell or other vehicle (e.g., a solid support, particle / nanoparticle, lipid-based or other formulation as described herein) presenting or loading a peptide, conjugate, or complex of the invention. Such compositions may further comprise (e.g., in admixture) a suitable diluent, carrier, excipient, and / or preservative (e.g., a pharma- ceutically acceptable diluent, carrier, excipient, and / or preservative). Thus, in some embodiments, the compositions are pharma-ceutically acceptable compositions.

[0158] In some embodiments, the peptides or epitopes of the invention are used (e.g., for therapeutic or detection or diagnostic use) in a "naked," unconjugated or uncomplexed form, such as an isolated or "free" peptide or epitope.

[0159] Compositions according to the invention may be provided, for example, in a form suitable for oral, nasal, parenteral, intravenous, topical, or rectal administration. In preferred embodiments, compositions according to the invention are provided in a form suitable for intravenous administration. In some embodiments, compositions according to the invention are provided in a form suitable for intraperitoneal (ip) administration. In some embodiments, compositions according to the invention are provided in a form suitable for injection.

[0160] The active compounds defined herein may be provided in conventional pharmacological administration forms, such as, for example, tablets, coated tablets, nasal sprays, solutions, emulsions, nanoformulations (nanoparticles), liposomes, powders, capsules, or sustained release forms. For the preparation of these forms, conventional pharmaceutical excipients and conventional production methods may be used.

[0161] The injection solutions may be produced in a conventional manner, for example by adding preservatives, such as p-hydroxybenzoic acid, or stabilizers, such as EDTA, etc. The solution may then be filled into injection vials or ampoules.

[0162] Nose drops may also be formulated as an aqueous solution and packaged in a spray container with an aerosol propellant or provided with a means for manual compression.

[0163] The pharmaceutical compositions (formulations) of the invention are preferably administered parenterally by any suitable means. Intravenous administration is preferred. In some embodiments, administration is intraperitoneal (ip) administration. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection with a syringe. Alternatively, parenteral administration may be performed by an infusion pump. A further option is a composition that may be a powder or liquid for administration of the peptide or peptide-containing complex or conjugate in the form of a nasal spray or pulmonal spray. Additionally, as a further option, the peptide or peptide-containing complex or conjugate of the invention may be administered transdermally, for example by a patch, optionally an iontophoretic patch, or transmucosally, for example buccally.

[0164] The appropriate dosage unit can be determined by one skilled in the art. The composition, e.g., a pharmaceutical composition, may additionally comprise further active ingredients (e.g., as described elsewhere herein) in the context of a co-administration or combination (combination therapy) regimen. For example, the therapeutic methods and uses of the present invention may be used in combination with any other suitable therapeutic regimen or therapeutic agent useful for the treatment or prevention of CeD.

[0165] Vaccines comprising one or more of the peptides, epitopes, complexes or conjugates of the invention, or nucleic acid molecules encoding such entities, or cells or other vehicles as described elsewhere herein of the invention, or compositions or formulations comprising such peptides, epitopes, complexes, conjugates, nucleic acid molecules, or cells or other vehicles as described elsewhere herein form further aspects of the invention. Such vaccines or vaccine compositions, formulations or vehicles optionally further comprise a pharma- ceutically acceptable carrier and / or adjuvant.

[0166] The epitopes or peptides or complexes or conjugates of the invention, in particular the various deamidated (or E residue-containing) epitopes or peptides or complexes or conjugates of the invention, may be associated with celiac disease, and therefore such epitopes or peptides or complexes or conjugates (or more particularly, detection of such epitopes or peptides or complexes or conjugates) may be used in the diagnosis of CeD.

[0167] The present invention further provides a method for diagnosing CeD in a subject, the method comprising contacting a sample from a subject with a peptide or epitope or complex or conjugate of the present invention and determining whether the peptide or epitope or complex or conjugate binds to (or is recognised by) a T cell in the sample or whether the sample contains an antibody that binds to (or recognises) the peptide or epitope or complex or conjugate, wherein binding of the peptide or epitope or complex or conjugate to T cells (or activation of T cells) or the presence of the antibody in the sample (e.g. as determined by binding of the antibody to the peptide or epitope or complex or conjugate) indicates that the subject has or is susceptible to CeD.

[0168] Thus, such methods or diagnostic methods can also be used to determine or monitor the progression of CeD in a subject, where binding of said peptide or epitope or complex or conjugate to T cells or the presence of said antibody in a sample indicates that CeD is present (or still present). Typically, such methods involve the analysis of samples at different time points, and a result showing that binding of said peptide or epitope or complex or conjugate to T cells (or activation of T cells) or the presence of said antibody in a sample (e.g., as determined by binding of said antibody to said peptide or epitope or complex or conjugate) is reduced, preferably measurably or significantly reduced, compared to a previous result for the same subject at a previous time point indicates that CeD is improving.

[0169] Similarly, such methods may be used to determine or monitor the effectiveness of a CeD therapy, such as a CeD therapy of the present invention or any other form of CeD therapy.

[0170] Thus, such methods or diagnostic methods can also be used to determine or monitor the effectiveness of CeD therapy in a subject, where binding of said peptide or epitope or complex or conjugate to T cells (or activation of T cells) or the presence of said antibody in the sample (e.g., as determined by binding of said antibody to said peptide or epitope or complex or conjugate) indicates that CeD is present (or still present). Typically, such methods include analysis of samples at different time points, e.g., before and / or after treatment and / or at several time points after treatment, where a result indicating that binding of said peptide or epitope or complex or conjugate to T cells (or activation of T cells) or the presence of said antibody in the sample (e.g., as determined by binding of said antibody to said peptide or epitope or complex or conjugate) is reduced, preferably measurably or significantly reduced, compared to a previous result for the same subject at a previous time point indicates that CeD has improved or is being effectively treated.

[0171] Such methods or diagnostic methods can be conveniently carried out in vitro, although any suitable method or assay can be used. Suitable in vitro assays can be carried out, for example, by immobilizing the peptide or epitope of the invention, or a suitable complex or conjugate thereof, such as a pMHC molecule, on a solid support and detecting the binding of T cells or antibodies by a suitable technique, such as an ELISA assay. Similarly, such solid supports can be used to detect the activation of T cells (see, for example, Frick et al., 2020, European J.Imm. 50(1):142-145). Measuring the activation of T cells is another convenient way to measure the binding of T cells to the peptide or epitope or complex or conjugate of the invention. Suitable methods for measuring such activation are well known to those skilled in the art, and any of these methods may be used.

[0172] In one embodiment, the present invention provides a method for diagnosing CeD in a subject, the method comprising the steps of: (a) contacting a test sample obtained from said subject with one or more of the peptides or epitopes or complexes or conjugates of the invention.

[0173] In a further embodiment, the present invention provides a method of diagnosing CeD in a subject, the method comprising the steps of: (a) contacting a test sample obtained from said subject with one or more of the peptides or epitopes or complexes or conjugates of the invention; (b) determining the presence and / or amount of T cells or antibodies in the test sample that bind to (or recognize) said peptide or epitope or complex or conjugate, and optionally (c) comparing the presence and / or amount of T cells or antibodies in the test sample to a control.

[0174] In the above methods, the contacting step is carried out under conditions that allow for the formation (e.g., detectable formation) of a T cell-peptide / epitope / complex / conjugate or antibody-peptide / epitope / complex / conjugate complex. Suitable conditions can be readily determined by one of skill in the art.

[0175] Any suitable test or biological sample may be used in the above methods, such as, for example, a blood or serum sample, a biopsy cell, material from a tissue or organ suspected of being affected by CeD (e.g., the small intestine) or a tissue section.

[0176] In certain of the above methods, the presence of any amount of T cell-peptide / epitope / complex / conjugate or antibody-peptide / epitope / complex / conjugate complex in the test sample will indicate the presence of CeD. Preferably, the amount of T cell-peptide / epitope / complex / conjugate or antibody-peptide / epitope / complex / conjugate complex in the test sample for a positive diagnosis is greater, preferably measurably or significantly greater, than the amount (control value or level) found in a suitable control sample. More preferably, the significantly greater level is statistically significant, preferably having a probability value of <0.05. Suitable methods for determining statistical significance are well known and documented in the art, and any of these methods may be used.

[0177] A suitable control sample can be easily selected by those skilled in the art. For example, a suitable control in the case of a diagnosis of CeD would be a sample from a subject without CeD, such as a healthy subject. In addition, a suitable control "value" or "level" can be easily determined, such as by referring to a range for a normal or healthy subject known in the art, without performing a control "sample" in every test. Thus, the control value or level may correspond to a level in a suitable control subject or sample, such as a cut-off level or range found in a control or reference population. Alternatively, the control value or level may correspond to a level in the same individual subject or a sample from the subject measured at an earlier time point (e.g., compared to the "baseline" level of that subject). This type of control level (i.e., a control level from an individual subject) is particularly useful for embodiments of the present invention in which continuous or periodic measurements are made in healthy or diseased individuals to find changes in the level, such as in embodiments involving subject monitoring. In this regard, a suitable control value or level may be the individual's own baseline, stable, zero, or previous level (as appropriate), rather than a control or cut-off level found in a general control (e.g., healthy) population. A control level may also be referred to as a "normal" level or a "reference" level. A control level may be a discrete number or a range.

[0178] Control values ​​or levels for comparison may be derived by testing a suitable set of control subjects, although the methods of the invention will not necessarily involve active testing of control subjects as part of the methods of the invention, and will typically involve comparison with control levels previously determined from control subjects and known to the person performing the methods of the invention.

[0179] In one embodiment, the method for diagnosing celiac disease is an in vitro method. In one embodiment, the method for diagnosing celiac disease is an in vivo method. Viewed alternatively, the present invention provides a method for screening for celiac disease in a subject.

[0180] In some embodiments, the epitopes or peptides or complexes or conjugates of the present invention may be used as companion diagnostics.

[0181] The present invention further provides a method for detecting or determining or measuring the presence or amount (level) of T cells or antibodies that bind to the peptide or epitope or complex or conjugate of the present invention in a sample from a subject, for example comprising contacting a sample from a subject with the peptide or epitope or complex or conjugate of the present invention and determining whether the peptide or epitope or complex or conjugate binds to (or is recognised by) T cells in the sample or whether the sample contains an antibody that binds to (or recognises) the peptide or epitope or complex or conjugate.

[0182] Alternatively viewed, the present invention provides a method for analyzing the presence or absence or level or amount of T cells or antibodies that bind to the peptide or epitope or complex or conjugate of the present invention in a sample from a subject. For example, the method comprises contacting a sample from a subject with the peptide or epitope or complex or conjugate of the present invention and determining whether the peptide or epitope or complex or conjugate binds to (or is recognized by) T cells in the sample or whether the sample contains an antibody that binds to (or recognizes) the peptide or epitope or complex or conjugate.

[0183] The present invention further provides methods for detecting or determining or measuring the presence, absence, amount (or level) of a peptide or epitope or complex or conjugate of the invention in a sample. For example, such methods may comprise detecting whether a sample, e.g. a biological sample, e.g. a sample from a subject, contains a peptide or epitope or complex or conjugate of the invention, or the amount (or level) of said peptide or epitope.

[0184] The features and discussions in this specification relating to the diagnostic method for CeD can be applied mutatis mutandis to the above-mentioned detection method of the present invention, etc.

[0185] In the above embodiments relating to diagnosis, monitoring, determination, analysis, screening or detection, the epitopes or peptides or complexes or conjugates of the invention may be provided in any suitable format suitable for binding to T cells or antibodies, such as a format suitable for enabling measurable or detectable binding. Thus, said peptides or epitopes may be provided as naked, free or isolated peptides. However, it may be useful to provide the epitopes or peptides as complexes or conjugates with one or more MHC molecules, such as provided as pMHC complexes or conjugates (e.g. isolated or synthetically or recombinantly produced complexes or conjugates), or as larger complexes in which multiple pMHC complexes are present, such as multimers, e.g. tetramers, of pMHC. Suitable pMHC-containing complexes are described elsewhere herein. As described elsewhere herein, the peptides or complexes may be conveniently attached to a solid support or other carrier, or may be expressed on the surface of a cell, e.g. as pMHC, or loaded onto the surface of a cell, e.g. a peptide or epitope of the invention may be loaded onto a cell which already expresses the appropriate MHC molecule.

[0186] In the above embodiments, the binding of the peptide or epitope or complex or conjugate to the T cell also includes the binding of the peptide or epitope or complex or conjugate to the TCR, e.g., on the surface of the T cell or in soluble or recombinant form. The manner of detecting the binding can be to measure the activation of the T cell (e.g., by monitoring suitable markers, e.g., IL-2 levels, intracellular IFN-gamma, e.g., using flow cytometry, e.g., as described in the Examples, or by monitoring other suitable cytokine levels, e.g., using flow cytometry, or by assessing the presence / upregulation of CD69, e.g., CD69 upregulation in a CD19 negative population, e.g., using flow cytometry, e.g., as described in the Examples).

[0187] In the above embodiments relating to diagnosis or detection, the epitopes or peptides or complexes or conjugates of the invention are typically epitopes associated with CeD, and therefore these are typically deamidated versions (or E residue-containing versions).

[0188] In the above embodiments relating to diagnosis or detection (or any other suitable embodiment described herein), the epitopes or peptides or complexes or conjugates of the invention may be labeled or otherwise modified, such as with a detectable label, or in other words may be labeled or modified epitopes or peptides or complexes or conjugates. The binding proteins of the invention described herein may also be labeled, such as with a detectable label, or in other words may be labeled binding proteins.

[0189] The invention further provides a method for determining whether a composition, such as a food or other ingestible material, can cause CeD, said method comprising detecting the presence of a peptide or epitope of the invention in said composition, the presence of which, particularly at a measurable or significant level, indicates that the composition can cause CeD.

[0190] A further aspect of the present invention provides a peptide or epitope or complex or conjugate of the present invention as defined herein (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate) for use in therapy. For example, epitope-specific immunotherapy is a form of antigen-specific immunotherapy that uses peptides instead of whole antigens to target and modify CD4+ T cells.

[0191] As used herein, "therapy" refers to the treatment of any medical condition. Treatment of a disease or condition (e.g., treatment of a pre-existing disease) according to the present invention includes curing the disease or condition, or any reduction or alleviation of the disease or symptoms of the disease (e.g., reducing the severity of the disease). The therapeutic methods and uses of the present invention are suitable for the prevention of disease and for the active treatment of disease (e.g., treatment of a pre-existing disease). Thus, such treatment may be prophylactic (i.e., preventative), therapeutic (or treatment intended to be therapeutic), or palliative (i.e., treatment designed to merely limit, alleviate, or ameliorate the symptoms of a condition).

[0192] Preferably, the peptide or epitope or complex or conjugate of the invention as defined herein is for use in the treatment or prevention of CeD. Thus, in one aspect, the invention provides a peptide or epitope of the invention as defined herein (eg a free or isolated peptide or epitope of the invention) for use in the treatment or prevention of CeD.

[0193] In another aspect, the present invention provides a conjugate or complex of the present invention, in particular a conjugate or complex of an epitope or peptide of the present invention and an MHC molecule (pMHC), for use in therapy, in particular for use in the treatment or prevention of CeD.

[0194] In the embodiments described herein, nucleic acid molecules encoding said peptides or epitopes or conjugates or complexes may be used for therapy as well.

[0195] The in vivo methods and uses, eg therapeutic uses, described herein are typically carried out in humans.

[0196] Thus, the terms "animal" or "patient" or "subject" as used herein typically refer to a human.

[0197] The therapeutic methods and uses of the invention may take any suitable form, some of which are discussed below. In addition, any suitable formulation (pharmaceutical formulation) of the peptide or epitope or conjugate or complex of the invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate) may be used, examples of which are described elsewhere herein.

[0198] The therapeutic methods and uses of the present invention may take the form of vaccination or tolerisation treatments. For example, the peptides of the invention, either as free or isolated peptides or when associated with an MHC molecule (pMHC), may be used to tolerize a subject to gluten or gliadin proteins, such as by suppressing or reducing an immune response, such as by reducing the production of a T cell response (e.g. a CD4 and / or CD8 T cell response) or an antibody (B cell) response to said peptide. Thus, a further embodiment provides a peptide, epitope, conjugate or complex of the invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate) for use in tolerizing a subject to gluten or gliadin peptides or for use in suppressing or reducing an immune response.

[0199] Such methods of tolerization or suppression may then be used to treat or prevent CeD. Tolerization results in a decrease in the recognition of the epitope or peptide by the immune system, e.g., T cells and / or B cells that recognize the epitope or peptide of the invention. Thus, after such tolerization, T cell activity responding to the epitope is decreased or the T cells become unresponsive (anergic). Alternatively or additionally, after such tolerization, the amount of antibodies produced against the epitope when the epitope is present is decreased. Such tolerization may also involve the generation or induction of Treg cells (e.g., antigen / epitope-specific or gluten-specific Treg cells), thereby further suppressing the immune response.

[0200] In such uses, the peptides and epitopes of the invention are presented to the immune system in a tolerizing context, for example, such epitopes can promote the generation and proliferation of antigen (epitope)-specific Tregs (gluten-specific Tregs) to induce immune tolerance. Methods for presenting antigens (e.g., peptides or epitopes of the invention) to the immune system in such contexts are known and reported in the art. In this regard, the peptides or epitopes of the invention may be in the form of free or isolated peptides (e.g., as peptides themselves, see, e.g., Goel et al., Lancet Gastroenterol. Hepatol., 2017, 2(7):479-493, etc.) or in the form of, e.g., pMHC molecules (see, e.g., Clemente-Casares et al., 2002, supra, etc.).

[0201] The peptides and epitopes of the invention are derived from (or are highly similar to) gluten proteins, are associated with (or are highly similar to peptides associated with) CeD, are capable of binding to HLA-DQ2.5 and / or HLA-DQ2.2, and because they (or substantially homologous peptides or epitopes) can be recognised by gluten-specific or gluten-reactive CD4+ T cells, such peptides or epitopes or complexes or conjugates of the invention may be used to engage such T cells and render them less responsive or unresponsive to further antigenic stimulation.

[0202] In such a method, for example, the peptide or epitope or complex or conjugate may be administered to CeD patients on a gluten-free diet at various doses, such as a predefined maximum tolerated dose, or gradually increasing doses, and then they are challenged with gluten. The first administration may result in symptoms similar to oral gluten challenge. However, with later or subsequent administrations, these symptoms should eventually cease to exist, for example only placebo-like symptoms or no symptoms will be observed.

[0203] In such methods, gluten-specific CD4+ T cells are driven into anergy, meaning that they stop responding to antigens or become non-responsive to antigens, e.g., do not produce, or produce significantly reduced levels of, inflammatory cytokines such as interferon-γ.

[0204] For such methods, the peptides or epitopes or complexes or conjugates of the present invention can be used alone or in combination with other T cell epitopes or peptides, such as other peptides or epitopes of the present invention (e.g., mixtures of such peptides, such as mixtures of 2, 3, 4, or 5 such peptides), or with different T cell epitopes or peptides known and reported in the art, in particular other T cell epitopes or peptides associated with or specific for CeD (e.g., those described in Non-Patent Document 7). Such T cell epitopes or peptides have the ability (or function) to link (or bind to or activate) CeD-specific T cells.

[0205] Such compositions comprising the peptides or epitopes or complexes or conjugates of the invention can be considered as tolerizing vaccines or vaccine compositions and can be used to treat or prevent CeD. Thus, compositions, such as vaccine compositions, comprising the peptides or epitopes or complexes or conjugates of the invention form a further aspect of the invention.

[0206] Other tolerization treatments may involve the use of nanoparticles or other types of nanoformulations (see, for example, Clemente-Casares et al., 2016, Nature 530, 434-4402; Freitag et al., 2020, Gastroenterology 158(6):1667-1681, etc.). For example, nanoparticles may be coated or associated with a peptide or epitope of the invention, e.g., alone, e.g., as an isolated, free, or uncomplexed peptide, or in association with an MHC molecule, in particular an MHC class II molecule. In other words, the nanoparticles are coated or associated with a peptide or epitope of the invention, or with a pMHC molecule, in which the peptide (p) is a peptide or epitope of the invention, and the MHC molecule is capable of binding to said peptide or epitope. Typically, such an MHC molecule according to the invention is therefore an HLA-DQ2 molecule, in particular HLA-DQ2.5 or HLA-DQ2.2. This description of pMHC molecules is appropriate for other aspects and embodiments of the invention described elsewhere herein.

[0207] Administration of such nanoparticles can promote the generation and proliferation of antigen-specific Tregs (such as TR1-like cells), which can then act, for example, to suppress autoantigen-loaded APCs, particularly APCs that contain MHC class II molecules that load or present the epitopes or peptides of the invention. Thus, the nanoparticles can act to suppress immune responses.

[0208] Thus, such nanoparticles as described above, coated or associated with a peptide or epitope of the invention, alone or associated with an MHC molecule, in particular an MHC class II molecule, or nanoparticles coated with a peptide or epitope of the invention or with a pMHC, in which the peptide (p) is a peptide or epitope of the invention and in which the MHC molecule is capable of binding to said peptide or epitope, form further preferred aspects of the invention.

[0209] Other types of formulations, such as pharmaceutical formulations or pharmaceutical carriers, such as nanoformulations, can be used in place of nanoparticles as well, such as lipid-based formulations, such as liposomes or micelles. The interior or core of such formulations can be loaded with peptides or epitopes of the invention, such as free or isolated peptides or epitopes, or pMHC complexes as described above and elsewhere herein. Similarly, said peptides or epitopes or pMHC complexes of the invention can be associated or conjugated with the outer surface of the lipid structure. Other entities to promote tolerance may also be included in the lipid formulation (or indeed other formulations of the invention). As known to those skilled in the art, micelles are aggregates of surfactants (e.g. fatty acids) in an aqueous liquid, with the hydrophilic heads of the surfactant forming the surface of the aggregate and the hydrophobic tails forming the core. Liposomes are spherical vesicles formed by a lipid bilayer surrounding an aqueous core.

[0210] Liposomes and micelles may be synthesized using any method known in the art. Suitable methods for liposome synthesis and drug loading are described, for example, in Akbarzadeh et al., Nanoscale Res Lett 8(1):102, 2013. Liposomes and micelles may be conjugated with suitable proteins using methods known in the art, for example, methods taught in Reulen et al., Bioconjug Chem 18(2):590-596, 2007; or Kung and Redemann, Biochim Biophys Acta 862(2):435-439, 1986.

[0211] Generally, when the peptides of the invention are used in pMHC formats, formats containing multiple pMHC units (multimers) may be produced, and may be preferred, for example dimeric or tetrameric pMHC formats are known in the art and may be used advantageously, for example in detection, diagnostic and therapeutic applications.

[0212] Thus, a further aspect of the invention provides the use of a peptide or epitope or complex or conjugate of the invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate) in the manufacture of a medicament or composition for use in the therapy, preferably the treatment or prevention of CeD.

[0213] A further aspect of the invention provides the use of said peptide or epitope or complex or conjugate (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate) in the manufacture of a medicament or composition for vaccination or tolerisation treatment, such as for use in tolerising a subject to the peptide or epitope or complex or conjugate of the invention or for suppressing or reducing an immune response to said peptide or epitope or complex or conjugate.

[0214] A further aspect of the present invention provides a method of treating or preventing CeD in a subject, said method comprising the step of administering to said subject an effective amount of a peptide or epitope or complex or conjugate of the present invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate).

[0215] A further aspect of the present invention provides a method of vaccination or tolerisation treatment in a subject, said method comprising the step of administering to said subject an effective amount of a peptide or epitope or complex or conjugate of the present invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate).

[0216] A further aspect of the invention provides a method of tolerizing a subject to a peptide or epitope or complex or conjugate of the invention, said method comprising the step of administering to said subject an effective amount of a peptide or epitope or complex or conjugate of the invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate).

[0217] A further aspect of the invention provides a method of suppressing or reducing an immune response in a subject to a peptide or epitope or complex or conjugate of the invention, said method comprising the step of administering to said subject an effective amount of a peptide or epitope or complex or conjugate of the invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate).

[0218] Said method or use preferably involves administering to a subject in need thereof a medicamentously or physiologically or therapeutically effective amount of said peptide or epitope or complex or conjugate of the invention (or a nucleic acid molecule encoding said peptide or epitope or complex or conjugate).

[0219] "Pharmaceutically or physiologically or therapeutically effective amount" means an amount sufficient to show a benefit to a subject's condition or to show a relevant physiological effect in a subject, such as tolerization or reduction of an immune response. Whether an amount is sufficient to show a benefit to a subject's condition or to show a relevant physiological effect in a subject can be determined by the subject himself or by a physician.

[0220] Alternative preferred embodiments and features of the invention described elsewhere herein equally apply to these methods of treatment and use of the invention. In some embodiments (e.g., methods of detection, diagnosis, or treatment), the subject (e.g., a human subject) is a subject at risk of developing or appearing CeD, such as a healthy subject or a subject not exhibiting any symptoms of celiac disease, or any other suitable "at risk" subject, such as a first-degree relative of a CeD patient, or a subject with an associated high-risk disorder, such as, for example, type I diabetes, selective IgA deficiency, autoimmune thyroiditis, Sjogren's syndrome, Down's syndrome, Addison's disease, Turner's syndrome, or Williams syndrome. In other embodiments, the subject (e.g., a human subject) is a subject who has CeD, or is suspected of having (or developing) CeD, or who may have (or develop) CeD.

[0221] In some embodiments, a suitable subject (eg, for detection, diagnosis, or treatment) is an HLA-DQ2.5 or HLA-DQ2.2 positive subject. In some embodiments, the methods (e.g., detection, diagnosis, or treatment methods) of the invention may further include an initial step of selecting a subject (e.g., a human subject), e.g., a subject at risk of developing CeD, or a subject at risk of developing celiac disease, or a subject with celiac disease, or a subject suspected of having (or developing) celiac disease, or a subject who may have (or be developing) CeD. For example, the subject may be selected, e.g., based on the subject (or a sample from the subject, e.g., a tissue biopsy or blood / serum sample) being positive for one or more CeD markers or risk factors.

[0222] In some embodiments, the diagnostic (or similar) method of the present invention is provided, which further comprises a step of treating CeD by therapy, such as by using the therapeutic methods described herein or any other suitable therapeutic methods, such as by using the peptides or epitopes of the present invention (or using different T cell epitopes or peptides, particularly alternative T cell epitopes or peptides related to or specific for CeD). For example, when the result of the diagnostic (or similar) method of the present invention indicates CeD in the subject (e.g., a positive diagnosis of CeD is made), then an additional step of treating CeD by therapy can be performed. Suitable therapeutic methods have been reported in the art, and include a gluten-free diet (GFD) or antibody therapy, such as an anti-CD20 antibody, such as rituximab.

[0223] In other embodiments, the diagnostic (or similar) method of the present invention may be used in conjunction with other suitable additional diagnostic methods for CeD, such as any such additional methods reported in the art, such as measurement or evaluation of TG2 antibodies, or measurement or evaluation of antibodies to other gluten peptides associated with CeD, or serological testing for CeD, including the use of small intestinal histological techniques or biopsies.

[0224] In other aspects, the therapeutic methods of the invention may be used in conjunction with other suitable additional therapeutic methods for CeD. Any such additional methods may be used, such as, for example, as reported in the art, e.g., gluten-free diet (GFD), or antibody therapy, e.g., with anti-CD20 antibodies, e.g., rituximab, or therapy with different T-cell epitopes or peptides (described elsewhere herein), in particular alternative T-cell epitopes or peptides related to or specific for CeD. In some embodiments, the therapeutic methods of the invention may be used as rescue therapy after accidental gluten exposure.

[0225] The invention further includes kits comprising one or more of the peptides, epitopes, complexes, conjugates (e.g. pMHC conjugates), vaccines, binding proteins, or compositions of the invention, or one or more nucleic acid molecules encoding such entities.

[0226] Preferably, the kit is for use in the methods and uses described herein, such as, for example, the therapeutic or detection or diagnostic methods described herein, or for use in the in vitro assays or methods described herein. Preferably, the kit includes suitable instructions for using the kit components in accordance with the present invention. Preferably, the kit is for the treatment or diagnosis of a disease or for the detection methods described elsewhere herein, and optionally includes instructions for using the kit components for the treatment or diagnosis or detection of such a disease.

[0227] In addition, the peptides or epitopes or complexes or conjugates of the invention as defined herein may be used as molecular tools for in vitro or in vivo applications and assays.

[0228] Thus, a further aspect of the present invention provides reagents comprising a peptide or epitope or complex or conjugate of the invention as defined herein and the use of such peptides or epitopes or complexes or conjugates as molecular tools, e.g. in in vitro or in vivo assays. Particularly preferred molecular tools and reagents may comprise or consist of a peptide or epitope of the invention associated with an MHC molecule as described herein (pMHC molecule), which pMHC molecule may be in multimeric form, e.g. in dimeric or tetrameric form.

[0229] The terms "a" and "an" as used throughout this application are used in the sense of meaning "at least one," "at least a first," "one or more," or "multiple" of the referenced components or steps, unless an upper limit is specifically stated hereafter. Thus, as used herein, "an epitope," or "a peptide," etc., means "at least a first epitope" or "at least a first peptide." The operable limits for the amounts of any single agent and combination parameters will be known to those of skill in the art in light of this disclosure.

[0230] In addition, when the terms "comprise," "comprises," "has," or "having," or other equivalent terms are used herein, in some more specific embodiments these terms include the terms "consisting of" or "consisting essentially of," or other equivalent terms. Methods comprising particular steps also include methods consisting of those steps, where appropriate.

[0231] The epitopes, peptides, complexes, conjugates, binding proteins, nucleic acid molecules, and cells, such as APCs, of the present invention are generally "isolated" or "purified" molecules, insofar as they are distinguished from any such components that may be present in situ in a human or animal body, or in a tissue sample derived from a human or animal body. However, the sequences may correspond to or be substantially homologous to sequences found in the human or animal body. Thus, the terms "isolated" or "purified" as used herein with reference to nucleic acid molecules or sequences and proteins, peptides, or polypeptides, such as epitopes, refer to such molecules when they are isolated, purified, or substantially free of their natural environment, such as, for example, isolated or purified from the human or animal body (when they are in fact naturally occurring), or when they are produced by technical processes, including molecules produced by recombinant and synthetic processes.

[0232] The term "increase" or "enhancement" (or equivalent terms) as used herein includes any measurable increase or elevation when compared to a suitable control. Suitable controls are readily identifiable by those skilled in the art, suitable examples of which are described herein. Preferably, the increase will be significant, e.g., clinically or statistically significant, e.g., having a probability value of ≦0.05, when compared to a suitable control level or value.

[0233] The term "reduction" or "reduction" (or equivalent terms) as used herein includes any measurable decrease or reduction when compared to a suitable control. Suitable controls are readily identifiable by those skilled in the art, suitable examples of which are described herein. Preferably, the decrease will be significant, e.g., clinically or statistically significant, e.g., having a probability value of ≦0.05, when compared to a suitable control level or value.

[0234] The method of determining the statistical significance of the difference between test groups of subjects or the difference in the level or value of a particular parameter is well known and established in the art.For example, generally, in this specification, a decrease or increase is considered to be statistically significant when a probability value of ≦0.05 is shown by statistical comparison using, for example, the Student's t-test, the Mann-Whitney U-rank test, the Chi-square test or the Fisher's exact test, one-way ANOVA or two-way ANOVA test, as appropriate.

[0235] A list of some of the nucleotide and amino acid sequences disclosed herein and their sequence identifiers (SEQ ID NOs). All nucleotide sequences herein are written 5' to 3' as is customary in the art. All amino acid sequences herein are written N-terminal to C-terminal as is customary in the art.

[0236] Amino acid sequence of Tritium urartu omega gliadin (SEQ ID NO:1) UPI000618D06A(A0A0E3SZN6) Omega-gliadins Triticum urartu MKTFLIFVLLAMAMNIATAARQLNPSNKELQSPQQSFSHQQQPFLQEPYPQQPYPSQQPYPSQQPFPTPQQQFSQQSQQPFPQTQQSFPLQPQQPFPQQPQQPFPQPQLPFPQQPEQIIPQQPQQPFPLQPQQPFPQQPQQPFPQPQQPI SVQPQQPFPQQSQQSQQPFPQPQQLFLELQQPIHQQPQQPFPQQPQQPFPQQPQQPFPQQPQQPFPLQPQQPFPQ QPQQSFLLGPQQPFPQQPQQSQQSFPQPQPQQPQQPSIMQPQQPLPQRPQQPFLLPQQQLSQQPEQTISQQPQQPH QPQQPYPQQQQPYGTSL TSIGGQ Mature alpha chain of HLA-DQ2.5MHC molecule (SEQ ID NO:2) (IMGT-HLA allele name: DQA1*05:01:01:01) IVADHVASYGVNLYQSYGPSGQYTHEFDGDEQFYVDLGRKETVWCLPVLRQFRFDPQFALTNIAVLKHNLNSLIKRSNSTAATNEVPEVTVFSKSPVTLGQPNILICLVDNIFPPVVNITWLSNGHSVTEGVSETSFLSKSDHSFFKISYLTLLPSAEESYDCKVEHWGLDKPLLKHWEPEIPAPMSELTETVVCALGLSVGLVGIVVGTVFIIRGLRSVGASRHQGPL The mature β chain of the HLA-DQ2.5 MHC molecule (SEQ ID NO:3) MGT - HLA allele name: DQB1*02:01:01) RDSPEDFVYQFKGMCYFTNGTERVRLVSRSIYNREEIVRFDSDVGEFRAVTLLGLPAAEYWNSQKDILERKRAAVDRVCRHNYQLELRTTLQRRVEPTVTISPSRTEALNHHNLLVCSVTDFYPAQIKVRWFRNDQEETAGVVSTPLIRNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSKMLSGIGGFVLGLIFLGLGLIIHHRSQKGLLH

[0237]

Table 3 - 1

[0238]

Table 3 - 2

[0239]

Table 3 - 3

[0240]

Table 3 - 4

[0241]

Table 3 - 5

[0242] [Table 4-1]

[0243] [Table 4-2]

[0244] [Table 4-3]

[0245] The invention will now be further described in the following non-limiting examples, with reference to the following figures: EXAMPLES

[0246] Example 1. Identification of novel T cell epitopes associated with celiac disease material and method Human PBMCs, peptides, and selected antibodies Frozen HLA-typed purified human PBMCs from confirmed celiac disease (CeD) patients and healthy controls (HC) were purchased from Hemacare-Cellero ( https: / / cellero.com / ).

[0247] All peptides were purchased from GenScript (http: / / www.genscript.com) with a purity of ≥85%. Pan anti-DQ (SPV-L3) and pan anti-DR (L243) antibodies were purchased from Beckman-Coulter and Thermo Scientific, respectively.

[0248] Expression, purification, and validation of recombinant pHLA T cell epitope DQ2.5-glial-α1a (QLQ PFPQPELPY (SEQ ID NO:53), underlined is the 9-mer core sequence), DQ2.5-glia-α2( PQPELPYPQPE (SEQ ID NO:57)), DQ2.5-Glia-NTP-001 (QPEQ PYPQQEQPY Recombinant HLA-DQ2.5 molecules bearing a covalently linked gluten-derived peptide containing (SEQ ID NO:31) were generated by GenScript (http: / / www.genscript.com) essentially as previously described (Karsten, H. et al., 2001, supra), with C-terminal FLAG and HIS tags added to the α and β chains to facilitate affinity purification and recombinant protein detection. Briefly, soluble in vivo biotinylated recombinant pMHC produced by Sf9 insect cells was affinity purified with anti-FLAG, concentrated, and proteins analyzed by SDS-PAGE and Western blot using standard protocols for molecular weight and purity determination.

[0249] Recombinant antibody expression, purification, and validation Recombinant human IgG1 proteins were produced by GenScript (http: / / www.genscript.com). Briefly, each antibody variable (V) gene was generated by gene synthesis and cloned in frame with human constant heavy (H) and light (L) genes in the eukaryotic expression vector pcDNA3.4. Proteins were produced in Expi293F cells and affinity purified with protein A and SEC, followed by SDS-PAGE and Western blot using standard protocols for molecular weight and purity determination. The V genes for clones 107 and 4.7C are shown in Table 1 and Table 2, respectively, and those for clones 1002-1E01 and 1002-1E03 from PDB IDs 5IHZ and 5IK3, respectively (Snir et al., 2017, JCI Insight, 2(16):e93961).

[0250] Searching and identifying candidate epitope patterns DQ.2.5-glial-α1a epitope ( P FPQPE LP Y To identify putative gliadin-derived sequences with distinct but similarities to DQ.2.5-glia-α1a (SEQ ID NO:4), the UniProtKB protein database was searched using the ScanProsite tool (https: / / prosite.expasy.org / scanprosite / ). Different motifs were used, apart from the conserved key residues of the DQ.2.5-glia-α1a epitope, and some searches also allowed variation in the length of the N- and C-terminal sequences beyond the 9-mer core. More specifically, proline (P) at p1, glutamine (Q) at p6, tyrosine (Y) at p9 were fixed, and P at position p10 after the Y at p9 (underlined) was not allowed. To focus the search output, the output was restricted to the Triticum taxon. 14-mer peptides of the putative hits were synthesized and tested for binding with 107 and 4.7C antibodies when loaded into Raji cells as described. In some cases, the Q residue was replaced with glutamic acid (E) in the peptide synthesis to mimic positional deamidation.

[0251] Expression, purification, and validation of recombinant gliadin Recombinant wheat gliadin proteins were produced essentially as described by GenScript (http: / / www.genscript.com) (Arentz-Hansen EH et al., Gut 2000;46:46-51). Briefly, each gliadin gene (Uniprot codes Q9M4L6, Q9FUW7, and A0A0E3SZN6) was generated by gene synthesis to add a C-terminal HIS tag and cloned in frame into the bacterial expression vector pET17b. Proteins were produced in E. coli BL21(DE3)pLysS cells and purified under denaturing conditions from whole cell lysates using a two-step purification by ethanol precipitation and salting out, followed by SDS-PAGE and Western blot using standard protocols for molecular weight and purity determination.

[0252] ELISA Peptide capture ELISA was performed as follows: Briefly, 96-well MaxiSorp microtiter plates (Nunc) were coated with NeutrAvidin (Avidity, 5 μg / ml in PBS) overnight at 4° C. and then blocked with 2% biotin-free nonfat dry milk in PBS (w / v). Various peptides (all synthesized with a biotinylated N-terminal GSGSGS extension) were diluted to 10 μg / ml in PBS™ (PBS plus 2% biotin-free nonfat dry milk (w / v) and 0.05% Tween-20 (v / v)) and captured on NeutrAvidin. 1E01 / 1E03 antibodies were diluted to 5 μg / ml in PBSTM and added to the wells and detected with either polyclonal rabbit anti-human (Sigma, 1:10000) in PBSTM, respectively, and developed by adding TMB solution (Calbiochem) followed by absorbance reading at 620 nm (450 nm if HCl was added). Assays were performed in duplicate wells at RT. Between each layer, plates were washed 3-5x with PBST.

[0253] pHLA-specific ELISA was performed as follows: Briefly, 96-well MaxiSorp microtiter plates (Nunc) were coated with neutravidin (Avidity, 5 μg / ml in PBS) overnight at 4° C., followed by blocking with 2% biotin-free nonfat dry milk in PBS (w / v). Different biotinylated pHLA were diluted to 20 μg / ml in PBSTM and captured on neutravidin. Different antibodies were diluted to 5 μg / ml in PBSTM, added to the wells and detected with either polyclonal rabbit anti-human (Sigma, 1:10000) or anti-mouse IgG-HRP (Sigma, 1:2000) in PBST, respectively, and developed by adding TMB solution (Calbiochem) followed by absorbance reading at 620 nm (450 nm if HCl was added), respectively. Assays were performed in duplicate wells at RT. Between each layer, plates were washed 3–5x with PBST.

[0254] Gliadin protein digestion and mass spectrometry (MS) Recombinant gliadin was digested in vitro with chymotrypsin essentially as described (Molberg O et al., Methods Mol Med. 2000;41:105-24). Briefly, approximately 1 mg of recombinant gliadin was digested with 200:1 (w:w) chymotrypsin (Sigma) in 0.1 M NH 4 HCO 3 and 2M urea at 37°C for 24 h, followed by enzyme inactivation at 95°C for 5 min and further subjecting to MS analysis essentially as described (Non-Patent Document 3). MS analysis was performed by the Proteomics Core Facility at the Department of Biosciences, University of Oslo (UiO). MS spectra were analyzed by searching against a customer database created by the respective gliadin Uniprot accession codes using PEAKS studio software (Bioinformatics Solutions Inc.).

[0255] Retroviral transduction and flow cytometry of human SKW3 T cells T cell receptor (TCR)-reconstituted SKW3 clones SKW3-380 and SKW3-364 have been reported (Flick R. et al., 2021, Sci. Immunol. 6(62):eabg4925). SKW3-S2 cells were generated in essentially the same manner using the TCR V gene sequence from PFB ID 4OZI (Petersen et al., Nat Struct Mol Biol 2014, 21(5), 480-488). Briefly, the TCR V gene sequence was reconstituted as a human / mouse chimeric TCR by gene synthesis using GenScript (http: / / www.genscript.com) and cloned into pMSCV (Clontech Laboratories). Retroviral transduction of SKW3 human T cells (CLS Cell Lines Service GmbH) was performed using the Retro-X Universal Packaging System (Clontech) according to the manufacturer's instructions. Stable and homogenous TCR-expressing SKW3 T cells were obtained by standard cell expansion and FACS sorting using a FACSAria II cytometer (BD Biosciences) based on TCR expression levels assessed by H57-Alexa647 (Thermo Fisher Scientific) antibody staining. Peptide-specific activation of TCR-transduced SKW3 cells was verified using Raji cells as antigen-presenting cells essentially as described (Flick, R. et al., 2021, supra). T cell activation was measured by CD69 upregulation assessed by anti-human CD69-APC (BD Biosciences) antibody staining. Data were acquired on a BD Accuri C6 cytometer (BD Biosciences) and analyzed using FlowJo software V10 (Tree Star).

[0256] T cell activation and inhibition assays For T cell activation assays, 50,000 Raji B cells were incubated with a defined amount of DQ2.5-glia-α1a (QLQPFPQPELPY (SEQ ID NO:53)) peptide in RPMI / 10% FCS at 37°C / ON, then washed to remove residual free peptide, and 40,000 SKW3 T cells were added. Cells were cultured at 37°C / ON and then analyzed by flow cytometry. As a control, a Cell Stimulation Cocktail containing PMA and ionomycin (eBioscience, 1:500) was added to wells containing only SKW3 T cells. Based on established dose-response in T cell activation, peptide concentrations estimated to result in approximately 60% T cell activation (measured as CD69 upregulation relative to the CD19neg population) were selected for inhibition assays. Following ON incubation with peptides as above and washing, either 4.7C or 3.C11 at 1 μM (final concentration) was added to Raji cells, followed by addition of T cells and continued ON incubation. Resulting T cell activation was measured as above. As control Abs, either pan anti-DR or pan anti-DQ at 0.1 μM (final concentration) was added in parallel.

[0257] Flow cytometric detection of intracellular IFN-γ in peptide-activated PBMCs Intracellular IFN-γ was assessed in peptide-stimulated PBMCs using reagents and standard protocols from BioLegend (https: / / www.biolegend.com / ). + PBMCs (Hemacare-Cellero) were gently thawed and washed in ice-cold PBS, then resuspended in RPMI 1640 supplemented with 10% FCS (v / v) and subsequently diluted in a volume of 1 ml (approximately 2 × 10 7Cells) were aliquoted into 24-well microtiter plates (Nunc). One set of wells received 20 μM of either peptide (A0A0E3SZN6_p6E_long (QPQQPYPQQEQPYGTSL (SEQ ID NO: 30)) or 33-mer (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 55))). The remaining cells did not receive any peptide. Cells were grown for 36 h at 37°C under standard conditions, after which Brefeldin A (Biolegend) and Monensin (Biolegend) were added and incubation continued for another 12 h. Cells were then assessed for intracellular IFN-γ by flow cytometer (BD Biosciences) and analyzed using FlowJo software V10 (Tree Star).

[0258] result We have previously identified a peptide-MHC (p-MHC) antibody capable of binding to HLA-DQ2.5:DQ2.5-glia-α1a, i.e., this antibody can bind to the MHC class II molecule HLA-DQ2.5 when associated with the coeliac disease (CeD)-associated glia-α1a epitope (PFPQPQLPY (SEQ ID NO:60)), i.e., this antibody can bind to the pMHC complex.

[0259] This antibody (called the 107 antibody) has been shown to react specifically with samples obtained from CeD patients and in an HLA-specific manner, i.e., the antibody is CeD-specific and also specific for the HLA molecule HLA-DQ2.5 (see Figure 1).

[0260] An affinity matured version of this antibody was also generated (called the 4.7C antibody), which has a higher affinity for binding to the pMHC complex than the parent antibody 107 (see Figure 2).

[0261] Detection of cell surface pMHC The above experiments were performed by assessing binding to soluble recombinant pMHC molecules. However, the 4.7C antibody also showed the ability to bind well and peptide-specifically to antigen-presenting cells (A20 mouse B cells) engineered to recombinantly express on their surface a pMHC complex in the form of HLA-DQ2.5 covalently linked to the α1a peptide and a control peptide (Figure 3A). A 12-mer peptide containing the minimal α1a peptide (underlined) was used (QLQ PFPQPELPY In this experiment, the CeD-related form of the epitope, i.e., an epitope containing an E rather than a Q residue (PFPQPQLPY (SEQ ID NO:60) vs. PFPQPQLPY (SEQ ID NO:61)) was used. E LPY (SEQ ID NO:4) was used.

[0262] Interestingly, when antibody 4.7C was tested against human antigen-presenting cells (Raji cells) with physiological levels of native MHC expression exogenously loaded with the same 12-mer containing the minimal epitope as a soluble peptide (i.e., peptide-pulsed cells), the binding was significantly weaker (Figure 3B). In addition, when the gluten 33-mer peptide was exogenously loaded, the antibody showed no binding to these antigen-presenting cells (Figure 3B). The α-gliadin 33-mer (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO:55)), which is thought to be a naturally occurring fragment and is strongly related to CeD, contains one copy of the minimal α1a epitope and three overlapping copies of the DQ2.5-glia-α2 epitope. These results were surprising given the results of binding to recombinantly expressed α1a peptide. However, there are several possible reasons for this, including that the interaction of 12-mers or 33-mers with HLA-DQ2.5 after loading of exogenous peptides may not be stable enough, or the fact that native Raji cells do not express as much MHC as cells recombinantly overexpressing pMHC and therefore there would be much fewer pMHC complexes on the cell surface (lower density of pMHC expression). Despite these possible confounding parameters, 33-mers are known to bind better to HLA molecules than shorter peptides, as evidenced, for example, in T cell activation assays (Gunnarsen et al., 2017, JCI Insight;2(17):e95193), and therefore it was expected that 33-mers would show better staining than 12-mers when compared to 12-mers, which was not the case.

[0263] Inhibition of T cell activation The ability of antibody 4.7C to inhibit T cell activation in vitro was also examined using a human T cell line (SKW380) expressing a TCR specific for DQ2.5-α1a. Raji cells loaded with a defined amount of stimulatory gliadin peptide (QLQPFPQPELPY (SEQ ID NO:53)) were co-cultured with SKW380 T cells, and T cell activation was measured by determining CD69 expression on SKW380 cells using flow cytometry (Figure 4A). Peptide concentrations inducing 60% T cell activation were selected for further experiments in which the T cell inhibitory abilities of the antibodies were evaluated by incubating peptide-loaded Raji cells with the antibodies and adding SKW380 T cells (Figure 4B).

[0264] Approximately 20% inhibition of T cell activation was observed and this inhibition was specific, although a higher level of inhibition would have been expected.

[0265] Staining of human small intestinal biopsies When CD19+CD45+ plasma cells derived from small intestinal biopsy samples obtained from inflamed mucosa of untreated, confirmed HLA-DQ2.5+ CeD patients and control subjects were examined, the high affinity (4.7C) and parental (107) antibodies showed good staining of the CeD material, indicating that they were detecting HLA-DQ2.5-associated gluten peptide presentation in cells from CeD patients (Figure 5, each patient shown as a separate circle).

[0266] These data were fully consistent with the previously reported extensive characterization of the 107 antibody and its ability to specifically detect peptide presentation in intact disease-, HLA-, and epitope-similar CeD patient material (Hoydahl et al., 2019, Gastroenterology 156(5), 1428-1439).

[0267] Interestingly, even though the 4.7C antibody was selected for its higher affinity binding to the DQ2.5-glia-α1a epitope (FIG. 2A), the levels of staining in patient samples were similar for the two antibodies (FIG. 5A).

[0268] Search for potential new T cell epitope sequences To gain better insight into this apparent difference in binding behavior and the less than expected ability to inhibit T cell activation, it was considered whether antibodies 4.7C and 107 might also recognize additional peptide sequences / T cell epitopes in CeD patients.

[0269] For example, when the experiments carried out were examined more carefully, the 12-mer used in the peptide pulsing experiments had a minimal T cell epitope of 9-mer at the C-terminus, i.e. (QLQ PFPQPELPY (SEQ ID NO:53)), whereas the 33-mer sequence is longer and has an additional sequence at the C-terminus (LQLQ PFPQPELPY It was noted that the 9-mer T cell epitope PFPQPELPY (SEQ ID NO:55) was a residue at position 10 or later (i.e., positions after the 9-mer minimal T cell epitope PFPQPELPY (SEQ ID NO:4)) and that the antibody might also recognize a similar T cell epitope sequence related to CeD but with alternative residues (other than P) at the C-terminus.

[0270] For example, other binding studies have shown that a Gly (G) residue at position 10 results in better levels of antibody binding with the 4.7C and 107 antibodies compared to having a naturally occurring Pro at p10 extending from the C-terminus of the minimal DQ2.5-glia-α1a epitope (data not shown).

[0271] Taken together, the data describing the binding properties of the two antibodies 107 and 4.7C, respectively, clearly demonstrated that both of them have the ability to specifically bind the DQ2.5-glia-α1a peptide epitope as soluble recombinant pHLA and pHLA on cells, including cells derived from CeD patient material. However, the data also showed that neither antibody has the ability to bind the epitope when a naturally occurring proline residue is present in the peptide version occupying the p10 position, including the predicted important 33-mer peptide from wheat α-gliadin (Dorm et al., J Immunol 2014 Nov. 1, 193(9)4497-4506). It has been recognized that there are gaps in the understanding of the epitopes of CeD, and that epitope elucidation may only account for up to 50% of patient reactivity (Non-Patent Document 10). Therefore, in light of the specific and well-defined binding profiles of the 107 and 4.7C antibodies, it was possible that their binding to additional, as yet undiscovered gluten epitopes might have led to somewhat unexpected similar staining levels in patient material.

[0272] To try and explore this, database searches were performed using the original 9-mer T cell epitope sequence PFPQPQLPY (SEQ ID NO:60) (and the deamidated version PFPQPELPY (SEQ ID NO:4)), along with the 12-mer sequence QLQPFPQPELPY (SEQ ID NO:53) (and the native / healthy version QLQPFPQPQLPY (SEQ ID NO:61)) used in the Raji cell peptide pulsing experiments, and various amino acid modifications to these sequences based on the known shared importance of specific positions in the epitope, such as the apparent invariant requirement of leucine at p7 for T cell reactivity (Petersen et al., Nature Structural and Molecular Biology 2014, 21(5), 480-488; Dahal-Koirala et al., Mucosal Immunology 2013, 23(5), 480-488). Immunol) 9, 587-596, 2016. In particular, we focused on finding naturally occurring gliadin peptides (i.e., peptides derived from wheat) that are similar to the α1a 9-mer / 12-mer but have alternative residues (i.e., other than proline) in the extension at position 10 and thus may potentially result in better binding by antibodies 107 and 4.7C.

[0273] During the course of this work, many candidate sequences were identified and eliminated. For example, it was observed that candidate sequences with tyrosine, serine, and alanine residues at position 10 did not show any significant binding with the 4.7C antibody. These observations were supported by the known binding profile outlined.

[0274] Finally, through a more liberal ScanProsite (https: / / prosite.expasy.org / scanprosite / ) pattern search of the UniProt Knowledge Base (https: / / www.uniprot.org / ), several further candidate sequences were identified that were less similar (but still plausible) to the 9-mer and had a Y residue at position 9 identified as potentially important for the conformation of the recognized epitope, but additionally had a G residue at position 10. These candidates were further tested for binding with the 4.7C antibody, and one of these sequences showed particularly promising results.

[0275] This candidate peptide (Figure 6A) is found as part of an omega gliadin protein in a wild form of wheat named red wild einkorn (Triticum uratu, UniProtKB ID: A0A0E3SZN6_TRIUA, SEQ ID NO: 1, Figure 6B).

[0276] This peptide showed some similarity to the α1a 9-mer / 12-mer, but in addition has a G residue at position 10 relative to the 9-mer sequence. This sequence is identical to the core 9-mer PYPQQ Q QPY (SEQ ID NO:8) (Figure 6A).

[0277] This sequence was considered to be a possible candidate for a CeD T cell epitope because it shares some similarity with the α1a epitope (although it is actually classified as an omega gliadin) and occurs naturally in wheat forms. In addition, there was a glutamine (Q) residue that could be targeted for deamidation to an E residue by the TG2 enzyme (targeting the QXP motif, Solid et al., 2002, Nat Rev Immunol 2, 647-655) in CeD patients. See position 6 of the 9-mer (underlined above) and Figure 6A. However, in other respects, this sequence does not correspond to a classical or known CeD T cell epitope. Indeed, using a recognized T cell epitope predictor (NetMHCIIpan4.0) against this T. Urartu sequence did not predict this sequence to be an HLA-DQ2.5 binding peptide.

[0278] Four different forms of this peptide (see FIG. 7A) were tested by loading onto Raji cells as described above and assessing binding of both the 107 and 4.7C antibodies by FACS (FIG. 7B). The peptides evaluated were: i) PQQPYPQQQQPYGT (SEQ ID NO:24) (referred to as A0A0E3SZN6_p4Q_p6Q or "QQ" and corresponding to the native / healthy form of the peptide containing Q residues at positions 4 and 6 of the candidate T cell epitope 9-mer); ii) PQQPYPEQQQPYGT (SEQ ID NO:62) (referred to as A0A0E3SZN6_p4E_p6Q or "EQ" and corresponding to the form of the peptide containing an E rather than a Q residue at position 4 of the candidate T cell epitope 9-mer); iii) PQQPYPQQEQPYGT (SEQ ID NO:29) (referred to as A0A0E3SZN6_p4Q_p6E or "QE" and corresponding to the form of the peptide containing an E rather than a Q residue at position 6 of the candidate T cell epitope 9-mer); iv) PQQPYPEQEQPYGT (SEQ ID NO: NO:63) (termed A0A0E3SZN6_p4E_p6E or "EE" and corresponding to a form of the peptide containing an E rather than a Q residue at positions 4 and 6 of the candidate T cell epitope 9-mer). Both antibodies were shown to bind well to the QE and EE peptides, but not to the QQ and EQ forms (Figure 7B). It is likely that peptides containing an E at position 6 of the T cell 9-mer epitope are more likely to be found in patients with CeD, since this residue is part of the classical TG2 target sequence, QXP (here QQP). In particular, the classical DQ2.5-glia-α1a epitope, as well as an artificial version containing the p10 glycine extension, were included as controls in this assay. As expected, the antibodies bound to the artificial version, whereas a low level of binding was seen to the classical form. No binding was observed to the homologous DQ2.5-glia-α2 epitope.

[0279] We also assessed how the antibody reacted with a more physiologically realistic form of this peptide, the version thought to be present in CeD patients, by performing an in silico trypsin / chymotrypsin digest of the T. uratu sequence (Figure 8A). As a control, we included in parallel the T. aestivum α-gliadin sequence encoding a 33-mer peptide (Figure 8B). This identified two possible physiological forms of the sequence, QPQQPYPQQQQPY (SEQ ID NO:20) (sometimes referred to herein as the "medium" peptide) and QPQQPYPQQQQPYGTSL (SEQ ID NO:22) (sometimes referred to herein as the "long" peptide). These two peptides were tested in the Raji peptide loading assay described above (p4Q_p6Q medium, p4Q_p6E medium, and p4Q_p6E long (Figure 8C)), which have a Gln (Q) to Glu (E) substitution in accordance with the likely TG2 activity (QXP) found in CeD patients.

[0280] Indeed, both 107 and 4.7C antibodies were shown to bind well to the E versions of these predicted physiologically relevant peptides, with the length being important for this binding, as the p6 deamidated nonamer showed no binding (Figure 8D). Importantly, the isotype-matched DQ2.5-glia-α2-specific 3.C11 antibody did not show any binding to any of the T. Urartu sequences, proving the specific binding of 107 and 4.7C (Figure 8D). What is interesting here is that the levels of binding that the results with both p6 deamidated forms showed for the 107 and 4.7C antibodies mirror those observed when these antibodies were tested on material from CeD patients (antigen presenting cells in the form of CD19+ plasma cells) (Figures 1 and 5). Thus, it appears that a new T cell epitope with the defined 9-mer core PYPQQQQPY (SEQ ID NO:8) associated with CeD has been identified, along with longer CeD-associated peptides containing this epitope.

[0281] It is well established that wheat intestinal proteolysis by gastric and pancreatic enzymes is heterogeneous and predicted enzyme specificity only partially reflects actual variation in digestion. Thus, to get a more realistic idea of ​​what the protein fragmentation patterns look like, A0A0E3SZN6_TRIUA, as well as the major wheat ω-gliadin Q9FUW7_WHEAT and α-gliadin Q9M4L6_WHEAT proteins were generated (Figure 9A-C) and subjected to in vitro chymotrypsin digestion essentially as described (Arents-Hansen EH et al., Gut 2000;46:46-51 and Molberg O et al., Methods Mol Med. 2000;41:105-24). These protein digests were then analyzed by mass spectrometry essentially as described (Dorm et al., 2014, supra), Figure 9D-F.

[0282] Indeed, the α-gliadin control protein Q9M4L6_WHEAT was fragmented into various smaller peptide fragments, including a well-characterized 33-mer multi-epitope segment (Shan L et al., Science 2002, 297(5590):2275-9) (Figure 9D). The extensive proteolysis observed is in good agreement with previous reports (Dorm et al., 2014, supra). When the same proteolysis was undertaken for A0A0E3SZN6_TRIUA, this protein was also fragmented into various smaller peptides (Figure 9E). Importantly, no destructive processing of this hypothesized novel 9-mer core peptide was observed, and the multiple species investigated were expected to have HLA-DQ2.5 binding capacity consistent with what was already seen in the previous analysis (Figure 8). The minimal proteolytic segment identified from A0A0E3SZN6_TRIUA corresponded to the hypothesized A0A0E3SZN6_p4Q_p6Q_medium 13-mer peptide (Figure 8C and Figure 9E). Because Triticum urartu is the diploid A genome ancestor of the modern hexaploid Triticum aestivum wheat crop (Marcussen et al., 2014, Science:345(6194), 1250092), the gene encoding A0A0E3SZN6_TRIUA is found as part of a highly complex cultivated crop genome and therefore likely represents a relevant food source for human consumption (Appels et al., 2018, Science 361(6403), eaar7191 and Juhasz et al., 2018, Science Advances 4(8), eaar8602). Indeed, signature motifs from food source analysis point to A0A0E3SZN6 as such a component (Spada et al., 2020, Front. Nutr. 7:98).Thus, the major known ω-gliadins appear to be unlikely sources of epitopes, since they encode only eight of the nine residues required to construct the HLA-binding nonameric core (Figure ​(Figure9F).9F).

[0283] In the A0A0E3SZN6_TRIUA MS analysis (Figure 9E), we also noticed that many peptides lacked the p10Gly residue that seems to be essential for efficient 4.7C binding to the DQ2.5-glia-α1a epitope (Figure 7). These data therefore strongly suggested that these two peptides adopt different shapes in the HLA groove and are additionally decoded into the 4.7C antibody in different ways. This was subsequently confirmed by alanine scanning experiments.

[0284] Extensive experimental evidence indicates two major mechanisms for how the human immune system acquires the ability to present pathogenic gluten peptides in CeD (Non-Patent Document 1). These observations support the central role that B cells appear to have in this disease, which can be divided into two main categories: tissue transglutaminase 2 (TG2) and gliadin-specific B cells. Regarding the latter, extensive characterization of the B cell receptors (BCRs) of these gliadin peptide-specific B cells has identified a particularly frequent sequence motif (QPQQPFP (SEQ ID NO:64)) that is part of the peptides most frequently of the omega gliadin type (Non-Patent Document 3) and that is expressed by CD4 +Based on in silico (Figure 8) and MS analysis (Figure 9) of both T. aestivum Q9M4L6_WHEAT and T. urartu A0A0E3SZN6_TRIUA proteins, we note that in contrast to the α-gliadin sequence of Q9M4L6_WHEAT, which lacks this BCR consensus sequence, the ω-gliadin sequence of A0A0E3SZN6_TRIUA has a closely related QPQQPYP (SEQ ID NO:37) motif at its N-terminus, and the Q / E position of the BCR epitope (which overlaps with the T cell epitope) corresponds to position p-2 as annotated according to the HLA 9-mer core. Therefore, to investigate whether the A0A0E3SZN6_TRIUA epitope has the ability to act as a target against these BCRs, we reconstituted prototype anti-gliadin peptide BCRs from two CeD patients (designated 1002-1E01 and 1002-1E03) as soluble antibodies and tested their binding to neutravidin-immobilized peptides in an ELISA essentially as described (Figure 10) (Sunil et al., 2017, JCI Insight, 2(16):e93961).

[0285] The two selected BCRs have been widely reported and their binding profiles to the target sequences are different (Figure 10A). Importantly, a key feature of the known QPQQPFP (SEQ ID NO:64) motif is its deamidation with TG2 to QPEQPFP (SEQ ID NO:65), a signature feature that separates healthy and CeD individuals. The two BCRs have different requirements for this TG2 modification, and therefore to validate our approach, we included two positive control peptides with either Gln (Q) or Glu (E) at this position (PC1 and PC2, Figure 10B). Indeed, the results showed strong reactivity of the BCRs to the appropriate PCs (Figure 10C), with the 1002-1E03 antibody clearly binding equally well to the A0A0E3SZN6_TRIUA epitope, but only with Glu (E) at p-2. The 1002-1E01 antibody reacts only with the PC peptide, which may be explained by the demonstrated strong requirement of this antibody for Phe (F) at position p2. Our results therefore indeed confirmed that the A0A0E3SZN6_TRIUA T cell epitope has an N-terminal QPEQPYP (SEQ ID NO:38) extension that serves as a good target for the TG2-sensitive prototypic gliadin peptide-specific BCR, which is repeatedly shared in CeD patients (Sunil et al., 2017, supra). Considering the difference in the described BCR epitope compared to that found in the A0A0E3SZN6_TRIUA sequence, where the Phe (F) residue is exchanged for a Tyr (Y) in the former, the question arose whether the 1002-1E03 antibody has a preference for any of the sequences. Therefore, we repeated the experiment with serial dilutions of the antibodies to estimate the individual EC50s. Indeed, the results showed that both sequences were recognized equally well (Figure 10D).

[0286] Taken together, these results point to a well-documented mechanism by which the A0A0E3SZN6_TRIUA BCR epitope can result in the generation of potent antibody responses by effectively presenting the T cell epitope and thereby establishing T cell help to B cells. This finding further strengthens the validity of our patient staining data using 107 and 4.7C antibodies (Figures 1 and 5) identifying B cells and their plasma cell (PC) progeny as the predominant gluten peptide-presenting cells in the inflamed intestine of CeD patients. Further strong evidence that B cells play an essential role in the GI tissue destruction characteristic of CeD was obtained in the only mouse CeD model that recapitulates the villous atrophy seen in CeD, where gluten-induced atrophy was completely suppressed by B cell depletion. Taken together, our data fully outline the main critical components necessary to fulfill the requirements for a pathogenic gluten epitope in CeD and point to the A0A0E3SZN6_TRIUA sequence protein as a food source.

[0287] Production of recombinant soluble pHLA molecules Recombinant soluble pHLA (rs-pHLA) is a valuable reagent in immunological studies, but unlike HLA class I, these reagents are still very difficult to produce for HLA class II in general, and HLA-DQ has proven to be particularly challenging for partly unknown reasons (Davis et al., 2011, Nature Reviews Immunol., 11, 551-558). In other reports, it has been well documented that native HLA-DQ2.5 has a very narrow peptide repertoire and the different phenotypes point to special requirements to be able to productively combine peptides with HLA (Fallang et al., 2009, Nature Immunol., 10, 1096-1101 and Bergseng, E. et al., 2015, Immunogenetics, 67, 73-84). To further validate the A0A0E3SZN6_TRIUA T cell epitope candidates, we attempted to generate rs-pHLA conjugates essentially as previously described (Karsten et al., J Immunol, 2001;167:4861). To evaluate their performance, we included two previously reported versions carrying the DQ2.5-glia-α1a and DQ2.5-glia-α2 T cell epitopes, respectively.

[0288] Indeed, it was possible to produce all three rs-pHLA complexes by this method, which involves covalently linking the T cell epitope to the N-terminus of the HLA β chain using a synthetic linker and simultaneous expression with engineered in vivo biotinylation in Sf9 insect cells. The yield and purity of affinity purified (FLAG tag purified) material from these expression cultures varied between each peptide variant as expected, but overall were within the usual ranges seen for such molecules (Figure 11D). Thus, the sheer ability to make these molecules to fairly good homogeneity (Figures 11A-C), including pHLA containing the T cell epitope of the present invention (DQ2.5-glia-NTP-001), strongly indicates that it is a true T cell epitope in the context of HLA-DQ2.5.

[0289] To further assess the integrity of these rs-pHLA complexes, we performed neutravidin capture ELISA binding experiments essentially as described for a panel of selected well-validated pan-HLA and TCR-like antibodies (Flick R. et al., 2021, Sci. Immunol. 6(62):eabg4925). All three versions showed good and concentration-dependent binding to the conformation-specific pan-DQ antibody SPV-L3, whereas no such binding was observed for the conformation-specific pan-DR antibody L243 (Figure 12A-C), strongly indicating a correctly folded molecule. Using rs-pHLA of HLA-DQ2.5:DQ2.5-glia-α1a, very similar to the version generated here, two TCR-like antibodies, 107 and 4.7C, were raised and tested for binding to this complex in an ELISA, which indeed showed highly specific and concentration-dependent binding as expected (Figure 12D), consistent with previous experiments (Figures 1A and 2B) and with a binding hierarchy in which the high affinity 4.7C outranks the low affinity mother clone 107. This binding profile was well mirrored when repeated with HLA-DQ2.5:DQ2.5-glia-NTP-001 (Figure 12E). In contrast, no binding to these complexes was observed by the 3.C11 antibody, which specifically binds only its own HLA-DQ2.5:DQ2.5-glia-α2 target (Figure 12F). Collectively, these results indicate that all three HLA complexes fold correctly and bind only strictly conformation-dependent ligands.

[0290] Thus, we conclude that the candidate DQ2.5-glia-NTP-001 (A0A0E3SZN6_p-2E_p6E_medium) epitope indeed fulfills the strict requirements for successful production as fully functional intact rs-pHLAII in the context of HLA-2.5, comparable to two other known immunodominant CeD epitopes DQ2.5-glia-α1a and DQ2.5-glia-α2.

[0291] T cell activation Through the series of experiments described above, the data clearly show that the novel DQ2.5-glia-NTP-001 peptide fulfills a strict set of requirements both to be the source of the TCR-like antibody reactivity observed in CeD patient material (Figure 5) and to do so by containing an HLA-DQ2.5-restricted CeD-specific T cell epitope that also crosstalks with the anti-DGP BCR, a well-documented part of CeD pathogenesis. We have not yet identified defined T cell clones (TCCs) from CeD tissue that would allow us to assess the potential for T cell reactivity in a highly controlled manner. However, it is well documented that a significant number of CeD TCCs are somewhat promiscuous and readily cross-react with multiple gluten epitopes (e.g., Dahal-Koirala, S. et al., 2016, supra). From previous studies, we have a panel of such TCCs in the SKW-3 reconstituted (Flick et al., 2021, supra, and Petersen et al., 2014, supra). We therefore chose to test different versions of the A0A0E3SZN6 peptide in these T cell activation assays against SKW-3 cells. We included two known DQ2.5-glia-α1a-reactive clones, 380 and S2, as well as the DQ2.5-glia-α2-reactive 364. Notably, the 380 clone is known not to distinguish between the two closely related epitopes DQ2.5-glia-α1a and DQ2.5-glia-ω1 (Gunnarsen et al., 2017, supra). We first estimated the peptide sensitivity and specificity of S2 and 380 for DQ2.5-glia-α1a, and both were confirmed to be specific, with the S2 clone being somewhat more sensitive (Figure 13A).

[0292] The assay was then repeated as outlined in Figure 8C, this time using a panel of different A0A0E3SZN6 versions. None of these peptides showed any reactivity, whereas all control peptides readily activated T cells (Figure 13B). Collectively, this again supports the notion that A0A0E3SZN6-derived peptides represent novel T cell epitopes.

[0293] The prevalence of CeD-specific T cells varies in spatial (tissue vs. periphery) and temporal (time) gradients between different patients and within each patient. However, it is well documented that their absolute frequency is low even in hyperinflammatory conditions (Risnes et al., J Clin Invest. 2018;128(6):2642-2650). With a tissue abundance of up to 1-2% or less, 30-50% of these T cells have unknown reactivity (Non-Patent Document 10 and Qiao SW et al., 2021, Frontiers in Immunology 12:646-163). Moreover, because some of these cells traffic between the blood and the gut at low frequency, we chose to use a highly sensitive intracellular IFNγ cytokine flow assay to assess whether we could detect clear CeD-specific reactivity against A0A0E3SZN6-derived candidates using HLA-DQ2.5-typed peripheral blood mononuclear cells (PBMCs) derived from either confirmed CeD patients or healthy controls (HCs) (Figure 14).

[0294] The total amount of T cells in PBMCs varies between 45-75%. Therefore, we first established baseline IFN-γ detection levels in CeD PBMC T cells (CD3+ / CD4+) in the absence of any exogenous peptide (Figure 14A). We then used 48 h cultured CeD and HC PBMCs from HLA-DQ2.5 positive CeD (donor 595) and HC (donor 557) in the presence of either a well-characterized deamidated 33-mer peptide from T. aestivum α-gliadin or a deamidated long version of T. urartu A0A0E3SZN6 ω-gliadin (Figure 14B). Indeed, here we saw a clear proliferation of IFN-γ positive CD4 T cells towards both peptides in CeD material, whereas no such proliferation was observed in HC (Figure 14B). This clear proliferation was slightly higher for the 33-mer compared to the A0A0E3SZN6 peptide (average 4.2% vs. 2.8%). Notably, this clear proliferation of INF-γ positive cells was not observed in the CD3+ / CD4- cell population (Figure 14C).

[0295] To better understand the individual variability of responses, the experiment was repeated with an expanded number of PBMC samples covering three different CeD donors and two HCs (Figure 15). This experiment is important because it is well known that T cell responses and amplitudes to gluten epitopes vary between CeD individuals, and responses in blood are usually more variable due to lower T cell abundance compared to intestinal biopsies. First, peptide-specific responses in CeD donor 595 were confirmed, and despite the often variability in absolute responses in such assays, the rank order of peptide reactivity remained essentially the same (Figure 15A). Furthermore, here we also observed a CeD donor-dependent variation in peptide reactivity, with donor 600 appearing to prefer the A0A0E3SZN6 peptide, donor 595 nearly equally to both (no statistical difference), and donor 585 appearing to prefer the known 33-mer from T. aestivum α-gliadin (Figure 15A). Thus, both of these epitopes indicate donor-specific responses, each present in 2 / 3 of the PBMC material tested. Importantly, no responses were seen in the two HCs (Figure 15B). Taken together, these data strongly suggest that the T. urartu A0A0E3SZN6 sequence is a bona fide T cell epitope in the context of HLA-DQ2.5 that appears to be restricted by CeD, comparable to the 33-mer peptide derived from T. aestivum α-gliadin.

Claims

1. an epitope comprising the amino acid sequence PYPQQQQPY (SEQ ID NO: 8), or An epitope comprising the amino acid sequence PYPQQQQPY (SEQ ID NO: 8) in which one or more Q residues are replaced by E residues. wherein said peptide is at most 40 amino acids in length.

2. the peptide comprises the amino acid sequence QPQQPYPQQQQPY (SEQ ID NO: 20), PQQPYPQQQQPYGT (SEQ ID NO: 24), or QPQQPYPQQQQPYGTSL (SEQ ID NO: 22), or an amino acid sequence in which one or more of the Q residues are replaced by an E residue, or an amino acid sequence substantially homologous thereto; the substantially homologous sequence includes a sequence having one, two, or three amino acid substitutions, additions, or deletions; 2. The peptide of claim 1, wherein the amino acid substitutions, additions, or deletions are located outside the core sequence PYPQQQQPY defined in claim 1.

3. 2. The peptide of claim 1, wherein the epitope comprises the amino acid sequence PYPQQEQPY (SEQ ID NO: 25), PYPEQEQPY (SEQ ID NO: 26), or PYPEQQQPY (SEQ ID NO: 27).

4. 2. The peptide of claim 1, wherein the peptide comprises a sequence obtainable by transglutaminase 2 (TG2) deamidating the amino acid sequence.

5. 2. The peptide of claim 1, wherein the peptide comprises the amino acid sequence PYPQQEQPY (SEQ ID NO: 25), preferably the peptide comprises QPQQPYPQQEQPY (SEQ ID NO: 28), QPQQPYPQQEQPYGTSL (SEQ ID NO: 30), or PQQPYPQQEQPYGT (SEQ ID NO: 29).

6. the peptide comprises the amino acid sequence QPQQPYPQQQQPY (SEQ ID NO: 20); and the Q at residue 3 is replaced by an E residue, and / or the Q at residue 10 is replaced by an E residue, and / or the Q at residue 8 is replaced with an E residue; or, The peptide comprises the amino acid sequence PQQPYPQQQQPYGT (SEQ ID NO: 24), and the Q in residue 2 is replaced with an E residue, and / or the Q at residue 9 is replaced by an E residue, and / or the Q at residue 7 is replaced with an E residue; or, The peptide comprises the amino acid sequence QPQQPYPQQQQPYGTSL (SEQ ID NO: 22), and the Q at residue 3 is replaced by an E residue, and / or the Q at residue 10 is replaced by an E residue, and / or the Q at residue 8 is replaced with an E residue; The peptide of claim 1.

7. The peptide has the amino acid sequence: QPQQPYPQQEQPY (SEQ ID NO: 28), QPEQPYPQQEQPY (SEQ ID NO: 31), QPEQPYPQQQQPY (SEQ ID NO: 32), QPQQPYPQQEQPYGTSL (SEQ ID NO: 30), QPEQPYPQQEQPYGTSL (SEQ ID NO: 33), QPEQPYPQQQQPYGTSL (SEQ ID NO: 34), PQQPYPQQEQPYGT (SEQ ID NO: 29), PEQPYPQQEQPYGT (SEQ ID NO: 35), or 2. The peptide of claim 1, comprising PEQPYPQQQQPYGT (SEQ ID NO: 36).

8. 2. The peptide of claim 1, wherein the peptide comprises the amino acid sequence QPQQPYP (SEQ ID NO: 37) or QPEQPYP (SEQ ID NO: 38).

9. 9. The peptide of claim 8, wherein the peptide comprises the amino acid sequence QPEQPYPQQEQPY (SEQ ID NO: 31) or QPEQPYPQQEQPYGTSL (SEQ ID NO: 33).

10. the peptide comprises a G residue at or corresponding to position 10; Preferably, the peptide comprises residues G and T at positions 10 and 11 or at positions corresponding to positions 10 and 11, or comprising residues G and L at positions 10 and 13, or at positions corresponding to positions 10 and 13; The peptide of claim 1, wherein the positions are defined relative to the 9-mer defined in claim 1.

11. A conjugate or complex comprising a peptide as defined in claim 1 bound or associated with an MHC molecule.

12. A peptide as defined in claim 1, or 10. One or more nucleic acid molecules comprising a nucleotide sequence encoding a conjugate or complex comprising a peptide as defined in claim 1 bound or associated with an MHC molecule.

13. An expression vector comprising the nucleic acid molecule of claim 12, or a cell comprising said expression vector, or a cell comprising the nucleic acid molecule of claim 12.

14. A composition comprising a peptide as defined in any one of claims 1 to 10, a conjugate or complex as defined in claim 11, a nucleic acid molecule as defined in claim 12, or an expression vector or cell as defined in claim 13.

15. The composition of claim 14, wherein the composition is a vaccine composition.

16. 1. A method for diagnosing celiac disease in a subject, the method comprising: contacting a sample from said subject with a peptide as defined in any one of claims 1 to 10 or a conjugate or complex as defined in claim 11; and whether the peptide, conjugate, or complex binds to T cells in the sample; or determining whether the sample contains an antibody that binds to the peptide, conjugate, or complex; wherein said binding of said peptide, conjugate or complex to T cells or said presence of said antibody in said sample indicates said subject has or is susceptible to celiac disease.

17. for use in therapy, preferably for use in the treatment or prevention of celiac disease, or for use in tolerizing a subject to said peptide, conjugate, or complex; or For use in suppressing or reducing an immune response to said peptide, conjugate, or complex, A peptide as defined in any one of claims 1 to 10, or a conjugate or complex as defined in claim 11, or a nucleic acid molecule as defined in claim 12, or an expression vector or cell as defined in claim 13.

18. for use in therapy, preferably for use in the treatment or prevention of celiac disease, or for use in tolerizing a subject to said peptide, conjugate, or complex; or For use in suppressing or reducing an immune response to said peptide, conjugate, or complex, Use of a peptide as defined in any one of claims 1 to 10, or a conjugate or complex as defined in claim 11, or a nucleic acid molecule as defined in claim 12, or an expression vector or cell as defined in claim 13 in the manufacture of a medicament or composition.

19. A binding protein that specifically binds to a conjugate or complex as defined in claim 11.

20. 20. The binding protein of claim 19, wherein the binding protein comprises a T cell receptor, or an antibody, or an antigen-binding domain of an antibody.

21. 21. The binding protein of claim 20, wherein the T cell receptor is a soluble T cell receptor.

22. 20. The binding protein of claim 19, wherein the binding protein is expressed on the surface of a cell, preferably a eukaryotic cell, more preferably a T cell or an NK cell.

23. 20. The binding protein of claim 19, wherein the binding protein is associated with a payload, such as a cytotoxic moiety or siRNA, or is associated with a second binding protein having specificity for an effector cell.

24. 24. A binding protein according to any one of claims 19 to 23 for use in therapy, preferably for use in the treatment or prevention of coeliac disease.

25. Use of a binding protein according to any one of claims 19 to 23 in the manufacture of a medicament or composition for use in therapy, preferably for use in the treatment or prevention of coeliac disease.

26. A method for producing a binding protein according to any one of claims 19 to 21, comprising:

12. A method, wherein the method comprises the use of a conjugate or complex as defined in claim 11.

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