CA2488218C

CA2488218C - Therapeutic epitopes and uses thereof

Info

Publication number: CA2488218C
Application number: CA2488218A
Authority: CA
Inventors: Robert Paul Anderson; Adrian Vivian Sinton Hill; Derek Parry Jewell
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2002-06-05
Filing date: 2003-06-05
Publication date: 2020-11-10
Anticipated expiration: 2023-06-05
Also published as: CA2488218A1; WO2003104273A2; AU2003244771A1; MXPA04012117A; US20060178299A1; EP1513873A2; SI1513873T1; EP2292649A2; WO2003104273A9; JP2013101146A; EP2292649B1; EP1513873B1; HK1205748A1; DK1513873T3; AU2003244771B2; GB0212885D0; JP5635302B2; JP6438423B2; US20180334484A1; MX338122B

Abstract

The invention herein disclosed is related to epitopes useful in methods of diagnosing, treating, and preventing coeliac disease. Therapeutic compositions which comprise at least one epitope are provided.

Description

THERAPEUTIC EPITOPES AND USES THEREOF
The invention relates to epitopes useful in the diagnosis and therapy of coeliac disease, including diagnostics, therapeutics, kits, and methods of using the foregoing.
An immune reaction to gliadin (a component of gluten) in the diet causes coeliac disease. It is known that immune responses in the intestinal tissue preferentially respond to gliadin which has been modified by an intestinal transglutaminase. Coeliac disease is diagnosed by detection of anti-endomysial antibodies, but this requires confirmation by the finding of a lymphocytic inflammation in intestinal biopsies. The taking of such a biopsy is inconvenient for the patient.
Investigators have previously assumed that only intestinal T cell responses provide an accurate indication of the immune response against gliadins.
Therefore they have concentrated on the investigatidn of T cell responses in intestinal tissue'.
Gliadin epitopes which require transglutaminase modification (before they are recognised by the immune system) are known2.
The inventors have found the immunodominant T cell A-gliadin epitope recognised by the immune system in coeliac disease, and have shown that this is recognised by T cells in the peripheral blood of individuals with coeliac disease (see WO 01/25793). Such T cells were found to be present at high enough frequencies to be detectable without restimulation (i.e. a 'fresh response' detection system could be used). The epitope was identified using a non-T cell cloning based method which provided a more accurate reflection of the epitopes being recognised. The immunodominant epitope requires transglutaminase modification (causing substitution of a particular glutamine to glutamate) before immune system recognition.
Based on this work the inventors have developed a test which can be used to diagnose coeliac disease at an early stage. The test may be carried out on a sample from peripheral blood and therefore an intestinal biopsy is not required. The test is more sensitive than the antibody tests which are currently being used.
The invention thus provides a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising:

2 (a) contacting a sample from the host with an agent selected from (i). the epitope comprising sequence which is: SEQ ID NO:1 (PQPELPY)or SEQ ID NO:2 (QLQPFPQPELPYPQPQS), or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO:3, (ii) an epitope comprising sequence comprising: SEQ ID NO:1, or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO:3 (shown in Table 1), which epitope is an isolated oligopeptide derived from a gliadin protein, (iii) an analogue of (i) or (ii) which is capable of being recognised by a T cell receptor that recognises (i) or (ii), which in the case of a peptide analogue is not more than 50 amino acids in length, or (iv) a product comprising two or more agents as defined in (i), (ii) or (iii), and (b) determining in vitro whether T cells in the sample recognise the agent, recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease.
Through comprehensive mapping of wheat gliadin T cell epitopes (see Example 13), the inventors have also found epitopes bioactive in coeliac disease in HLA-DQ2+ patients in other wheat gliadins, having similar core sequences (e.g., SEQ ID NOS:18-22) and similar full length sequences (e.g., SEQ ID NOS:31-36), as well as in rye secalins and barley hordeins (e.g., SEQ ID NOS:39-41); see also Tables 20 and 21. Additionally, several epitopes bioactive in coeliac disease in HLA-DQ8+ patients have been identified (e.g., SEQ ID NOS:42-44, 46). This comprehensive mapping thus provides the dominant epitopes recognized by T
cells in coeliac patients. Thus, the above-described method and other methods of the invention described herein may be performed using any of these additional identified epitopes, and analogues and equivalents thereof; (i) and (ii) herein include these additional epitopes. That is, the agents of the invention also include these novel epitopes.
The invention also provides use of the agent for the preparation of a diagnostic means for use in a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual, said method comprising determining whether T
cells of the individual recognise the agent, recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease.

3 The finding of an immunodominant epitope which is modified by transglutaminase (as well as the additional other epitopes defined herein) also allows diagnosis of coeliac disease based on determining whether other types of immune response to this epitope are present. Thus the invention also provides a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising determining the presence of an antibody that binds to the epitope in a sample from the individual, the presence of the antibody indicating that the individual has, or is susceptible to, coeliac disease.
The invention additionally provides the agent, optionally in association With a carrier, for use in a method of treating or preventing coeliac disease by tolerising T
cells which recognise the agent. Also provided is an antagonist of a T cell which has a T cell receptor that recognises (i) or (ii), optionally in association with a carrier, for use in a method of treating or preventing coeliac disease by antagonising such T
cells. Additionally provided is the agent or an analogue that binds an antibody (that binds the agent) for use in a method of treating or preventing coeliac disease in an individual by tolerising the individual to preVent the production of such an antibody.
The invention provides a method of determining whether a composition is capable of causing coeliac disease comprising determining whether a protein capable of being modified by a transglutaminase to an oligopeptide sequence as defined above is present in the composition, the presence of the protein indicating that the composition is capable of causing coeliac disease.
The invention also provides a mutant gliadin protein whose wild-type sequence can be modified by a transglutaminase to a sequence that comprises an epitope comprising sequence as defined above, but which mutant gliadin protein has been modified in such a way that it does not contain sequence which can be modified by a transglutaminase to a sequenue that comprises such an epitope comprising sequence; or a fragment of such a mutant gliadin protein which is at least 15 amino acids long and which comprises sequence which has been modified in said way.
The invention also provides a protein that comprises a sequence which is able to bind to a T cell receptor, which T cell receptor recognises the agent, and which sequence is able to cause antagonism of a T cell that carries such a T cell receptor.

4 Additionally the invention provides a food that comprises the proteins defined above.
SUMMARY OF THE INVENTION
The present invention provides methods of preventing or treating coeliac disease comprising administering to an individual at least one agent selected from: a) a peptide comprising at least one epitope comprising a sequence selected from the group consisting of SEQ ID NOs:18-22, 31-36, 39-44, and 46, and equivalents thereof; and b) an analogue of a) which is capable of being recognised by a T
cell receptor that recognises the peptide of a) and which is not more than 50 amino acids in length; and c) optionally, in addition to the agent selected from a) and b), a peptide comprising at least one epitope comprising a sequence selected from SEQ ID
NO:1 and SEQ ID NO:2. In some embodiments, the agent is HLA-DQ2-restricted, HLA-DQ8-restricted or one agent is BLA-DQ2-restricted and a second agent is 1-ILA-DQ8-restricted. In some embodiments, the agent comprises a wheat epitope, a rye epitope, a barley epitope or any combination thereof either as a single agent or as multiple agents.
The present invention also provides methods of preventing or treating coeliac disease comprising administering to an individual a pharmaceutical composition comprising an agent above and pharmaceutically acceptable carrier or diluent.
The present invention also provides methods of preventing or treating coeliac disease comprising administering to an individual a pharmaceutical composition comprising an antagonist of a T cell which has a T cell receptor as defined above, and a phan-naceutically acceptable carrier or diluent.
The present invention also provides methods of preventing or treating coeliac disease comprising administering to an individual a composition for tolerising an individual to a gliadin protein to suppress the production of a T cell or antibody response to an agent as defined above, which composition comprises an agent as defined above. , The present invention also provides methods of preventing or treating coeliac disease by 1) diagnosing coeliac disease in an individual by either: a) contacting a sample from the host with at least one agent selected from: i) a peptide comprising at least one epitope comprising a sequence selected from the group consisting of:
SEQ
ID NOS:18-22, 31-36, 39-44, and 46, and equivalents thereof; and ii) an analogue of i) which is capable of being recognised by a T cell receptor that recognises i) and which is not more than 50 amino acids in length; and iii) optionally, in addition to the

5 agent selected from i) and ii), a peptide comprising at least one epitope comprising a sequence selected from SEQ ID NOS:1 and 2; and determining in vitro whether T
cells in the sample recognise the agent; recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease; orb) administering an agent as defined above and determining in vivo whether T cells in the individual recognise the agent, recognition of the agent indicating that the individual has or is susceptible to r coeliac disease; and 2) administering to an individual diagnosed as having, or being susceptible to, coeliac disease a therapeutic agent for preventing or treating coeliac disease.
The present invention also provides agents as defined above, optionally in association with a carrier, for use in a method of treating or preventing coeliac disease by tolerising T cells which recognise the agent.
The present invention also provides antagonists of a T cell which has a T cell receptor as defined above, optionally in association with a carrier, for use in a method of treating or preventing coeliac disease by antagonising such T cells.
The present invention also provides proteins that comprises a sequence which is able to bind to a T cell receptor, which T cell receptor recognises an agent as defined above, and which sequence is able to cause antagonism of a T cell that carries such a T cell receptor.
The present invention also provides pharmaceutical compositions comprising an agent or antagonist as defined and a pharmaceutically acceptable carrier or diluent.
The present invention also provides compositions for tolerising an individual to a gliadin protein to suppress the production of a T cell or antibody response to an agent as defined above, which composition comprises an agent as defined above.
The present invention also provides compositions for antagonising a T cell response to an agent as defined above, which composition comprises an antagonist as defined above.

6 The present invention also provides mutant gliadin proteins whose wild-type sequence can be modified by a transglutaminase to a sequence which is an agent as defined in claim 1, which mutant gliadin protein comprises a mutation which prevents its modification by a transglutaminase to a sequence which is an agent as defined above; or a fragment of such a mutant gliadin protein which is at least 15 amino acids long and which comprises the mutation.
The present invention also provides polynucleotides that comprises a coding sequence that encodes a protein or fragment as defined above.
The present invention-also provides cells comprising a polynucleotide as defined above or which has been transformed with such a polynucleotide.
The present invention also provides mammals that expresses a T cell receptor as defined above.
The present invention also provides methods of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising: a) contacting a sample from the host with at least one agent selected from i) a peptide comprising at least one epitope'comprising a sequence selected from the group consisting of: SEQ
ID
NOS:18-22, 31-36, 39-44, and 46, and equivalents thereof; and ii) an analogue of i) which is capable of being recognised by a T cell receptor that recognises i) and which is not more than 50 amino acids in length; and iii) optionally, in addition to the agent selected from i) and ii), a peptide comprising at least one epitope comprising a sequence selected from SEQ ID NOS:1 and 2; and b) determining in vitro whether T
cells in the sample recognise the agent; recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease.
The present invention also provides methods of determining whether a composition is capable of causing coeliac disease comprising determining whether a protein capable of being modified by a transglutaminase to an oligopeptide sequence is present in the composition, the presence of the protein indicating that the composition is capable of causing coeliac disease.
The present invention also provides methods of identifying an antagonist of a T cell, which T cell recognises an agent as defined above, comprising contacting a candidate substance with the T cell and detecting whether the substance causes a decrease in the ability of the T cell to undergo an antigen specific response, the

7 detecting of any such decrease in said ability indicating that the substance is an antagonist.
The present invention also provides kits for carrying out any of the method described above comprising an agent as defined above and a means to detect the recognition of the peptide by the T cell.
The present invention also provides methods of identifying a product which is therapeutic for coeliac disease comprising administering a candidate substance to a mammal as defined above which has, or which is susceptible to, coeliac disease and deten-nining whether substance prevents or treats coeliac disease in the mammal, the prevention or treatment of coeliac disease indicating that the substance is a therapeutic product.
The present invention also provides processes for the production of a protein encoded by a coding sequence as defined above which process comprises: a) cultivating a cell described above under conditions that allow the expression of the protein; and optionally b) recovering the expressed protein.
The present invention also provides methods of obtaining a transgenic plant cell comprising transforming a plant cell with a vector as described above to give a transgenic plant cell.
The present invention also provides methods of obtaining a first-generation transgenic plant comprising regenerating a transgenic plant cell transformed with a vector as described above to give a transgenic plant.
The present invention also provides methods of obtaining a transgenic plant seed comprising obtaining a transgenic seed from a transgenic plant obtainable as described above.
The present invention also provides methods of obtaining a transgenic progeny plant comprising obtaining a second-generation transgenic progeny plant from a first-generation transgenic plant obtainable by a method as described above, and optionally obtaining transgenic plants of one or more further generations from the second-generation progeny plant thus obtained.
The present invention also provides transgenic plant cells, plants, plant seeds or progeny plants obtainable by any of the methods described above.

8 The present invention also provides transgenic plants or plant seeds comprising plant cells as described above.
The present invention also provides transgenic plant cell calluses comprising plant cells as described above obtainable from a transgenic plant cell, first-generation plant, .. plant seed or progeny as defined above.
The present invention also provides methods of obtaining a crop product comprising harvesting a crop product from a plant according to any method described above and optionally further processing the harvested product.
The present invention also provides food that comprises a protein as defined above.
The present invention as claimed relates to:
(A) A peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID
NO: 19;
.. (B) Use of at least one agent for the preparation of a medicament for treatment or prevention of coeliac disease, wherein the agent is: (a) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (b) the peptide of (a) comprising a modification that corresponds to a natural post translational modification present on the N or .. C terminus; and (c) optionally, in addition to the peptide of (a) or (b), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
(C) A pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier or diluent, wherein the agent is: (a) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (b) the peptide of (a) comprising a modification that corresponds to a natural post translational modification present on the N or 8a C terminus; and (c) optionally, in addition to the peptide of (a) or (b), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
(D) A method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising: (a) contacting a sample from the individual with at least one agent that is: (i) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID
NO: 19; or (ii) the peptide of (i) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (iii) optionally, in addition to the peptide of (i) or (ii), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2; and (b) determining in vitro whether T-cells in the sample recognise the agent; whereby recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease;
(E) Use of at least one agent which is: (i) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said 'I-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (ii) the peptide of (i) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (iii) optionally, in addition to the peptide (i) or (ii), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
for the preparation of a diagnostic means for use in a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual, said method comprising determining whether T-cells of the individual recognise the agent, wherein recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease;
(F) A method of determining whether a composition is capable of causing coeliac disease comprising determining whether a protein comprising the sequence set forth in SEQ ID
NO: 19 is present in the composition, the presence of the protein indicating that the composition is capable of causing coeliac disease;
(G) A kit for carrying out the method according to (D) or the use according to (E) above, comprising: (a) at least one agent that is: (i) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of 8b transglutaminase-deamidated SEQ ID NO: 19; or (ii) the peptide of (i) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (iii) optionally, in addition to the peptide of (i) or (ii), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
and (b) a means to detect the recognition of the peptide by T-cells;
(H) Use of a peptide comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19, to produce an antibody specific to the peptide;
(I) Use of at least one agent for the treatment or prevention of coeliac disease, wherein the agent is: (a) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID
NO: 19; or (b) the peptide of (a) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (c) optionally, in addition to the peptide of (a) and (b), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2;
(J) A peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID
NO:796);
(K) Use of a peptide for the treatment or prevention of coeliac disease, wherein the peptide is a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796);
(L) A pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier or diluent, wherein the agent is a peptide of not more than 50 amino acids in length comprising at least one T cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796);
(M) A method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising: (a) contacting a sample from the individual with at least one agent that is a peptide of not more than 50 amino acids in length comprising at least one T cell epitope, 8c said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796); and (b) determining in vitro whether T-cells in the sample recognise the agent;
whereby recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease;
(N) Use of at least one agent which is a peptide of not more than 50 amino acids in length comprising at least one T cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796) for the preparation of a diagnostic means for use in a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual, said method comprising determining whether T-cells of the individual recognise the agent, wherein recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease; and (0) A kit for carrying out the method of (M) or the use of (N) above, comprising: (a) at least one agent that is a peptide of not more than 50 amino acids in length comprising at least one T
cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID
NO:796);
and (b) a means to detect the recognition of the peptide by T-cells.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is illustrated by the accompanying drawings in which:
Figure 1 shows freshly isolated PBMC (peripheral blood mononuclear cell) IFNy ELISPOT responses (vertical axis shows spot forming cells per 106 PBMC) to transglutaminase (tTG)-treated and untreated peptide pool 3 (each peptide 10 jig/m1) .. including five overlapping 15mers spanning A-gliadin 51-85 (see Table 1) and a-chymotrypsin-digested gliadin (40 jig/ml) in coeliac disease Subject 1, initially in remission following a gluten free diet then challenged with 200g bread daily for three days from day 1 (a). PBMC IFNy ELISPOT responses by Subject 2 to tTG-treated A-gliadin peptide pools 1-10 spanning the complete A-gliadin protein during ten day bread challenge (b).
The horizontal axis shows days after commencing bread.
Figure 2 shows PBMC IFNy ELISPOT responses to tTG-treated peptide pool 3 (spanning A-gliadin 51-85) in 7 individual coeliac disease subjects (vertical axis shows spot 8d forming cells per 106 PBMC), initially in remission on gluten free diet, challenged with bread for three days (days 1 to 3). The horizontal axis shows days after commencing bread. (a).
PBMC IFNy Elispot responses to tTG-treated overlapping 15mer peptides included in pool 3;
bars represent the mean ( SEM) response to individual peptides (10 g/m1) in 6 Coeliac disease subjects on day 6 or 7(b). (In individual subjects, ELISPOT responses to peptides were calculated as a % of response elicited by peptide 12 - as shown by the vertical axis.)

9 Figure 3 shows PBMC IFNy ELISPOT responses to tTG-treated truncations of A-gliadin 56-75 (0.1 JAM). Bars represent the mean ( SEM) in 5 Coeliac disease subjects. (In individual subjects, responses were calculated as the % of the maximal response elicited by any of the peptides tested.) Figure 4 shows how the minimal structure of the dominant A-gliadin epitope was mapped using tTG-treated 7-17mer A-gliadin peptides (0.1 M) including the sequence, PQP2LPY (SEQ ID NO:4) (A-gliadin 62-68) (a), and the same peptides without tTG treatment but with the substitution Q--->E65 (b). Each line represents PBMC IFNy ELISPOT responses in each of three Coeliac disease subjects on day 6 or 7 after bread was ingested on days 1-3. (In individual subjects, ELISPOT
responses were calculated as a % of the response elicited by the 17mer, A-gliadin 57-73.) Figure 5 shows the amino acids that were deamidated by tTG. A-gliadin 56-75 LQLQPFPQPQLPYPQPQSFP (SEQ ID NO:5) (0.111M) was incubated with tTG
(501.1.g/m1) at 37 C for 2 hours. A single product was identified and purified by reverse phase HPLC. Amino acid analysis allowed % deamidation (Q¨>E) of each Gin residue in A-gliadin 56-75 attributable to tTG to be calculated (vertical axis).
Figure 6 shows the effect of substituting Q--),E in A-gliadin 57-73 at other positions in addition to Q65 using the 17mers: QLQPFPQPELPYPQPES (SEQ ID
NO:6) (E57,65), QLQPFPQPELPYPQPES (SEQ ID NO:7) (E65,72), ELQPFPQPELPYPQPES (SEQ ID NO:8) (E57, 65, 72), and QLQPFPQPELPYPQPQS (SEQ ID NO:2) (E65) in three Coeliac disease subjects on day 6 or 7 after bread was ingested on days 1-3. Vertical axis shows % of the response.
Figure 7 shows that tTG treated A-gliadin 56-75 (0.1 [iM) elicited IFN-g ELISPOT responses in (a) CD4 and CD8 magnetic bead depleted PBMC. (Bars represent CD4 depleted PBMC responses as a % of CD8 depleted PBMC responses;
spot forming cells per million CD8 depleted PBMC were: Subject 4: 29, and Subject 6: 535). (b) PBMC IFNy ELISPOT responses (spot forming cells/million PBMC) after incubation with monoclonal antibodies to ITLA-DR (L243), -DQ (L2) and -DP
(B7.21) (10 1.tg/m1) lh prior to tTG-treated 56-75 (0.1 RM) in two coeliac disease subjects homozygous for HLA-DQ al*0501,131*0201.

Figure 8 shows the effect of substituting Glu at position 65 for other amino acids in the immunodominant epitope. The vertical axis shows the % response in the 3 subjects in relation to the immunodominant epitope.
Figure 9 shows the immunoreactivity of naturally occurring gliadin peptides 5 (measuring responses from 3 subjects) which contain the sequence PQLPY
(SEQ ID
NO:12) with (shaded) and without (clear) transglutaminase treatment.
Figure 10 shows CDS, CD4, p7, and aE -specific immunomagnetic bead depletion of peripheral blood mononuclear cells from two coeliac subjects 6 days after commencing gluten challenge followed by interferon gamma ELISPot. A-

10 gliadin 57-73 QE65 (25mcg/m1), tTG-treated chymotrypsin-digested gliadin (100 meg/nil) or PPD (10 mcg/m1) were used as antigen.
Figure 11 shows the optimal T cell epitope length. ' Figure 12 shows a comparison of A-gliadin 57-73 QE65 with other peptides in a dose response study.
Figure 13 shows a comparison of gliadin and A-gliadin 57-73 QE65 specific responses.
Figure 14 shows the bioactivity of gliadin polymorphisms in coeliac subjects.
Figures 15 and 16 show the defining of the core epitope sequence.
Figures 17 to 27 show the agonist activity of A-gliadin 57-73 QE65 variants.
Figure 28 shows responses in different patient groups.
Figure 29 shows bioactivity of prolamin homologues of A-gliadin 57-73.
Figure 30 shows, for healthy HLA-DQ2 subjects, the change in IFN-gamma ELISpot responses to tTG-deamidated gliadin peptide pools.
Figure 31 shows, for coeliac HLA-DQ2 subjects, the change in EN-gamma ELISpot responses to tTG-deamidated gliadin peptide pools.
Figure 32 shows individual peptide contributions to "summed" gliadin peptide response.
Figure 33 shows, for coeliac FILA-DQ2/8 subject C08, gluten challenge induced IFNy ELISpot responses to tTG-deamidated gliadin peptide pools.
Figure 34 shows, for coeliac FILA-DQ2/8 subject C07, gluten challenge induced IFNy ELISpot responses to tTG-deamidated gliadin peptide pools.

11 Figure 35 shows, for coeliac PILA-DQ8/7 subject C12, gluten challenge induced IFNy ELISpot responses to tTG-deamidated gliadin peptide pools.
Figure 36 shows, for coeliac HLA-DQ6/8 subject C11, gluten challenge induced IFNy ELISpot responses to tTG-deamidated gliadin peptide pools.
Detailed Description of the Invention The term "coeliac disease" encompasses a spectrum of conditions caused by varying degrees of gluten sensitivity, including a severe form characterised by a flat small intestinal mucosa (hyperplastic villous atrophy) and other forms characterised by milder symptoms.
The individual mentioned above (in the context of diagnosis or therapy) is human. They may have coeliac disease (symptomatic or asymptomatic) or be suspected of having it. They may be on a gluten free diet. They may be in an acute phase response (for example they may have coeliac disease, but have only ingested gluten in the last 24 hours before which they had been on a gluten free diet for 14 to 28 days). =
The individual may be susceptible to coeliac disease, such as a genetic susceptibility (determined for example by the individual having relatives with coeliac disease or possessing genes which cause predisposition to coeliac disease).
The agent The agent is typically a peptide, for example of length 7 to 50 amino acids, such as 10 to 40, or 15 to 30 amino acids in length.
SEQ ID NO:1 is PQPELPY. SEQ ID NO:2 is QLQPFPQPELPYPQPQS.
SEQ ID NO:3 is shown in Table 1 and is the sequence of a whole A-gliadin. The glutamate at position 4 of SEQ ID NO:1 (equivalent to position 9 of SEQ ID
NO:2) is generated by transglutaminase treatment of A-gliadin. =
The agent may be the peptide represented by SEQ ID NO:1 or 2 or an epitope comprising sequence that comprises SEQ ID NO:1 which is an isolated oligopeptide derived from a gliadin protein; or an equivalent of these sequences from a naturally occurring gliadin protein which is a homologue of SEQ ID NO:3. Thus the epitope may be a derivative of the protein represented by SEQ ID NO:3. Such a derivative is

12 typically a fragment of the gliadin, or a mutated derivative of the whole protein or fragment. Therefore the epitope of the invention does not include this naturally occurring whole gliadin protein, and does not include other whole naturally occurring gliadins.
The epitope may thus be a fragment of A-gliadin (e.g. SEQ ID NO:3), which comprises the sequence of SEQ ID NO:1, obtainable by treating (fully or partially) with transglutaminase, i.e. with 1, 2, 3 or more glutamines substituted to glutamates (including the substitution within SEQ ID-NO:1).
Such fragments may be or may include the sequences represented by 0 positions 55 to 70, 58 to 73, 61 to 77 of SEQ ID NO:3 shown in Table 1.
Typically such fragments will be recognised by T cells to at least the same extent that the peptides represented by SEQ ID NO:1 or 2 are recognised in any of the assays described herein using samples from coeliac disease patients.
Additionally, the agent may be the peptide represented by any of SEQ ID
NOS:18-22, 31-36, 39-44, and 46 or a protein comprising a sequence corresponding to any of SEQ ID NOS:18-22, 31-36, 39-44, and 46 (such as fragments of a gliadin comprising any of SEQ ID NOS:18-22, 31-36, 39-44, and 46, for example after the gliadin has been treated with transglutaminase). Bioactive fragments of such sequences are also agents of the invention. Sequences equivalent to any of SEQ
ID
NOS:18-22, 31-36, 39-44, and 46 or analogues of these sequences are also agents of the invention.
In the case where the epitope comprises a sequence equivalent to the above epitopes (including fragments) from another gliadin protein (e.g. any of the gliadin proteins mentioned herein or any gliadins which cause coeliac disease), such equivalent sequences will correspond to a fragment of a gliadin protein typically treated (partially or fully) with transglutaminase. Such equivalent peptides can be determined by aligning the sequences of other gliadin proteins with the gliadin from which the original epitope derives, such as with SEQ ID NO:3 (for example using any of the programs mentioned-herein). Transglutaminase is commercially available (e.g. Sigma T-5398). Table 4 provides a few examples of suitable equivalent sequences.

13 The agent which is an analogue is capable of being recognised by a TCR
which recognises (i) or (ii). Therefore generally when the analogue is added to T
cells in the presence of (i) or (ii), typically also in the presence of an antigen presenting cell (APC) (such as any of the APCs mentioned herein), the analogue inhibits the recognition of (i) or (ii), i.e. the analogue is able to compete with (i) or (ii) in such a system.
The analogue may be one whiCh is capable of binding the TCR which recognises (i) or (ii). Such binding can be tested by standard techniques.
Such TCRs can be isolated from T cells which have been shown to recognise (i) or (ii) (e.g. using lo the method of the invention). Demonstration of the binding of the analogue to the TCRs can then shown by determining whether the TCRs inhibit the binding of the analogue to a substance that binds the analogue, e.g. an antibody to the analogue.
Typically the analogue is bound to a class II MHC molecule (e.g. HLA-DQ2) in such an inhibition of binding assay.
Typically the analogue inhibits the binding of (i) or (ii) to a TCR. In this case the amount of (i) or (ii) which can bind the TCR in the presence of the analogue is decreased. This is because the analogue is able to bind the TCR and therefore competes with (i) or (ii) for binding to the TCR.
T cells for use in the above binding experiments can be isolated from patients with coeliac disease, for example with the aid of the method of the invention.
Other binding characteristics of the analogue may also. be the same as (i) or (ii), and thus typically the analogue binds to the same MHC class II molecule to which the peptide binds (HLA-DQ2 or -DQ8). The analogue typically binds to antibodies specific for (i) or (ii), and thus inhibits binding of (i) or (ii) to such antibodies.
The analogue is typically a peptide. It may have homology with (i) or (ii), typically at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99%
homology with (i) or (ii), for example over a region of at least 15 more (such as the entire length of the analogue and/or (i) or (ii), or across the region which contacts the TCR or binds the MI-IC molecule) contiguous amino acids. Methods of measuring protein homology are well known in the art and it will be understood by those of skill

14 in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology").
For example the UWC1CG Package provides the BBSTFIT program which . can be used to -calculate homology (for example used on its default settings) (Devareux et al (1984 Nucleic Acids Research 12, p387-395).. The PILEUP and BLAST algorithms can be used to calculate homology or line up Sequences (typically on their default setting.), for example as described in Altschul S. F. (1993)3 Mol =
Evol 36:290-300; Altschul, 5, F at al (1990) .1 Mol Bid l 215:403-10.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information on the world wide web through the internet. This algorithm involves first identifying high Scoring sequence pair (HSPa) by identifying Short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T
is is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPa = containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, duo to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment The = BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Heralroff and Henikoff (1992) Proc. Natl. Acad ScL USA 89: 10915-10919) alignments (B) of 50, expectation (B) d 10, Ni=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc: Natl. AcaeL
Sc!.
USA 90: 5873-5787. One measure of similarity provided by the BLAST algoritinn is the smallest sum probability MI which provides an indication of the probability by which a match between two nucleotide or amino acid sequences =
= =

would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
5 The homologous peptide analogues typically differ from (i) or (ii) by 1, 2, 3, 4, 5, 6, 7, 8 or more mutations (which may be substitutions, deletions or insertions).
These mutations may be measured across any of the regions mentioned above in relation to calculating homology. The substitutions are preferably 'conservative'.
These are defined according to the following Table. Amino acids in the same block 10 in the second column and preferably in the same line in the third column may be substituted for each other:
ALIPHATIC Non-polar G A P
=
I L V
Polar ¨ uncharged CSTM
N
Polar ¨ charged D E
KR
AROMATIC HENVY
Typically the amino acids in the analogue at the equivalent positions to amino

15 acids in (i) or (ii) that contribute to binding the MHC molecule or are responsible for the recognition by the TCR, are the same or are conserved.
Typically the analogue peptide comprises one or more modifications, which may be natural post-translation modifications or artificial modifications. The modification may provide a chemical moiety (typically by substitution of a hydrogen, e.g. of a C-H bond), such as an amino, acetyl, hydroxy or halogen (e.g.
fluorine) group or carbohydrate group. Typically the modification is present on the N or C terminus.
The analogue may comprise one or more non-natural amino acids, for example amino acids with a side chain different from natural amino acids.

16 Generally, the non-natural amino acid will have an N terrninus and/or a C
terminus.
The non-natural amino acid may be an L- or a D- amino acid.
The analogue typically has a shape, size, flexibility or electronic configuration thatis substantially similar to (i) or (ii). It is typically a derivative of (i) or (ii). In one embodiment the analogue is a fusion protein comprising the sequence of SEQ ID NO:1 or 2, or any of the other peptides mentioned herein;
and non-gliadin sequence.
In one embodiment the analogue is or mimics (i) or (ii) bound to a MHC class II molecule. 2, 3, 4 or more of such complexes may be associated or bound to each other, for example using a biotin/streptavidin based system, in which typically 2, 3 or 4 biotin labelled MHC molecules bind to a streptavidin moiety. This analogue typically inhibits the binding of the (i) or (ii)/MHC Class II complex to a TCR or antibody which is specific for the complex.
The analogue is typically an antibody or a fragment of an antibody, such as a Fab or (Fab)2 fragment. The analogue may be immobilised on a solid support, particularly an analogue that mimics peptide bound to a MHC molecule.
The analogue is typically designed by computational means and then synthesised using methods known in the art. Alternatively the analogue can be selected from a library of compounds. The library may be a combinatorial library or a display library, such as a phage display library. The library of compounds may be expressed in the display library in the form of being bound to a MHC class II
molecule, such as HLA-DQ2 or -DQ8. Analogues are generally selected from the library based on their ability to mimic the binding characteristics (i) or (ii). Thus they may be selected based on ability to bind a TCR or antibody which recognises (i) or (ii).
Typically analogues will be recognised by T cells to at least the same extent as any of the agents (i) or (ii), for example at least to the same extent as the equivalent epitope and preferably to the same extent as the peptide represented by SEQ ID NO:2, is recognised in any of the assays described herein, typically using T
cells from coeliac disease patients. Analogues may be recognised to these extents in vivo and thus may be able to induce coeliac disease symptoms to at least the same

17 extent as any of the agents mentioned herein (e.g. in a human patient or animal model).
Analogues may be identified in a method comprising determining whether a candidate substance is recognised by a T cell receptor that recognises an epitope of the invention, recognition of the substance indicating that the substance is an analogue. Such TCRs may be any of the TCRs mentioned herein, and may be present on T cells. Any suitable assay mentioned herein can be used to identify the analogue. In one embodiment this method is carried out in vivo. As mentioned above preferred analogues are recognised to at least the same extent as the peptide SEQ ID NO:2, and so the method may be used to identify analogues which are recognised to this extent.
In one embodiment the method comprises determining whether a candidate substance is able to inhibit the recognition of an epitope of the invention, inhibition of recognition indicating that the substance is an analogue.
The agent may be a product comprising at least 2, 5, 10 or 20 agents as defined by (i), (ii) or (iii). Typically the composition comprises epitopes of the invention (or equivalent analogues) from different gliadins, such as any of the species or variety of or types of gliadin mentioned herein. Preferred compositions comprise at least one epitope of the invention, or equivalent analogue, from all of the gliadins present in any of the species or variety mentioned herein, or from 2, 3, 4 or more of the species mentioned herein (such as from the panel of species consisting of wheat, rye, barley, oats and triticale). Thus, the agent may be monovalent or multivalent.
Diagnosis As mentioned above the method of diagnosis of the invention may be based on the detection of T cells that bind the agent or on the detection of antibodies that recognise the agent.
The T cells that recognise the agent in the method (which includes the use mentioned above) are generally T cells that have been pre-sensitised in vivo to gliadin. As mentioned above such antigen-experienced T cells have been found to be present in the peripheral blood.

18 In the method the T cells can be contacted with the agent in vitro or in vivo, and determining whether the T cells recognise the agent can be performed in vitro or in vivo. Thus the invention provides the agent for use in a method of diagnosis practiced on the human body. Different agents are provided for simultaneous, separate or sequential use in such a method.
The in vitro method is typically carried out in aqueous solution into which the agent is added. The solution will also comprise the T cells (and in certain embodiments the APCs discussed below). The term 'contacting' as used herein includes adding the particular substance to the solution.
0 Determination of whether the T cells recognise the agent is generally accomplished by detecting a change in the state of the T cells in the presence of the agent or determining whether the T cells bind the agent. The change in state is generally caused by antigen specific functional activity of the T cell after the TCR
binds the agent. The change of state may be measured inside (e.g. change in intracellular expression of proteins) or outside (e.g. detection of secreted substances) the T cells.
The change in state of the T cell may be the start of or increase in secretion of a substance from the T cell, such as a cytokine, especially IFN-y, IL-2 or TNF-ot.
Determination of IFN-y secretion is particularly preferred. The substance can typically be detected by allowing it to bind to a specific binding agent and then measuring the presence of the specific binding agent/substance complex. The specific binding agent is typically an antibody, such as polyclonal or monoclonal antibodies. Antibodies to cytokines are commercially available, or can be made using standard techniques.
Typically the specific binding agent is immobilised on a solid support. After the substance is allowed to bind the solid support can optionally be washed to remove material which is not specifically bound to the agent. The agent/substance complex may be detected by using a second binding agent that will bind the complex. Typically the second agent binds the substance at a site which is different from the site which binds the first agent. The second agent is preferably an antibody and is labelled directly or indirectly by a detectable label.

19 Thus the second agent may be detected by a third agent that is typically labelled directly or indirectly by a detectable label. For example the second agent may comprise a biotin moiety, allowing detection by a third agent which comprises a streptavidin moiety and typically alkaline phosphatase as a detectable label.
In one embodiment the detection system which is used is the ex-vivo ELISPOT assay described in W0,98/23960. In that assay IFN-y secreted from the T
cell is bound by a first IFN-7 specific antibody that is immobilised on a solid support.
The bound IFN-y is then detected using a second IFNI specific antibody which is labelled with a detectable label. Such a labelled antibody can be obtained from MABTECH (Stockholm, Sweden). Other detectable labels which can be used are discussed below.
The change in state of the T cell that can be measured may be the increase in the uptake of substances by the T cell, such as the uptake of thymidine. The change in state may be an increase in the size of the T cells, or proliferation of the T cells, or a change in cell surface markers on the T cell.
In one embodiment the change of state is detected by measuring the change in the intracellular expression of proteins, for example the increase in intracellular expression of any of the cytokines mentioned above. Such intracellular changes may be detected by contacting the inside of the T cell with a moiety that binds the expressed proteins in a specific manner and which allows sorting of the T
cells by flow cytometry.
In one embodiment when binding the TCR the agent is bound to an MHC
class II molecule (typically HLA-DQ2 or -DQ8), which is typically present on the surface of an antigen presenting cell (APC). However as mentioned herein other =
agents can bind a TCR without the need to also bind an MHC molecule.
Generally the T cells which are contacted in the method are taken from the individual in a blood sample, although other types of samples which contain T
cells can be used. The sample may be added directly to the assay or may be processed first. Typically the processing may comprise diluting of the sample, for example with water or buffer. Typically the sample is diluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10 fold.

The processing may comprise separation of components of the sample.
Typically mononuclear cells (MCs) are separated from the samples. The MCs will comprise the T cells and APCs. Thus in the method the APCs present in the separated MCs can present the peptide to the T cells. In another embodiment only T
5 cells, such as only CD4 T cells, can be purified from the sample. PBMCs, MCs and T cells can be separated from the sample using techniques known in the art, such as those described in Lalvani et al (1997) J. Exp. Med. 186, p859-865.
In one embodiment, the T cells used in the assay are in the form of unprocessed or diluted samples, or are freshly isolated T cells (such as in the form of D freshly isolated MCs or PBMCs) which are used directly ex vivo, i.e. they are not cultured before being used in the method. Thus the T cells have not been restimulated in an antigen specific manner in vitro. However the T cells can be cultured before use, for example in the presence of one or more of the agents, and generally also exogenous growth promoting cytokines. During culturing the agent(s) 15 are typically present on the surface of APCs, such as the APC used in the method.
Pre-culturing of the T cells may lead to an increase in the sensitivity of the method.
Thus the T cells can be converted into cell lines, such as short tem' cell lines (for example as described in Ota eta! (1990) Nature 346, p183-187).
The APC that is typically present in the method may be from the same

20 individual as the T cell or from a different host. The APC may be a naturally occurring APC or an artificial APC. The APC is a cell that is capable of presenting the peptide to a T cell. It is typically a B cell, dendritic cell or macrophage. It is typically separated from the same sample as the T cell and is typically co-purified with the T cell. Thus the APC may be present in MCs or PBMCs: The APC is typically a freshly isolated ex vivo cell or a cultured'cell. It may be in the form of a cell line, such as a short term or immortalised cell line. The APC may express empty MI-IC class II molecules on its surface.
In the method one or more (different) agents may be used. Typically the T
cells derived from the sample can be placed into an assay with all the agents which it . 30 is intended to test or the T cells can be divided and placed into separate assays each of which contain one or more of the agents.

21 The invention also provides the agents such as two or more of any of the agents mentioned herein (e.g. the combinations of agents which are present in the composition agent discussed above) for simultaneous separate or sequential use (eg.
for in vivo use).
In one embodiment agent per se is added directly to an assay comprising T
cells and APCs. As discussed .above the T cells and APCs in such an assay could be in the form of MCs. When agents that can be recognised by the T cell without the need for presentation by APCs are used then APCs are not required. Analogues which mimic the original (i) or (ii) bound to a MHC molecule are an example of such an agent.
In one embodiment the agent is provided to the APC in the absence of the T
= cell. The .APC is then provided to the T cell, typically after being allowed to present the agent on its surface. The peptide may have been taken up inside the APC
and presented, or simply be taken up onto the surface without entering inside the APC.
The duration for which the agent is contacted with the T cells will vary depending on the method used for determining recognition of the peptide.
Typically 105 to 107, preferably 5x105 to 106 PBMCs are added to each assay. In the case where agent is added directly to the assay its concentration is from 10-1 to 1034ml, preferably 0.5 to 504m1 or 1 to 10m/m1.
90 Typically the length of time for which the T cells are incubated with the agent is from 4 to 24 hours, preferably 6 to 16 hours. When using ex vivo PBMCs it has been found that 0.3x106 PBMCs can be incubated in 1 Ogg/m1 of peptide for 12 hours at 37 C.
The determination of the recognition of the agent by the T cells may be done by meastiring the binding of the agent to the T cells (this can be carried out using any suitable binding assay format discussed herein). Typically T cells which bind the agent can be sorted based on this binding, for example using a FACS machine.
The presence of T cells that recognise the agent will be deemed to occur if the frequency of cells sorted using the agent is above a "control" value. The frequency of antigen-experienced T cells is generally 1 in 106 to 1 in 103, and therefore whether or not the sorted cells are antigen-experienced T cells can be determined.
=

22 The determination of the recognition of the agent by the T cells may be measured in vivo. Typically the agent is administered to the host and then a response which indicates recognition of the agent may be measured. The agent is typically administered intradermally or epidermally. The agent is typically administered by = 5 contacting with. the outside of the skin, and may be retained at the site with the aid of a plaster or dressing. Alternatively the agent may be administered by needle, such as by injection, but can also be administered by other methods such as ballistics (e.g.
the ballistics techniques which have been used to deliver nucleic acids). EP-A-0693119 describes techniques that can typically be used to administer the agent.
to Typically from 0.001 to 1000 rig, for example from 0.01 to 100 1.T or 0.1 to 101..Lg of agent is administered.
In one embodiment a product can be administered which is capable of providing the agent in vivo. Thus a polynucleotide capable of expressing the agent can be administered, typically in any of the ways described above for the 15 administration of the agent. The polynucleotide typically has any of the characteristios of the polynucleotide provided by the invention which is discussed below. The agent is expressed from the polynucleotide in vivo. Typically from 0.001 to 1000 pig, for example from 0.01 to 100 p.g or 0.1 to 10 jig of polynucleotide is administered.
20 Recognition of the agent administered to the skin is typically indicated by the occurrence of inflammation (e.g. induration, erythema or oedema) at the site of administration. This is generally measured by visual examination of the site.
The method of diagnosis based on the detection of an antibody that binds the . agent is typically carried out by contacting a sample from the individual (such as any 25 of the samples mentioned here, optionally processed in any manner mentioned herein) with the agent and determining whether an antibody in the sample binds the agent, such a binding indicating that the individual has, or is susceptible to coeliac disease. Any suitable format of binding assay may be used, such as any such foiniat mentioned herein.
Therapy

23 The identification of the immunodominant epitope and other epitopes described herein allows therapeutic products to be made which target the T
cells which recognise this epitope (such T cells being ones which participate in the immune response against gliadin). These findings also allow the prevention or treatment of coeliac disease by suppressing (by tolerisation) an antibody or T
cell response to the epitope(s).
Certain agents of the invention bind the TCR that recognises the epitope of the invention (as measured using any of the binding assays discussed above) and cause tolcrisation of the T cell that carries the TCR. Such agents, optionally in association with a carrier, can therefore be used to prevent or treat coeliac disease.
Generally tolerisation can be caused by the same peptides which can (after being recognised by the TCR) cause antigen specific functional activity of the T cell (such as any such activity mentioned herein, e.g. secretion of cytokines).
Such agents cause tolerisation when they are presented to the immune system in a IS 'tolerising' context.
Tolerisation leads to a decrease in the recognition of a T cell or antibody epitope by the immune system. In the case of a T cell epitope this can be caused by the deletion or anergising of T cells that recognise the epitope. Thus T cell activity (for example as measured in suitable assays mentioned herein) in response to the .
epitope is decreased. Tolerisation of an antibody response means that a decreased amount of specific antibody to the epitope is produced when the epitope is administered.
Methods of presenting antigens-to the immune system in such a context are known and are described for example in Yoshida et al. Clin. Immunol.
Immunopathol. 82, 207-215 (1997), Thurau et al. Clin. Exp. Immunol. 109, 370-6 (1997), and Weiner et al. Res. Immunol. 148, 528-33 (1997). In particular certain . routes of administration can cause tolerisation, such as oral, nasal or intraperitoneal.
Tolerisation may also be accomplished via dendritic cells and tetramers presenting peptide. Particular products which cause tolerisation may be administered (e.g. in a composition that also comprises the agent) to the individual. Such products include cytokines, such as cytokines that favour a Th2 response (e.g. IL-4, TGF-I3 or IL-10). =
Products or agent may be administered at a dose that causes tolerisation.

24 =
The invention provides a protein that comprises a sequence able to act as an antagonist of the T cell (which T cell recognises the agent). Such proteins and such antagonists can also be used to prevent or treat coeliac disease. The antagonist will cause a decrease in the T cell response. In one embodiment, the antagonist binds the TCR of the T cell (generally in the form of a complex with HLA-DQ2 or -DQ8) but instead of causing normal functional activation causing an abnormal signal to be passed through the TCR intracellular signalling cascade, which causes the T
cell to have decreased function activity (e.g. in response to recognition of an epitope, typically as measured by any -suitable assay mentioned herein).
In one embodiment the antagonist competes with epitope to bind a Component of MHC processing and presentation pathway, such as an MHC molecule (typically HLA-DQ2 or -DQ8). Thus the antagonist may bind HLA-DQ2 or -DQ8 (and thus be a peptide presented by this MHC molecule), such as peptide TP
(Table 10) or a homologue thereof.
Methods of causing antagonism are known in the art. In one embodiment the antagonist is a homologue of the epitopes mentioned above and may have any of the sequence, binding or other properties of the agent (particularly analogues).
The antagonists typically differ from any of the above epitopes (which are capable of causing a normal antigen specific function in the T cell) by 1, 2, 3, 4 or more mutations (each of which may be a substitution, insertion or deletion). Such antagonists are termed "altered peptide ligands" or "APL" in the art. The mutations are typically at the amino acid positions that contact the TCR.
The antagonist may differ ,from the epitope by a substitution within the sequence that is equivalent to the sequence represented by amino acids 65 to 67 of A-gliadin (such antagonists are shown in Table 9). Thus preferably the antagonist has a substitution at the equivalent of position 64, 65 or 67. Preferably the substitution is 64W, 67W, 67M or 65T.
Since the T cell immune response to the epitope of the invention in an individual is polyclonal, more than one antagonist may need to be administered to cause antagonism of T cells of the response which have different TCRs.
Therefore the antagonists may be administered in a composition which comprises at least 2, 4, 6 or more different antagonists, which each antagonise different T cells.

The invention also provides a method of identifying an antagonist of a T cell (which recognises the agent), comprising contacting a candidate substance with the T
cell and detecting whether the substance causes a decrease in the ability of the T cell to undergo an antigen specific response (e.g. using any suitable assay mentioned 5 herein), the detecting of any such decrease in said ability indicating that the substance is an antagonist.
In one embodiment, the antagonists (including combinations of antagonists to a particular epitope) or tolerising (T cell and antibody tolerising) agents are present in a composition comprising at least 2, 4, 6 or more antagonists or agents which 10 antagonise or tolerise to different epitopes of the invention, for example to the combinations of epitopes discussed above in relation to the agents which are a product comprising more than one substance.
Testing whether a composition is capable of causing coeliac disease 15 As mentioned above the invention provides a method of determining whether a composition is capable of causing coeliac disease comprising detecting the presence of a protein sequence which is capable of being modified by a transglutaminase to as sequence comprising the agent or epitope of the invention (such transglutaminase activity may be a human intestinal transglutaminase activity).
20 Typically this is performed by using a binding assay in which a moiety which binds to the sequence in a specific manner is contacted with. the composition and the formation of sequence/moiety complex is detected and used to ascertain the presence of the agent. Such a moiety may be any suitable substance (or type of substance) mentioned herein, and is typically a specific antibody. Any suitable format of

25 binding assay can be used (such as those mentioned herein).
In one embodiment, the composition is contacted with at least 2, 5, 10 or more antibodies which are specific for epitopes of the invention from different gliadins, for example a panel of antibodies capable of recognising the combinations of epitopes discussed above in relation to agents of the invention which are a product comprising more than one substance.
The composition typically comprises material from a plant that expresses a gliadin which is capable of causing coeliac disease (for example any of the gliadins

26 or plants mentioned herein). Such material may be a plant part, such as a harvested product (e.g. seed). The material may be processed products of the plant material (e.g. any such product mentioned herein), such as a flour or food that comprises the gliadin. The processing of food material and testing in suitable binding assays is routine, for example as mentioned in Kricka LT, J. Biolumin. Chemilumin. 13, 93 (1998).
Binding assays The determination of binding between any two substances mentioned herein may be done by measuring a characteristic of either or both substances that changes upon binding, such as a spectroscopic change.
The binding assay format may be a 'band shift' system. This involves determining whether the presence of one substance (such as a candidate substance) advances or retards the progress of the other substance during gel electrophoresis.
The format may be a competitive binding method which determines whether the one substance is able to inhibit the binding of the other substance to an agent which is known to bind the other substance, such as a specific antibody.
Mutant gliadin proteins The invention provides a gliadin protein in which an epitope sequence of the invention, or sequence which can be modified by a transglutaminase to provide such a sequence has been mutated so that it no longer causes, or is recognised by, a T cell response that recognises the epitope. In this context the term recognition refers to the TCR binding the epitope in such a way that normal (not antagonistic) antigen-specific functional activity of the T cell occurs.
Methods of identifying equivalent epitopes in other gliadins are discussed above. The wild type of the mutated gliadin is one which causes coeliac disease.
Such a gliadin may have homology with SEQ ID NO:3, for example to the degree mentioned above (in relation to the analogue) across all of SEQ ID NO:3 or across 15, 30, 60, 100 or 200 contiguo,us amino acids of SEQ ID NO:3. Likewise, for other non-A-gliadins, homology will be present between the mutant and the native form of that gliadin. The sequences of other natural gliadin proteins are known in the art.

27 The mutated gliadin will not cause coeliac disease or will cause decreased =
symptoms of coeliac disease. Typically the mutation decreases the ability of the epitope to induce a T cell response. The mutated epitope may have a decreased binding to HLA-DQ2 or -DQ8, a decreased ability to be presented by an APC or a decreased ability to bind to or to be recognised (i.e. cause antigen-specific functional activity) by T cells that recognise the agent. The mutated gliadin or epitope will therefore show no or reduced recognition in any of the assays mentioned herein in relation to the diagnostic aspects of the invention.
The mutation may be one-or more deletions, additions or substitutions of length 1 to 3, 4 to 6, 6 to 10, 11 to 15 or more in the epitope, for example across sequence SEQ ID NO:2 or across any of SEQ ID NOS: 18-22, 31-36, 39-44, and 46;

or acioss equivalents thereof. Preferably the mutant gliadin has at least one mutation in the sequence SEQ ID NO: 1. A preferred mutation is at position 65 in A-gliadin (or in an equivalent position in other gliadins). Typically the naturally occurring glutamine at this position is substituted to any of the amino acids shown in Table 3, preferably to histidine, tyrosine, tryptophan, lysine, proline, or arginine.
The invention thus also provides use of a mutation (such any of the mutations in any of the sequences discussed herein) in an epitope of a gliadin protein, which epitope is an epitope of the invention, to decrease the ability of the gliadin protein to cause coeliac disease.
In one embodiment the mutated sequence is able to act as an antagonist.
Thus the invention provides a protein that comprises a sequence which is able to bind to a T cell receptor, which T cell receptor recognises an agent of the invention, and which sequence is able to cause antagonism of a T cell that carries such a T
cell receptor.
The invention also provides proteins which are fragments of the above mutant gliadin proteins, which are at least 15 amino acids long (e.g. at least 30, 60, 100, 150, 200, or 250 amino acids long) and which comprise the mutations discussed above which decrease the ability of the gliadin to be recognised. Any of the mutant proteins (including fragments) mentioned herein may also be present in the form of fusion proteins, for example with other gliadins or with non-gliadin proteins.

28 The equivalent wild type protein to the mutated gliadin protein is typically from a g-raminaceous monocotyledon, such as a plant of genus Triticum, e.g.
wheat, rye, barley, oats or triticale. The protein is typically an a, af3, f3, y or a) gliadin. The gliadin may be an A-gliadin.
Kits The invention also provides a kit for carrying out the method comprising one or more agents and optionally a means to detect the recognition of the agent by the T
cell. Typically the different agents are provided for simultaneous, separate or sequential use. Typically the means to detect recognition allows or aids detection based on the techniques discussed above.
Thus the means may allow detection of a substance. secreted by the T cells after recognition. The kit may thus additionally include a specific binding moiety for the substance, such as an antibody. The moiety is typically specific for IFNI.
The moiety is typically immobilised on a solid support. This means that after binding the moiety the substance will remain in the vicinity of the T cell which secreted it. Thus "spots" of substance/moiety complex are formed on the support, each spot representing a T cell which is secreting the substance. Quantifying the spots, and typically comparing against a control, allows determination of recognition of the agent.
The kit may also comprise a means to detect the substance/moiety complex.
A detectable change may occur in the moiety itself after binding the substance, such as a colour change. Alternatively a second moiety directly or indirectly labelled for detection may be allowed to bind the substance/moiety complex to allow the determination of the spots. As discussed above the second moiety may be specific for the substance, but binds a different site on the substance than the first moiety.
The immobilised support may be a plate with wells, such as a microtitre plate.

Each assay can therefore be carried out in a separate well in the plate.
The kit may additionally comprise medium for the T cells, detection moieties or washing buffers to be used in the detection steps. The kit may additionally comprise reagents suitable for the separation from the sample, such as the separation of PBMCs or T cells from the sample. The kit may be designed to allow detection of

29 the T cells directly in the sample without requiring any separation of the components of the sample.
The kit may comprise an instrument which allows administration of the agent, such as intradermal or epidermal administration. Typically such an instrument comprises plaster, dressing or one or more needles. The instrument may allow ballistic delivery of the agent. The agent in the kit may be in the form of a pharmaceutical composition.
The kit may also comprise controls, such as positive or negative controls.
The positive control may allow the detection system to be tested. Thus the positive control typically mimics recognition of the agent in any of the above methods.
Typically in the kits designed to determine recognition in vitro the positive control is a cytokine. In the kit designed to detect in vivo recognition of the agent the positive control may be antigen to which most individuals should response.
The kit may also comprise a means to take a sample containing T cells from the host, such as a blood sample. The kit may comprise a means to separate mononuclear cells or T cells from a sample from the host.
Polynucleotides,.cells, transgenic mammals and antibodies The invention also provides a polynucleotide which is capable of expression to provide the agent or mutant gliadin proteins. Typically the polynucleotide is DNA
, or RNA, and is single or double stranded. The polynucleotide will preferably comprise at least 50 bases or base pairs, for example 50 to 100, 100 to 500, 500 to 1000 or 1000 to 2000 or more bases or base pairs. The polynucleotide therefore comprises a sequence which encodes the sequence of SEQ ID NO: 1 or 2 or any of the other agents mentioned herein. To the 5' and 3' of this coding sequence the polynucleotide of the invention has sequence or codons which are different from the sequence or codons 5' and 3' to these sequences in the corresponding gliadin gene.
5' and/or 3 to the sequence encoding the peptide the polynucleotide has coding or non-coding sequence. Sequence 5' and/or 3' to the coding sequence may comprise sequences which aid expression, such as transcription and/or translation, of the sequence encoding the agent. The polynucleotide may be capable of expressing the agent prokaryotic or eukaryotic cell. In one embodiment the polynucleotide is capable of expressing the agent in a mammalian cell, such as a human, primate or rodent (e.g. mouse or rat) cell.
A polynucleotide of the invention may hybridise selectively to a polynucleotide that encodes SEQ ID NO:3 at a level significantly above background.
5 Selective hybridisation is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50 C to about 60 C). However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al (1989), Molecular Cloning: A Laboratory Manual). -For example, if high stringency is required, suitable 10 conditions include 0.2 x SSC at 60 C. If lower stringency is required, suitable conditions include 2 x SSC at 60 C.
Agents or proteins of the invention may be encoded by the polynucleotides described herein.
The polynucleotide may form or be incorporated into a replicable vector.
15 Such a vector is able to replicate in a suitable cell. The vector may be an expression vector. In such a vector the polynucleotide of the invention is operably linked to a control sequence which is capable of providing for the expression of the polynucleotide. The vector may contain a selectable marker, such as the ampicillin resistance gene.
20 The polynucleotide or vector may be present in a cell. Such a cell may have been transformed by the polynucleotide or vector. The cell may express the agent.
The cell will be chosen to be compatible with the said vector and may for example be a prokaryotic (bacterial), yeast, insect or mammalian cell. The polynucleotide or.
vector may be introduced into host cells using conventional techniques including 25 calcium phosphate precipitation, DEAE-dextran transfection, or electroporation.
The invention provides processes for the production of the proteins of the invention by recombinant means. This may comprise (a) cultivating a transformed cell as defined above under conditions that allow the expression of the protein; and preferably (b) recovering the expressed polypeptide. Optionally, the polypeptide

30 may be isolated and/or purified, by techniques known in the art.
The invention also provides TCRs which recognise (or bind) the agent, or fragments thereof which are capable of such recognition (or binding). These can be

31 present in the any form mentioned herein (e.g. purity) discussed herein in relation to the protein of the invention. The invention also provides T cells which express such TCRs which can be present in any form (e.g. purity) discussed herein for the cells of the invention.
The invention also provides monoclonal or polyclonal antibodies which specifically recognise the agents (such as any of the epitopes of the invention) and which recognise the mutant gliadin proteins (and typically which do not recognise the equivalent wild-type gliadins) of the invention, and methods of making such antibodies. Antibodies of the- invention bind specifically to these substances of the invention.-For the purposes of this invention, the term "antibody" includes antibody fragments such as Fv, F(ab) and F(ab)2 fragments, as well as single-chain antibodies.
A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immuno globulins from the serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG
fraction purified. A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with.
tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).
An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.
The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified. =

32 The polynucleotide, agent, protein or antibody of the invention, may carry a detectable label. Detectable labels. which allow detection of the secreted substance by visual inspection, optionally with the aid of an optical magnifying means, are preferred. Such a system is typically based on an enzyme label which causes colour change in a substrate, for example alkaline phosphatase causing a colour change in a substrate. Such substrates are commercially available, e.g. from BioRad. Other suitable labels include other enzymes such as peroxidase, or protein labels, such as biotin; or radioisotopes, such as 32P or 35S. The above labels may be detected using known techniques. .
Polynucleotides, agents, proteins, antibodies or cells of the invention may be in substantially purified form. They may be in substantially isolated form, in which case they will generally comprise at least 80% e.g. at least 90, 95, 97 or 99%
of the polynucleotide, peptide, antibody, cells or dry mass in the preparation. The polynucleotide, agent, protein or antibody is typically substantially free of other cellular components. The polynucleotide, agent, protein or. antibody may be used in such a substantially isolated, purified or free form in the method or be present in such - forms in the kit.
The invention also provides a transgenic non-human mammal which expresses a TCR of the invention. This may be any of the mammals discussed herein (e.g. in relation to the production of the antibody). Preferably the mammal has, or is susceptible, to coeliac disease. The mammal may also express HLA-DQ2 or -DQ8 or HLA-DR3-DQ2 and/or may be given a diet comprising a gliadin which cause coeliac disease (e.g. any of the gliadin proteins mentioned herein). Thus the mammal may act as an animal model for coeliac disease.
The invention also provides a method of identifying a product which is therapeutic for coeliac disease comprising administering a candidate substance to a mammal of the invention which has, or which is susceptible to, coeliac disease and determining whether substance prevents or treats coeliac disease in the mammal, the prevention or treatment of coeliac disease indicating that the substance is a therapeutic product. Such a product may be used to treat or prevent coeliac disease.
The invention provides therapeutic (including prophylactic) agents or diagnostic substances (the agents, proteins and polynucleotides of the invention).

33 These substances are formulated for clinical administration by mixing them with a pharmaceutically acceptable carrier or diluent. For example they can be formulated for topical, parenteral, intravenous, intramuscular,, subcutaneous, intraocular, intrademial, epidermal or transdermal administration. The substances may be mixed with any vehicle which is pharmaceutically acceptable and appropriate for the desired route of administration. The pharmaceutically carrier or diluent for injection may be, for example, a sterile or isotonic solution such as Water for Injection or physiological saline, or a carrier particle for ballistic delivery.
The dose of the substances may be adjusted according to various parameters, especially according to the agent used; the age, weight and condition of the patient to be treated; the mode of administration used; the severity of the condition to be . treated; and the required clinical regimen. As a guide, the amount of substance administered by injection is suitably from 0.01 mg/kg to 30 mg/kg, preferably from = = 0.1 mg/kg to 10 mg/kg.
The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.
The substances of the invention may thus be used in a method of treatment of the human or animal body, or in a diagnostic method practised on the human body.
In particular they may be used in a method of treating or preventing coeliac disease.
The invention also provide the agents for use in a method of manufacture of a medicament for treating or preventing coeliac disease. Thus the invention provides a method of preventing or treating coeliac disease comprising administering to a human in need thereof a substance of the invention (typically a non-toxic effective amount thereof).
The agent of the invention can be made using standard synthetic chemistry techniques, such as by use of an automated synthesizer. The agent may be made from a longer polypeptide e.g. a fusion protein, which polypeptide typically comprises the sequence of the peptide. The peptide may be derived from the polypeptide by for example hydrolysing the polypeptide, such as using a protease; or by physically breaking the polypeptide. The polynucleotide of the invention can be made using standard techniques, such as by using a synthesiser.

34 Plant cells and plants that express mutant gliadin proteins or express proteins comprising sequences which can act as antagonists The cell of the invention may be a plant cell, such as a cell of a graminaceous monocotyledonous species. The species may be one whose wild-type form expresses gliadins, such as any of the gliadin proteins mentioned herein (including gliadins with any degree of homology to SEQ ID NO:3 mentioned herein). Such a gliadin may cause coeliac disease in humans. The cell may be of wheat, maize, oats, rye,.
rice, barley, triticale, sorghum, or sugar cane. Typically the cell is of the Triticum genus, such aS aestivum, spelta, polonicum or monococcum.
The plant cell of the invention is typically one which does not express a wild-type gliadin (such as any of the gliadins Mentioned herein which May cause coeliac disease), or one which does not express a gliadin comprising a sequence that can be recognised by a T cell that recognises the agent. Thus if the wild-type plant cell did express such a gliadin then it may be engineered to prevent or reduce the expression of such a gliadin or to change the amino acid sequence of the gliadin so that it no longer causes coeliac disease (typically by no longer expressing the epitope of the invention).
This can be done for example by introducing mutations into 1, 2, 3 or more or all of such gliadin genes in the cell, for example into coding or non-coding (e.g.
promoter regions). Such mutations can be any of the type or length of mutations discussed herein (e.g., in relation to homologous proteins). The mutations can be introduced in a directed manner (e.g., using site directed mutagenesis or homologous recombination techniques) or in a random manner (e.g. using a mutagen, and then typically selecting for mutagenised cells which no longer express the gliadin (or a gliadin sequence which causes coeliac disease)).
In the case of plants or plant cells that express a protein that comprises a sequence able to act as an antagonist such a plant or plant cell may express a wild-type gliadin protein (e.g. one which causes coeliac disease). Preferably though the presence of the antagonist sequence will cause reduced coeliac disease symptoms (such as no symptoms) in an individual who ingests a food comprising protein from the plant or plant cell.

The polynucleotide which is present in (or which was transformed into) the plant cell will generally comprise promoter capable of expressing the mutant gliadin protein the plant cell. Depending on the pattern of expression desired, the promoter may be cOnstitutive, tissue- or stage-specific; and/or inducible. For example, strong 5 constitutive expression in plants can be obtained with the CAMV 35S, Rubisco ssu, or histone promoters. Also, tissue-specific or stage-specific promoters may be used to target expression of protein of the invention to particular tissues in a transgenic plant or to particular stages in its development. Thus, for example seed-specific, root-specific, leaf-specific, flower-specific etc promoters may be used. Seed-specific 10 promoters include those described by Dalta et al (Biotechnology Ann. Rev. (1997), 3, pp.269-296). Particular examples of seed-specific promoters are napin promoters (EP-A-0 255, 378), phaseolin promoters, glute_nine promoters, helianthenine promoters (W092/17580), albumin promoters (W098/45460), Oleosin promoters (W098/45461) and ATS1 and ATS3 promoters (PCT11JS98/06798).
. 15 The cell may be in any form. For example, it may be an isolated cell, e.g. a protoplast, or it may be part of a plant tissue, e.g. a callus, or a tissue excised from a plant, or it may be part of a whole plant. The cell may be of any type (e.g.
of any type of plant part). For example, an undifferentiated cell, such as a callus cell; or a differentiated cell, such as a cell of a type found in embryos, pollen, roots, shoots or 20 leaves. Plant parts include roots; shoots; leaves; and parts involved in reproduction, such as pollen, ova, stamens, anthers, petals, sepals and other flower parts.
The invention provides a method of obtaining a transgenic plant cell comprising transforming a plant cell with a polynucleotide or vector of the invention to give a transgenic plant cell. Any suitable transformation method may be used (in 25 the case of wheat the techniques disclosed in Vasil V et al, Biotechnology 10, 667-674 (1992) may be used). Preferred transformation techniques include electroporation of plant protoplasts and particle bombardment. Transformation may thus give rise to a chimeric tissue or plant in which some cells are transgenic and some are not.
30 The cell of the invention or thus obtained cell may be regenerated into a transgenic plant by techniques known in the art. These may involve the use of plant growth substances such as auxins, giberellins and/or cytokinins to stimulate the growth and/or division of the transgenic cell. Similarly, techniques such as somatic embryogenesis and meristem culture may be used. Regeneration techniques are well known in the art and examples can be found in, e.g. US 4,459,355, US
4,536,475, US
5,464,763, US 5, 177,010, US 5, 187,073, EP 267,159, EP 604, 662, EP 672, 752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US
5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US
5,554,798, US 5,489,520, US 5,510,318, US. 5,204,253, US 5,405,765, EP
442,174, EP 486,233, EP 486,234, EP 539,563, EP 674,725, W091/02071 and W095/06128.
In many such techniques,nne step is the formation of a callus, i.e. a plant tissue comprising expanding and/or dividing cells. Such calli are a further aspect of the invention as are other types of plant cell cultures and plant parts. Thus, for exanaple, the invention provides transgenic plant tissues and parts, including embryos, meristems, seeds, shoots, roots, stems, leaves and flower parts.
These may be chimeric in the sense that some of their cells are cells of the invention and some are not. Transgenic plant parts and tissues, plants and seeds of the invention may be of any of the plant species mentioned herein.
Regeneration procedures will typically involve the selection of transformed cells by means of marker genes.
The regeneration step gives rise to a first generation transgenic plant. The .. invention also provides methods of obtaining transgenic plants of further generations from this first generation plant. These are known as progeny transgenic plants.
Progeny plants of second, third, fourth, fifth, sixth and further generations may be obtained from the first generation transgenic plant by any means known in the art.
Thus, the invention provides a method of obtaining a transgenic progeny plant comprising obtaining a second-generation transgenic progeny plant from a first-generation transgenic plant of the invention, and optionally obtaining transgenic plants of one or more further generations from the second-generation progeny plant thus obtained.
Progeny plants may be produced from their predecessors of earlier generations by any known technique. In particular, progeny plants may be produced by:

obtaining a transgenic seed from a transgenic plant of the invention belonging to a previous generation, then obtaining a transgenic progeny plant of the invention belonging to a new generation by growing up the transgenic seed; and/or propagating clonally a transgenic plant of the invention belonging to a previous generation to give a transgenic progeny plant of the invention belonging to a new generation; and/or crossing a first-generation transgenic plant of the invention belonging to a previous generation with another compatible plant to give a transgenic progeny plant of the invention belonging to -a. new generation; and optionally obtaining transgenic progeny plants of one or more further generations from . the progeny plant thus obtained.
These techniques may be used in any combination. For example, clonal propagation and sexual propagation may be used at different points in a process that gives rise to a transgenic plant suitable for cultivation. In particular, repetitive back-crossing with a plant taxon with agronomically desirable characteristics may be undertaken. Further steps of removing cells from a plant and regenerating new plants therefrom may also be carried out.
Also, further desirable characteristics may be introduced by transforming the cells, plant tissues, plants or seeds, at any suitable stage in the above process, to introduce desirable coding sequences other than the'polynucleotides of the invention.
This may be carried out by the techniques described herein for the introduction of polynucleotides of the invention.
For example, further transgenes may be selected from those coding for other herbicide resistance traits, e.g. tolerance to: Glyphosate (e.g. using an EPSP
synthase gene '(e.g. EP-A-0 293,358) or a glyphosate oxidoreductase (WO 92/000377) gene);
or tolerance to fosametin; a dihalobenzonitrile; glufosinate, e.g. using a phosphinothrycin acetyl transferase (PAT) or glutamine synthase gene (cf. EP-A-242,236); asulam, e.g. using a dihydropteroate synthase gene (EP-A-0 369,367);
or a sulphonylurea, e.g. using an ALS gene); diphenyl ethers such as acifluorfen or oxyfluorfen, e.g. using a protoporphyrogen oxidase gene); an oxadiazole such as oxadiazon; a cyclic it-nide such as chlorophthalim; a phenyl pyrazole such as TNP, or a phenopylate or carbamate analogue thereof similarly, genes for beneficial properties other than herbicide tolerance may be introduced. For example, genes for insect resistance may be introduced, notably genes encoding Bacillus thuringiensis (Bt) toxins. Likewise, genes for disease resistance may be introduced, e.g. as in W091/02701 or W095/06128.
Typically, a protein of the invention is expressed in a plant of the invention.
Depending on the promoter used, this expression may be constitutive or inducible.
Similarly, it may be tissue- or stage-specific, i.e. directed towards a particular plant tissue (such as any of the tissues mentioned herein) or stage in plant development.
The invention also provides methods of obtaining crop products by harvesting, and optionally processing further, transgenic plants of the invention. By crop product is meant any useful product obtainable from a crop plant.
Products that contain mutant gliadin proteins or proteins that comprise sequence capable of acting as an antagonist The invention provides a product that comprises the mutant gliadin proteins or protein that comprises sequence capable of acting as an antagonist. This is typically derived from or comprise plant parts from plants mentioned herein which express such proteins. Such a product may be obtainable directly by harvesting or indirectly, by harvesting and further processing the plant of the invention.
Directly obtainable products include grains. Alternatively, such a product may be obtainable indirectly, by harvesting and further processing. Examples of products obtainable by further processing are flour or distilled alcoholic beverages; food products made from directly obtained or further processed material, e.g. baked products (e.g. bread) made from flour. Typically such food products, which are ingestible and digestible (i.e. non-toxic and of nutrient value) by human individuals.
In the case of food products that comprise the protein which comprises an antagonist sequence the food product may also comprise wild-type gliadin, but .
preferably the antagonist is able to cause a reduction (e.g. completely) in the coeliac disease symptoms after such food is ingested.
The invention is illustrated by the following nonlimiting Examples:
Example 1 We carried out epitope mapping in Coeliac disease by using a set of 51 synthetic 15-mer peptides that span the complete sequence of a fully characterized a-gliadin, "A-gliadin" (see Table 1). A-Gliadin peptides were also individually treated with tTG to generate products that might mimic those produced in vivo3. We also sought to study Coeliac disease patients at the point of initiation of disease relapse to avoid the possibility that epitope "spreading" or "exhaustion" may have occurred, as described in experimental infectious and autoimmune diseased.
=
Clinical and A-gliadin specific Pcell responses with 3 and 10 day bread challenge In a pilot study, two subjects with Coeliac disease in remission, defined by absence of serum anti-endomysial antibody (EMA), on a gluten free diet were fed four slices of standard gluten-containing white bread daily in addition to their Usual gluten free diet. Subject 1 ceased bread because of abdominal pain, mouth ulcers and mild diarrhoea after three days, but Subject 2 continued for 10 days with only mild nausea at one week. The EMA became positive in Subject 2 one week after the bread challenge, indicating the bread used had caused a relapse of Coeliac disease.
But in Subject 1, EMA remained negative up to two months after bread challenge. In both subjects, symptoms that appeared with bread challenge resolved within two days after returning to gluten free diet.
PBMC responses in IFNy ELISPOT assays to ,A-gliadin peptides were not found before or during bread challenge. But from the day after bread withdrawal (Day 4) in Subject 1 a single pool of 5 overlapping peptides spanning A-gliadin 51-85 (Pool 3) treated with tTG showed potent IFNy responses (see Figure la). In Subject 1, the PBMC IFNy response to A-gliadin peptide remained targeted to Pool 3 alone and was maximal on Day 8. The dynamics and magnitude of the response to Pool 3 was similar to that elicited by a-chymotrypsin digested gliadin. PBMC

responses to tTG-treated Pool 3 were consistently 5 to 12-fold greater than Pool 3 not treated with tTG, and responses to a-chymotrypsin digested gliadin were 3 to 10-fold greater if treated with tTG. In Subject 2, Pool 3 treated with tTG was also the only immunogenic set of A-gliadin peptides on Day 8, but this response was weaker than Subject 1, was not seen on Day 4 and by Day 11 the response to Pool 3 had diminished and other tTG-treated pools of A-gliadin peptides elicited stronger IFNa responses (see Figure lb).
The pilot study indicated that the initial T cell response in these Coeliac disease subjects was against a single tTG-treated A-gliadin pool of five peptides and was readily measured in peripheral blood. But if antigen exposure is continued for 5 ten days instead of three, T cell responses to other A-gliadin peptides appear, consistent with epitope spreading.
Coeliac disease-specific .1-FN-g induction by 11'G-treated A-gliadin peptides In five out of six further Coeliac disease subjects on gluten free diet (see 10 Table 1), bread challenge for three days identified tTG-treated peptides in Pool 3, and in particular, peptides corresponding to 56-70 (12) and 60-75 (13) as the sole A-gliadin components eliciting IFI\lry from PBMC (see Figure 2). IL-10 ELISPOT
assays run in parallel to IFNy ELISPOT showed no IL-10 response to tTG-treated peptides 12 or 13. In one subject, there were no 'FM/ responses to any A-gliadin 15 .. peptide or a-chymotrypsin digested gliadin before, during or up to four days after = bread challenge. In none of these Coeliac disease subjects did EMA status change from baseline when measured for up to two months after bread challenge.
PBMC from four healthy, BMA-negative subjects with the HLA-DQ alleles a 1*0501, p,1*0201 (ages 28-52, 2 females) who had been challenged for three days 20 with bread after following a gluten free diet for one month, showed no IFNy responses above the negative control to any of the A-gliadin peptides with or without tTG treatment. Thus, induction of IFNy in PBMC to tTG-treated Pool 3 and A-gliadin peptides 56-70 (12) and 60-75 (13) were Coeliac disease specific (7/8 vs. 0/4, .
p<0.01 by Chi-squared analysis).
Fine mapping of the minimal A-gliadin T cell epitope tTG-treated peptides representing truncations of A-gliadin 56-75 revealed that the same core peptide sequence QPQLP (SEQ ID NO:9) was essential for antigenicity in all of the five Coeliac disease subjects assessed (see Figure 3). PBMC
IFNy responses to tTG-treated peptides spanning this core sequence beginning with the 7-mer PQPQLPY (SEQ ID NO:4) and increasing in length, indicated that the tTG-treated 17-mer QLQPFPQPQLPYPQPQS (SEQ ID NO:10) (A-gliadin 57-73) possessed optimal activity in the IFN'y ELISPOT (see Figure 4).
Deamidation of Q65 by tTG generates the inummodominant T cell epitope in A-gliadin HPLC analysis demonstrated that tTG treatment of A-gliadin 56-75 generated a single product that eluted marginally later than the parent peptide. Amino acid sequencing indicated that out of the six glutamine (Q) residues contained in A-gliadin 56-75, Q65 was preferentially deamidated by tTG (see Figure 5).
Bioactivity of peptides corresponding to serial expansions from the core A-gliadin 62-68 sequence in which glutamate (E) replaced Q65, was equivalent to the same peptides with Q65 after tTG-treatment (see Figure 4a). Replacement of Q57 and Q72 by E
together or alone, with E65 did not enhance antigenicity of the 17-mer in the three Coeliac disease subjects studied (see Figure 6). Q57 and Q72 were investigated because glutamine residues followed by proline in gliadin peptides are not deamidated by tTG in vitro (W. Vader et al, Proceedings 8th International .Symposium Coeliac Disease). Therefore, the immunodominant T cell epitope was defined as QLQPFPQPELPYPQPQS (SEQ ID NO:2).
Inununodominant T cell epitope response is DQ2-restricted and CD4 dependent In two Coeliac disease subjects homozygous for HLA-DQ a 1*0501, 131*0201, anti-DQ monoclonal antibody blocked the ELISPOT IFN'y response to tTG-treated A-gliadin 56-75, but anti-DP and -DR antibody did not (see Figure 7).
Anti-CD4 and anti-CD8 magnetic bead depletion of PBMC from two Coeliac disease subjects indicated the IFNI/ response to tTG-treated A-gliadin 56-75 is CD4 T
cell-mediated.
Discussion In this study we describe a rather simple dietary antigen challenge using standard white bread to elicit a transient population of CD4 T cells in peripheral blood of Coeliac disease subjects responsive to a tTG-treated A-gliadin 17-mer with the sequence: QLQPFPQPELPYPQPQS (SEQ ID NO:2) (residues 57-73). The immune response to A-gliadin 56-75 (Q--)E65) is restricted to the Coeliac disease-associated HLA allele, DQ a1*0501, 131*0201. Tissue transglutaminase action in vitro selectively deamidates Q65. Elicited peripheral blood IFNg responses to synthetic A-gliadin peptides with the substitution Q-->E65 is equivalent to tTG-treated Q65 A-gliadin peptides; both stimulate up to 10-fold more T cells in the IFNg ELISPOT than unmodified Q65 A-gliadin peptides.
We have deliberately defined this Coeliac disease-specific T cell epitope using in vivo antigen challenge and short-term ex vivo immune assays to avoid the possibility of methodological artifacts that may occur with the use of T cell clones in = epitope mapping. Our findings indicate that peripheral blood T cell responses to ingestion of gluten are rapid but short-lived and can be utilized for epitope mapping.
In vivo antigen challenge has also shown there is a temporal hierarchy of immune responses to A-gliadin peptides; A-gliadin 57-73 modified by tTG not only elicits the strongest IFNg response in PBMC but it is also the first IFNg resPonse to appear.
Because we have assessed only peptides spanning A-gliadin, there may be other epitopes in other gliadins of equal or greater importance in the pathogenesis of Coeliac disease. Indeed, the peptide sequence at the core of the epitope in A-gliadin that we have identified PQPQLPY (SEQ ID NO:4) is shared by several other gliadins (SwissProt and Trembl accession numbers: P02863, Q41528, Q41531, Q41533, Q9ZP09, P04722, P04724, P18573). However, A-gliadin peptides that have previously been shown to possess bioactivity in biopsy challenge and in vivo studies (for example: 31-43, 44-55, and 206-217)4'5 did not elicit IFNg responses in PBMC
following three day bread challenge in Coeliac disease subjects. These peptides may be "secondary". T cell epitopes that arise with spreading of the immune response.
Example 2 The effect on T cell recognition of substitutions in the immunodominant epitope The effect of substituting the glutamate at position 65 in the 57-73 A-gliadin epitope was determined by measuring peripheral blood responses against the substituted epitopes in an IFNy ELISPOT assay using synthetic peptides (at 50 tg/m1). The responses were measured in 3 Coeliac disease subjects 6 days after commencing gluten challenge (4 slices bread daily for 3 days). Results are shown in table 3 and Figure 8. As can be seen substitution of the glutamate to histidine, tyrosine, tryptophan, lysine, proline or arginine stimulated a response whose magnitude was less than 10% of the magnitude of the response to the immunodominant epitope. Thus mutation of A-gliadin at this position could be used to produce a mutant gliadin with reduce or absent immunoreactivity.
Example 3 Testing the immunoreactivity of equivalent peptides from other naturally occurring gliadins The immunoreactivity of -equivalent peptides form other naturally occurring wheat gliadins was assessed using synthetic peptides corresponding to the naturally occurring sequences which were then treated with transglutaminase. These peptides were tested in an ELISPOT in the same manner and with PBMCs from the same subjects as described in Example 2. At least five of the peptides show immunoreactivity comparable to the A-gliadin 57-73 E65 peptide (after transglutaminase treatment) indicating that other gliadin proteins in wheat are also likely to induce this Coeliac disease-specific immune response (Table 4 and Figure 9).
Methods Subjects: Patients used in the study attended a Coeliac Clinic in Oxford, United Kingdom. Coeliac disease was diagnosed on the basis of typical small intestinal histology, and normalization of symptoms and small intestinal histology with gluten free diet.
=
Tissue typing: Tissue typing was performed using DNA extracted from EDTA-anticoagulated peripheral blood. HLA-DQA and DQB genotyping was performed by PCR using sequence-specific primer mixes6-8.
Anti-endonzysial antibody assay: EMA were detected by indirect immunofiuorescence using patient serum diluted 1:5 with monkey oesophagus, followed by FITC-conjugated goat anti-human IgA. IgA was quantitated prior to EMA, none of the subjects were IgA deficient.

Antigen Challenge: Coeliac disease subjects following a gluten free diet, consumed 4 slices of gluten-containing bread (50g/slice, Sainsbury's "standard white sandwich bread") daily for 3 or 10 days. EMA was assessed the week before and up to two months after commencing the bread challenge. Healthy subjects who had followed a gluten free diet for four weeks, consumed their usual diet including four slices of gluten-containing bread for three days, then returned to gluten free diet for a further six days.
-IFNy and IL-.10 ELISPOT: PBMC were prepared from 50-100 ml of venous blood by Ficoll-Hypaque density centrifugation. After three washes, PBMC were resuspended in complete RPMI containing 10% heat inactivated human AB serum. ELISPOT
assays for single cell secretion of IFI\Ty and IL-10 were performed using commercial = kits (Mabtech; Stockholm, Sweden) with 96-well plates (MAIP-S-45;
Millipore, Bedford, MA) according to the manufacturers instructions (as described elsewhere9) with 2-5x105 (IFNy) or 0.4-1x105 (IL-10) PBMC in each well. Peptides were assessed in duplicate wells, and Mycobacterium tuberculosis purified protein derivative (PPD RT49) (Serum Institute; Copenhagen, Denmark) (20 ftg/m1) was included as a positive control in all assays.
Peptides: Synthetic peptides were purchased from Research Genetics (Huntsville, Alabama) Mass-spectroscopy and HPLC verified peptides' authenticity and >70%
purity. Digestion of gliadin (Sigma; G-3375) (100 mg/ml) with a-chymotrypsin (Sigma; C-3142) 200:1 (w/w) was performed at room temperature in 0.1 M
NH4HCO3 with 2M urea and was halted after 24 h by heating to 98 C for 10 minutes.
After centrifugation (13,000g, 10 minutes), the gliadin digest supernatant was filter-sterilized (0.2 mm). Digestion of gliadin was verified by SDS-PAGE and protein concentration assessed. a-Chymotrypsin-digested gliadin (640 ,ug/m1) and synthetic gliadin peptides (15-mers: 160 pig/ml, other peptides: 0.1 inM) were individually treated with tTG (Sigma; T-5398) (50 1.tg/m1) in PBS + CaCl2 1 mM for 2 h at 37 C.
Peptides and peptide pools were aliquotted into sterile 96-well plates and stored frozen at -20 C until use.

Amino acid sequencing of peptides: Reverse phase HPLC was used to purify the peptide resulting from tTG treatment of A-gliadin 56-75. A single product was identified and subjected to amino acid sequencing (automated sequencer Model 5 494A, Applied Biosystems, Foster City, California). The sequence of unmodified 056-75 was confirmed as: LQLQPFPQPQLPYPQPQSFP (SEQ ID NO:5), and tTG
treated G56-75 was identified as: LQLQPFPQPELPYPQPQSFP (SEQ ID NO:11).
Deamidation of glutamyl residues was defined as the amount (pmol) of glutamate recovered expressed as a percent-of the combined amount of glutamine and lo glutamate recovered in cycles 2,4, 8, 10, 15 and 17 of the amino acid sequencing.
Deamidation attributable to tTG was defined as (% deamidation of glutamine in the tTG treated peptide - % deamidation in the untreated peptide) / (100 - %
deamidation in the untreated peptide).
CD4/CD8 and HLA Class II Restriction: Anti-CD4 or anti-CD8 coated magnetic 15 beads (Dynal, Oslo, Norway) were washed four times with RPMI then incubated with PBMC in complete RPMI containing 10% heat inactivated human AB serum (5x106 cells/ml) for 30 minutes on ice. Beads were removed using a magnet and = cells remaining counted. In vivo HLA-class II restriction of the immune response to tTG-treated A-gliadin 56-75 was established by incubating PBMC (5x106cells/m1) 20 with anti-HLA-DR (L243), -DQ (L2), and -DP (B7.21) monoclonal antibodies (10 i_tg/m1) at room temperature for one hour prior to the addition of peptide.
Example 4 NIucosal integrin expression by gliadin -specific peripheral blood lymphocytes 25 Interaction between endothelial and lymphocyte adressins facilitates homing of organ-specific lymphocytes. Many adressins are known. The heterodimer 47 is specific for lamina propria gut and other mucosa] lymphocytes, and aE137 is specific and intra-epithelial lymphocytes in the gut and skin. Approximately 30% of peripheral blood CD4 T cells express a4137 and are presumed to be in transit to a 30 mucosal site, while 5% of peripheral blood T cells express aEf37.
Immunomagnetic beads coated with antibody specific for a E or 137 deplete PBMC of cells expressing aE137 or aE07 and a4137, respectively. In combination with ELISpot assay, immunomagnetic bead depletion allows determination of gliadin-specific T cell addressin expression that may identify these cells as homing to a mucosal surface.
Interestingly, gluten challenge in vivo is associated with rapid influx of CD4 T cells to the small intestinal lamina propria (not intra-epithelial sites), where over 90%
lymphocytes express 0437.
Immunomagnetic beads were prepared and used to deplete PBMC from coeliac subjects on day 6 or 7 after commencing 3 day gluten challenge. FACS
analysis demonstrated a. beads depleted approximately 50% of positive CD4 T
cells, while 13 7 beads depleted all- p 7 positive CD4 T cells. Depletion of PBMC using CD4- or 137-beads, but not CD8- or cc E -beads, abolished responses in the interferon gamma ELISpot. tTG gliadin and PPD responses were abolished by CD4 depletion, but consistently affected by integrin-specific bead depletion.
Thus A-gliadin 57-73 QE65-specific T cells induced after gluten challenge in coeliac disease express the integrin, ct437, present on lamina propria CD4 T
cells in =
the small intestine.
Example 5 ¨
Optimal T cell Epitope Length Previous data testing peptides from 7 to 17 amino acids in length spanning the core of the dominant T cell epitope in A-gliadin indicated that the 17mer, A-gliadin 57-73 QE65 (SEQ ID NO:2) induced maximal responses in the interferon gamma Elispot using peripheral blood mononuclear cells (PBMC) from coeliac volunteers 6 days after commencing a 3-day gluten challenge.
Peptides representing expansions form the core sequence of the dominant T
cell epitope in A-gliadin were assessed in the IFN gamma ELISPOT using peripheral blood mononuclear cells (PBMC) from coeliac volunteers in 6 days after commencing a 3-day gluten challenge (n=4). Peptide 13: A-gliadin 59-71 QE65 (13mer), peptide 15: 58-72 QE65 (15mer), ..., peptide 27: 52-78 SE65 (27mer).
As shown in Figure 11 expansion of the A-gliadin 57-73 QE65 sequence does not substantially enhance response in the IFNgamma Elispot. Subsequent Examples characterise the agonist and antagonist activity of A-gliadin 57-73 QE65 using 17mer peptides.
Example 6 Comparison of A-gliadin 57-73 QE65 with other DQ2-restricted T cell epitopes in coeliac disease Dose response studies were perfon-ned using peptides corresponding to unmodified and transglutaminase-treated peptides corresponding to T cell epitopes of gluten-specific T cell clones and lines from intestinal biopsies of coeliac subjects.
Responses to peptides were expressed as percent of response to A-gliadin 57-73 QE65. All subjects were HLA-DQ2+ (none were DQ8+).
The studies indicate that A-gliadin 57-73 QE65 is the most potent gliadin peptide for induction of interferon gamma in the ELISpot assay using coeliac PBMC
after gluten challenge (see Figure 12a-h, and Tables 5 and 6). The second and third epitopes are suboptimal fragments of larger peptides i.e. A-gliadin 57-73 QE65 and GDA4 WHEAT P04724-84-100 QE92. The epitope is only modestly bioactive (approximately 1/20th as active as A-gliadin 57-73 QE65 after blank is subtracted).
A-gliadin 57-73 QE65 is more potent than other known T cell epitopes in coeliac disease. There are 16 polymorphism of A-gliadin 57-73 (including the sequence PQLPY (SEQ ID NO:12)) amongst sequenced gliadin genes, their bioactivity is assessed next.
Example 7 Comparison of gliadin- and A-gliadin 57-73 QE65-specific responses in peripheral blood The relatiVe contribution of the dominant epitope, A-gliadin 57-73 QE65, to the total T cell response to gliadin in coeliac disease is a critical issue.
Pepsin-trypsin and chymotrypsin-digested gliadin have been traditionally used as antigen for development of T cell lines and clones in coeliac disease. However, it is possible that these proteases may cleave through certain peptide epitopes. Indeed, chymotrypsin digestion of recombinant a9-gliadin generates the peptide QLQPFPQPELPY (SEQ ID NO:13), that is a truncation of the optimal epitope sequence QLQPFPQPELPYPQPQS (SEQ ID NO:2) (see above).
Transglutaminase-treatment substantially increases the potency of chymotrypsin-digested gliadin in proliferation assays of gliadin-specific T cell clones and lines.
Hence, transglutaminase-treated chymotrypsin-digested gliadin (tTG gliadin) may not be an ideal antigen, but responses against this mixture may approximate the "total" number of peripheral blood lymphocyte specific for gliadin. Comparison of responses against A-gliadin 57-73 QE65 and tTG gliadin in the ELISpot assay gives an indication of the contribution of this dominant epitope to the overall immune response to gliadin in coeliac disease, and also be a measure of epitope spreading.
PBMC collected on day 6 or 7 after commencing gluten challenge in 4 coeliac subjects were assessed in dose response studies using chymotrypsin-digested gliadin +/- tTG treatment and compared with ELISpot responses to an optimal concentration of A-gliadin 57-73 QE65 (25mcg/m1). TTG treatment of gliadin enhanced PBMC responses in the ELISpot approximately 10-fold (tTG was comparable to blank when assessed alone) (see Figure 13a-c). In the four coeliac subjects studied, A-gliadin 57-73 QE65 (25 mcg/m1) elicited responses between and 115% those of tTG gliadin (500 meg/nil), and the greater the response to A-gliadin 57-73 QE65 the greater proportion it represented of the tTG gliadin response.
Relatively limited data suggest that A-gliadin 57-73 QE65 responses are comparable to tTG gliadin in some subjects. Epitope spreading associated with more evolved anti-gliadin T cell responses may account for the smaller contribution of A=
-gliadin 57-73 QE65 to "total" gliadin responses in peripheral blood in some =
individuals. EpitOpe spreading may be maintained in individuals with less strictly gluten free diets.
Example 8 Definition of gliadin peptides bioactive in coeliac disease: polymoiphisms of A-gliadin 57-73 Overlapping 15mer peptides spanning the complete sequence of A-gliadin were assessed in order to identify the immunodominant sequence in coeliac disease.
A-gliadin was the first fully sequenced alpha gliadin protein and gene, but is one of approximately 30-50 related alpha gliadin proteins in wheat. Twenty five distinct alpha-gliadin genes have been identified by searching protein data bases, Swiss-Prot and TREMBL describing a further 8 alpha-gliadins. Contained within these 25 alpha-gliadins, there are 16 distinct polymorphisms of the sequence corresponding to A-gliadin 57-73 (see Table 7).
Synthetic peptides corresponding to these 16 polymorphisms, in an unmodified form, after treatment with transglutaminase in vitro, as well as with glutamate substituted at position 10 (equivalent to QE65 in A-gliadin 57-73) were assessed using PBMC from coeliac subjects, normally following a gluten free diet, day 6 or 7 after gluten challenge in interferon gamma ELISpot assays.
Glutamate-.. substituted peptides were compared at three concentrations (2.5, 25 and 250 meg/nil), unmodified peptide and transglutaminase-treated peptides were assessed at 25 meg/nil only. Bioactivity was expressed as % of response associated with A-gliadin 57-73 QE65 25 megiml in individual subjects (n=4). (See Fig 14).
Bioactivity of "wild-type" peptides was substantially increased (>5-fold) by treatment with transglutaminase. Transglutaminase treatment of wild-type peptides resulted in bioactivity similar to that of the same peptides substituted with glutamate at position 10. Bioactivities of five glutamate-substituted peptides (B, C, K, L, M), were >70% that of A-gliadin 57-73 QE65 (A), but none was significantly more bioactive than A-gliadin 57-73 QE65. PBMC responses to glutamate-substituted peptides at concentrations of 2.5 and 250 mcg/ml were comparable to those at mcg/ml. Six glutamate-substituted gliadin peptides (H, I, J, N, 0, P) were <15% as bioactive as A-gliadin 57-73 QE65. Other peptides were intermediate in bioactivity.
At least six gliadin-derived peptides are equivalent in potency to A-gliadin 57-73 QE65 after modification by transglutaminase: Relatively non-bioactive .. polymorphisms of A-gliadin 57-73 also exist. These data indicate that transglutaminase modification of peptides from several gliadins of Triticum aestivum, T. ziartu and T. spelta may be capable of generating the immunodominant T cell epitope in coeliac disease.
Genetic modification of wheat to generate non-coeliac-toxic wheat may likely require removal or modification of multiple gliadin genes. Generation of wheat containing gliadins or other proteins or peptides incorporating sequences defining altered peptide ligand antagonists of A-gliadin 57-73 is an alternative strategy to generate genetically modified wheat that is therapeutic rather than "non-toxic" in coeliac disease.
Example 9 5 Definition of Core Epitope Sequence:
Comparison of peptides corresponding to truncations of A-gliadin 56-75 from the N- and C-terminal indicated that the core sequence of the T cell epitope is PELPY (A-gliadin 64-68). Attempts to define non-agonists and antagonists will focus on variants of A-gliadin that are substituted at residues that substantially 10 contribute to its bioactivity.
Peptides corresponding to A-gliadin 57-73 QE65 with alanine (Figure 15) or lysine (Figure 16) substituted for residues 57 to 73 were compared in the IFN
gamma ELISPOT using peripheral blood mononuclear cells (PBMC) from coeliac volunteers 6 days after commencing a 3-day gluten challenge (n=8). (BL is blank, E is A-15 gliadin 57-73 QE65: QLQPFPQPELPYPQPQS (SEQ ID NO:2)).
It was found that residues corresponding to A-gliadin 60-70 QE65 (PFPQPELPYPQ (SEQ ID NO:14)) contribute substantially to the bioactivity in A-' gliadin 57-73 QE65. Variants of A-gliadin .57-73 QE65 substituted at positions 60-70 are assessed in a 2-step procedure. Initially, A-gliadin 57-73 QE65 substituted at 20 positions 60-70 using 10 different amino acids with contrasting properties are assessed. A second group of A-gliadin 57-73 QE65 variants (substituted with all other naturally occurring amino acids except cysteine at positions that prove are sensitive to modification) are assessed in a second round.
25 Example 10 Agonist activity of substituted variants ofA-gliadin 57-73 0E65 A-gliadin 60-70 QE65 is the core sequence of the dominant T cell epitope in A-gliadin. Antagonist and non-agonist peptide variants of this epitope are most likely generated by modification of this core sequence. Initially, A-gliadin 30 QE65 substituted at positions 60-70 using 10 different amino acids with contrasting properties will be assessed in the IFNgamma ELISPOT using PBMC from coeliac subjects 6 days after starting 3 day gluten challenge. A second group of A-gliadin 5743 QE65 variants (substituted with all other naturally occurring amino acids except cysteine) at positions 61-70 were also assessed. Both groups of peptides (all at 50 mcg/ml, in duplicate) were assessed using PBMC from 8 subjects and compared to the unmodified peptide (20 replicates per assay). Previous studies indicate that the optimal concentration for A-gliadin 57-73 QE65 in this assay is between 10 and 100 mcg/ml.
Results are expressed as mean response in spot forming cells (95%
confidence interval) as % A-G 57-73 QE65 mean response in each individual.
Unpaired t-tests will be used to compare ELISPOT responses of modified peptides with A-G 57-73 QE65. Super-agonists were defined as having a greater response than A-G 57-73 QE65 at a level of significance of p<0.01; partial agonists as having a response less than A-G 57-73 QE65 at a level of significance of p<0.01, and non-agonists as being not significantly different (p>0.01) from blank (buffer without peptide). Peptides with agonist activity 30% or less that of A-gliadin 57-73 were considered "suitable" partial or non-agonists to assess for antagonistic activity (see Table 8 and Figures 17-27).
The IFNgamma ELISPOT response of PBMC to A-gliadin 57-73 QE65 is highly specific at a molecular level. Proline at position 64 (P64), glutamate at 65 (E65) and leucine at position 66 (L66), and to a lesser extent Q63, P67, Y68 and P69 are particularly sensitive to modification. The substitutions Y61 and Y70 both generate super-agonists with 30% greater bioactivity than the parent peptide, probably by enhancing binding to HLA-DQ2 since the motif for this HLA molecule indicates a preference for bulky hydrophobic resides at positions 1 and 9.
Eighteen non-agonist peptides were identified. Bioactivities of the variants (50 mcg/m1): P65, 1(64, 1(65 and Y65 (bioactivity 7-8%) were comparable to blank (7%). In total, mutated variants of A-gliadin 57-73 QE65 were 30% or less bioactive than A-gliadin 57-73 QE65.
The molecular specificity of the peripheral blood lymphocyte (PBL) T cell response to the dominant epitope, A-gliadin 57-73 QE65, is consistently reproducible amongst TILA-DQ2+ coeliac subjects, and is highly specific to a restricted number of amino acids in the core 7 amino acids. Certain single-amino acid variants of A-gliadin 57-73 QE65 are consistently non-agonists in all HLA-DQ2+ coeliac subjects.

Example 11 Antagonist activity of substituted variants The homogeneity of the PBL T cell response to A-gliadin 57-73 QE65 in HLA-DQ2+ coeliac disease suggests that altered peptide ligands (APL) capable of antagonism in PBMC ex vivo may exist, even though the PBL T cell response is likely to be poly- or oligo-clonal. APL antagonists are generally weak agonists.
Fifty:seven single amino acid-substituted variants of A-gliadin 57-73 QE65 with agonist activity 30% or less have-been identified and are suitable candidates as APL
antagonists. In addition, certain weakly bioactive naturally occurring polymorphisms of A-gliadin 57-73 QE65 have also been identified (see below) and may be "naturally occurring" APL antagonists. It has also been 'suggested that competition for binding MHC may also antagonise antigen-specific T cell immune. Hence, non-gliadin peptides that do not induce IFNgamma responses in coeliac PBMC after gluten challenge but are known to bind to HLA-DQ2 may be capable of reducing T
cell responses elicited by A-gliadin 57-73 QE65. Two peptides that bind avidly to HLA-DQ2 are HLA class 1 a 46-60 (}{LA la) (PRAPWIEQEGPEYW (SEQ ID
NO:15)) and thyroid peroxidase (tp) 632-645Y (IDVWLGGLLAENFLPY (SEQ ID
NO:16)).
Simultaneous addition of peptide (50p.g/m1) or buffer and A-gliadin 57-73 QE65 (10pg/m1) in IFNgamma ELISPOT using PBMC from coeliac volunteers 6 days after commencing 3 day gluten challenge (n=5). Results were expressed as response with peptide plus A-G 57-73 QE65 (mean of duplicates) as % response with buffer plus A-G 57-73 QE65 (mean of 20 replicates). (See Table 9).
Four single amino acid-substituted variants of A-gliadin 57-73 QE65 reduce the interferon gamma PBMC ELISPOT response to A-gliadin 57-73 QE65 (p<0.01) by between 25% and 28%, 13 other peptide variants reduce the ELISPOT response by between 18% and 24% (p<0.06). The FILA-DQ2 binder, thyroid peroxidase (tp) 632-645Y reduces PBMC interferon gamma responses to A-gliadin 57-73 QE65 by 31% (p<0.0001) but the other HLA-DQ2 binder, HLA class 1 a 46-60, does not alter responses (see Tables 9 and 10). The peptide corresponding to a transglutaminase-modified: polymorphism of A-gliadin 57-73, SwissProt accession no.: P04725 82-QE90 (PQPQPFPPELPYPQPQS (SEQ ID NO:17)) reduces responses to A-gliadin 57-73 QE65 by 19% (p<0.009) (see Table 11).
Interferon gamma responses of PBMC to A-gliadin 57-73 QE65 in ELISPOT
assays are reduced by co-administration of certain single-amino acid A-gliadin QE65 variantsõ a polymorphism of A-gliadin 57-73 QE65, and an unrelated peptide known to bind HLA-DQ2 in five-fold excess. These finding suggest that altered peptide ligand antagonists of A-gliadin 57-73 QE65 exist. Not only putative APL
antagonists but also certain peptides that bind HLA-DQ2 effectively reduce PBL
T
cell responses to A-gliadin 57-73' QE65.
These findings support two strategies to interrupt the T cell response to the dominant A-gliadin epitope in HLA-DQ2+ coeliac disease.
1. Optimisation of APL antagonists by substituting amino acids at more than one position (64-67) for use as "traditional" peptide pharmaceuticals or for specific genetic modification of gliadin genes in wheat.
2. Use of high affinity HLA-DQ2 binding peptides to competitively inhibit presentation of A-gliadin 57-73 QE65 in association with HLA-DQ2.
These two approaches may be mutually compatible. Super-agonists were generated by replacing F61 and Q70 with tyrosine residues. It is likely these super-agonists resulted from improved binding to HLA-DQ2 rather than enhanced contact with the T cell receptor. By combining these modifications with other substitutions that generate modestly effective APL antagonists might substantially enhance the inhibitory effect of substituted A-gliadin 57-73 QE65 variants.
Example 12 Development of interferon gamma ELISpot using PBMC and A-gliadin 57-73 QE65 and P04724 84-100 QE92 as a diagnostic for coeliac disease: Definition of immune-responsiveness in newly diagnosed coeliac disease Induction of responsiveness to the dominant A-gliadin T cell epitope in PBMC measured in the interferon gamma ELISpot follows gluten challenge in almost all DQ2+ coeliac subjects following a long term strict gluten free diet (GFD) but not in healthy DQ2+subjects after 4 weeks following a strict GFD. A-gliadin =

57-73 QE65 responses are not measurable in PBMC of coeliac subjects before gluten challenge and pilot data have suggested these responses could not be measured in PBMC of untreated coeliacs. These data suggest that in coeliac disease immune-responsiveness to A-gliadin 57-73 QE65 is restored following antigen exclusion (GFD). If a diagnostic test is to be developed using the ELISpot assay and PBMC, it is desirable to define the duration of GFD required before gluten challenge is capable of inducing responses to A-gliadin 57-73 QE65 and other immunoreactive gliadin peptides in blood.
Newly diagnosed DQ2+ -coeliac subjects were recruited from the gastroenterology outpatient service. PBMC were prepared and tested in interferon gamma ELISpot assays before subjects commenced GFD, and at one or two weeks after commencing GFD. In addition, gluten 'challenge (3 days consuming 4 slices standard white bread, 200g/day) was performed at one or two weeks after starting GFD. PBMC were prepared and assayed on day six are after commencing gluten challenge. A-gliadin 57-73 QE65 (A), P04724 84-100 QE92 (B) (alone and combined) and A-gliadin 57-73 QP65 (P65) (non-bioactive variant, see above) (all mcg/m1) were assessed.
All but one newly diagnosed coeliac patient was DQ2+ (one was DQ8+) (n=11). PBMC from newly diagnosed coeliacs that were untreated, or after 1 or 20 weeks following GFD did not show responses to A-gliadin 57-73 QE65 and 84-100 QE92 (alone or combined) that were not significantly different froth blank or A-gliadin 57-73 QP65 (n=9) (see Figure 28). Gluten challenge in coeliacs who had followed GFD for only one week did not substantially enhance responses to A-gliadin 57-73 QE65 or P04724 84-100 QE92 (alone or *combined). But gluten . 25 challenge 2 weeks after commencing GFD did induce responses to A-gliadin 57-73 QE65 and P04724 84-100 QE92 (alone or combined) that were significantly greater than the non-bioactive variant A-gliadin 57-73 QP65 and blank. Although these responses after gluten challenge at 2 weeks were substantial they appear to be less than in subjects >2 months after commencing GFD. Responses to A-gliadin 57-73 QE65 alone were equivalent or greater than responses to P04724 84-100 QE92 alone or when mixed with A-gliadin 57-73 QE65. None of the subjects experienced troubling symptoms with gluten challenge.

Immune responsiveness (as measured in PBMC after gluten challenge) to A-gliadin is partially restored 2 weeks after commencing GFD, implying that "immune unresponsiveness" to this dominant T cell epitope prevails in untreated coeliac disease and for at least one week after starting GFD. The optimal timing of a 5 diagnostic test for coeliac disease using gluten challenge and measurement of responses to A-gliadin 57-73 QE65 in the ELISpot assay is at least 2 weeks after commencing a GFD.
Interferon gamma-secreting T cells specific to A-gliadin 57-73 QE65 cannot = be measured in the peripheral blood in untreated coeliacs, and can only be induced 0 by gluten challenge after at least 2 weeks GFD (antigen exclusion).
Therefore, timing of a diagnostic test using this methodology is crucial and further studies are needed for its, optimization. These finding are consistent with functional ariergy of T
cells specific for the dominant epitope, A-gliadin 57-73 QE65, reversed by antigen exclusion (GFD). This phenomenon has not been previously demonstrated in a 15 human disease, and supports the possibility that T cell anergy may be inducible with peptide therapy in coeliac disease.
Example 13 Comprehensive Mapping of Wheat Gliadin T Cell Epitopes 20 Antigen challenge induces antigen-specific T cells in peripheral blood.
In coeliac disease, gluten is the antigen that maintains this immune-mediated disease. =
Gluten challenge in coeliac disease being treated with a gluten free diet leads to the appearance of gluten-specific T cells in peripheral blood, so enabling determination of the molecular specificity of gluten T cell epitopes. As described above, we have 25 identified a single dominant T cell epitope in a model gluten protein, ALgliadin (57-73 deamidated at Q65). In this Exainpie, gluten challenge in coeliac patients was used to test all potential 12 amino acid sequences in every known wheat gliadin protein derived from 111 entries in Genbank. In total, 652 20mer peptides were tested in HLA-DQ2 and HLA-DQ8 associated coeliac disease. Seven of the 9 30 coeliac subjects with the classical HLA-DQ2 complex (HLA-DQA1*05,,HLA-DQB1*02) present in over 90% of coeliacs had an inducible A-gliadin 57-73 QE65-and gliadin-specific T cell response in Peripheral blood. A-gliadin 57-73 was the only significant a-gliadin T cell epitope, as well as the most potent gliadin T cell epitope, in HLA-DQ2-associated coeliac disease. In addition, there were as many as families of structurally related peptides that were between 10 and 70% as potent as A-gliadin 57-73 in the interferon-'y ELISpot assay. These new T cell epitopes were 5 derived from y- and co-gliadins and included common sequences that were structurally very similar, but not identical to the core sequence of A-gliadin (core sequence: FPQPQLPYP (SEQ ID NO:18)),.for example: FPQPQQPFP (SEQ
ID NO:19) and PQQPQQPFP (SEQ ID NO:20). Although no homologues of A-gliadin 57-73 have been found inrye or barley, the other two cereals toxic in coeliac disease, the newly defined T cell epitopes in 7- and co-gliadins have exact matches in rye and barley storage proteins (secalins and hordeins, respectively).
. Coeliac disease not associated with HLA-DQ2 is almost always associated with HLA-DQ8. None of the seven HLA-DQ8+ coeliac subjects had inducible A-.
gliadin 57-73-specific T cell responses following gluten challenge, unless they also possessed the complete HLA-DQ2 complex. Two of 4 ITLA-DQ8+ coeliac subjects who did not possess the complete HLA-DQ2 complex, had inducible gliadin peptide-specific T cell responses following gluten challenge. In one HLA-DQ8 subject, a novel dominant T cell epitope was identified with the core sequence LQPQNPSQQQPQ (SEQ ID NO:21). The transglutaminase-deamidated version of this peptide was more potent than the non-deamidated peptide. Previous studies suggest that the transglutaminase-deamidated peptide would have the sequence LQPENPSQEQPE (SEQ ID NO:22); but further studies are required to confirm this sequence. Amongst the healthy HLA-DQ2 (10) and HLA-DQ8 (1) subjects who followed a gluten free diet for a month, gliadin peptide-specific T cell responses were uncommon, seldom changed with gluten challenge, and were never potent T
cell epitopes revealed with gluten challenge in coeliac subjects. In conclusion, there are unlikely to be more than six important T cell epitopes in HLA-DQ2-associated coeliac disease, of which A-gliadin 57-73 is the most potent. HLA-DQ2- and 1-ILA-DQ8-associated coeliac disease do not share the same T cell specificity.
We have shown that short-term gluten challenge of individuals with coeliac disease following a gluten free diet induces gliadin-specific T cells in peripheral blood. The frequency of these T cells is maximal in peripheral blood on day 6 and then rapidly wanes over the following week. Peripheral blood gliadin-specific T
cells express the integrin a4437 that is associated with homing to the gut lamina propria. We exploited this human antigen-challenge design to map T cell epitopes relevant to coeliac disease in the archetypal gluten a-gliadin protein, A-gliadin.
Using 15naer peptides overlapping by 10 amino acids with and without deamidation by transglutaminase (tTG), we demonstrated that T cells induced in peripheral blood initially target only one A-gliadin peptide, residues 57-73 in which glutamine at position 65 is deamidated. The epitope is HLA-DQ2-restricted, consistent with the intimate association of coeliac disease with HLA-DQ2.
Coeliac disease is reactivated by wheat, rye and barley exposure. The a/p-gliadin fraction of wheat glutenis consistently toxic in coeliac disease, and most studies have focused on these proteins. The gene cluster coding for a/3-gliadins is located on wheat chromosome 6C. There are no homologues of a/13-gliadins in rye or barley. However, all three of the wheat gliadin subtypes (a/13, y, and co) are toxic .. in coeliac disease. They- and co-gliadin genes are located on chromosome 1A
in wheat, and are homologous to the secalins and hordeins in rye and barley.
There are now genes identified for 61 a-gliadins in wheat.(Triticum aestivum). The a-gliadin sequences are closely homologous, but the dominant epitope in A-gliadin derives from the most polymorphic region in the a-gliadin sequence. Anderson et al (1997) have estimated that there are a total of about distinct a-gliadin genes in T. aestivum, but many are psuedogenes. Hence, it is unlikely that T-cell epitopes relevant to coeliac disease are not included within known a-gliadin Sequences.
Our work has identified a group of deamidated a-gliadin peptides almost identical to A-gliadin 57-73 as potent T cell epitopes specific to coeliac disease.
Over 90% of coeliac patients are HLA-DQ2+, and so far, we have only assessed HLA-DQ2+ coeliac subjects after gluten challenge. However, coeliac patients who do not express HLA-DQ2 nearly all carry HLA-DQ8. Hence, it is critical to know .
whether A-gliadin 57-73 and its homologues in other wheat, rye and barley gluten proteins are the only T-cell epitopes recognized by T cells induced by gluten challenge in both HLA-DQ2+ and HLA-DQ8+ coeliac disease. If this were the case, design of peptide therapeutics for coeliac disease might only require one peptide.

Homologues of A-gliadin 57-73 as T-cell epitopes Initial searches of SwissProt and Trembl gene databases for cereal genes coding for the core sequence of A-gliadin 57-73 (PQLPY <SEQ ID NO:12>) only revealed a/13-gliadins. However, our fine-mapping studies of the A-gliadin 57-QE65 epitope revealed a limited number of permissive point substitutions in the core region (PQLP) (note Q65 is actually deamidated in the epitope). Hence, we extended our search to genes in SwissProt or Trembl databases encoding for peptides with the sequence XXXXXXXPQ[ILMPT[PST]XXXXXX (SEQ ID NO:23). Homologues were identified amongst y-gliadins, glutenins, hordeins and secalins (see Table 12).
A further homologue was identified in w-gliadin by visual search of the three w-gliadin entries in Genbank.
These homologues of A-gliadin 57-73 were assessed after deamidation by tTG (or synthesis of the glutamate(QE)-substituted variant in four close homologues) using the IFNy ELISpot assay with peripheral blood mononuclear cells after gluten challenge in coeliac subjects. The 10-g1iadin sequence (AAG17702 141-157) was the only bioactive peptide, approximately half as potent as A-gliadin 57-73 (see Table 12, and Figure 29). Hence, searches for homologues of the dominant A-gliadin epitope failed to account for the toxicity of 'y-gliadin, secalins, and hordeins.
Methods Design of a set of peptides spanning all possible wheat gliadin T-cell epitopes In order to identify all possible T cell epitopes coded by the known wheat (Triticum aestivum) gliadin genes or gene fragments (61 a/f3-, 47 y-, and 3 co-gliadin entries in Genbank), gene-derived protein sequences were aligned using the CustalW
software (MegAlign) and arranged into phylogenetic groupings (see Table 22) Many entries represented truncations of longer sequences, and many gene segments were identical except for the length of polyglutamine repeats or rare substitutions.
Hence, it was possible to rationalize all potential unique 12 amino acid sequences encoded by known wheat genes to be included in a set of 652 20mer peptides.
(Signal peptide sequences were not included). Peptide sequences are listed in Table 23.
Comprehensive epitope mapping Healthy controls (HLA-DQ2+ n--10, and HLA-DQ8+ n=1) who had followed a gluten free diet for 4 weeks, and coeliac subjects (six HLA-DQ2, four complex heterozygotes HLA-DQ2/8, and three HLA-DQ8/X) (see Table 13) following long-term gluten free diet were studied before and on day 6 and 7 after 3-day gluten challenge (four 50g slices of standard white bread ¨ Sainsbury's sandwich bread, each day). Peripheral blood (a total of 300m1 over seven days) was collected and peripheral blood mononuclear cells (PBMC) were separated by Lymphoprep density gradient. PBMC were incubated with pools of 6 or 8 20mer peptides, or single peptides with or without deamidation by tTG in overnight interferon gamma (IFNy) ELISpot assays.
Peptides were synthesized in batches of 96 as Pepsets (Mimotopes Inc., Melbourne Australia). Approximately 0.6 micromole of each of 652 20mers was provided. Two marker 20mer peptides were included in each set of 96 (VLQQHNIAHGSSQVLQESTY ¨ peptide 161 (SEQ ID NO:24), and IKDFHVYFRESRDALWKGPG (SEQ ID NO:25)) and were characterized by reverse phase-HPLC and amino acid sequence analysis. Average purities of these marker peptides were 50% and 19%, respectively. Peptides were initially dissolved in acetonitrile (10%) and Hepes 100mM to 10mg/ml.
The final concentration of individual peptides in pools (or alone) incubated with PBMC for the IFNy ELISpot assays was 201,tg/ml. Five-times concentrated solutions of peptides and pools in PBS with calcium chloride 1mM were aliquotted and stored in 96-well plates according to the template later used in ELISpot assays.
Deamidated peptides and: pools of peptides were prepared by incubation with guinea pig tissue tTG (Sigma T5398) in the ratio 100:32 1.1g/m1 for two hours at 37 C.
Peptides solutions were stored at ¨20 C and freshly thawed prior to use.
Gliadin (Sigma G3375) (100 mg/m1) in endotoxin-free water and 2M urea was boiled for 10 minutes, cooled to room temperature and incubated with filter (0.2 um)-sterilised pepsin (Sigma P6887) (2 mg/ml) in HC1 0.02M or chymotrypsin (C3142) (4mg/m1) in ammonium bicarbonate (0.2M). After incubation for 4 hours, pepsin-digested gliadin was neutralized with sodium hydroxide, and then both pepsin- and chymotrypsin-digested gliadin were boiled for 15 minutes.
Identical incubations with protease in which gliadin was omitted were also perfonued.

Samples were centrifuged at 15 000g, then protein concentrations were estimated in supernatants by the BCA method (Pierce, USA). Before final use in IFNy ELISpot assays, aliquots of gliadin-protease were incubated with tTG in the ratio 2500:64 ttg/ml.
5 IFNy ELISpot assays (Mabtech, Sweden) were performed in 96-well plates (MAIP S-45, Millipore) in which each well contained 250 of peptide solution and 100u1 of PBMC (2-8x105/well) in RPM' containing 10% heat inactivated human AB
serum. Deamidated peptide pools were assessed in one 96-well ELISpot plate, and peptides pools without deamidation in a second plate (with an identical layout) on 10 both day 0 and day 6. All wells in the plate containing deamidated peptides included tTG (64 !.ig/m1). In each ELISpot plate there were 83 wells with peptide pools (one unique pool in each well), and a series of wells for "control" peptides (peptides all >90% purity, characterized by MS and HPLC, Research Genetics): P04722 77-93 (QLQPFPQPQLPYPQPQP (SEQ ID NO:26)), P04722 77-93 QE85 (in duplicate) 15 (QLQPFPQPELPYPQPQP (SEQ ID NO:27)), P02863 77-93 (QLQPFPQPQLPYSQPQP (SEQ ID NO:28)), P02863 77-93 QE85 (QLQPFPQPELPYSQPQP (SEQ ID NO :29)), and chymotrypsin-digested gliadin (500 tg/m1), pepsin-digested gliadin (500 [tg/m1), chymotrypsin (20 ig/m1) alone, pepsin (10 p.g/m1) alone, and blank (PBS+/-tTG) (in triplicate).
20 After development and drying, IFNy ELISpot plates were assessed using the MAIP automated ELISpotplate counter. In HLA-DQ2 healthy and coeliac subjects, = induction of spot forming cells (sfc) by peptide pools in the IFNy ELISpot assay was tested using a one-tailed Wilcoxon Matched-Pairs Sig-ned-Ranks test (using SPSS
software) applied to spot forming cells (sfc) per million PBMC minus blank on day 6 25 versus day 0 ("net response"). Significant induction of an IF1\17 response to peptide pools in PBMC by in vivo gluten challenge was defined as a median "net response"
of at least 10 sfc/million PBMC and p<0.05 level of significance. Significant response to a particular pool of peptides on day 6 was followed by assessment of individual peptides within each pool using PBMC drawn the same day or on day 7.
30 For IFN7 ELISpot assays of individual peptides, bioactivity was expressed as a percent of response to P04722 77-93 QE85 assessed in the same ELISpot plate.

Median response to blank (PBS alone) was 0.2 (range 0-5) sfc per well, and the =
positive control (P04722 77-93 QE85) 76.5 (range: 25-282) sfc per well using a median of 0.36 million (range: 0.3-0.72) PBMC. Hence, median response to blank expressed as a percentage of P04722 77-93 QE65 was 0.2% (range: 0-6.7).
Individual peptides with mean bioactivity greater than10% that of P04722 QE85 were analyzed for common structural motifs.
Results Healthy HLA-DQ2 subjects None of the healthy HLA-DQ2+ subjects following a gluten free diet for a month had IFNI/ ELISpot responses to homologues of A-gliadin 57-73 before or after gluten challenge. However, in 9/10 healthy subjects, gluten challenge was associated with a significant increase in IFNy responses to both peptic- and chymotryptic-digests of gliadin, from a median of 0-4 sfc/million on day 0 to a median of sfc/million (see Table 14). Gliadin responses in healthy subjects were unaffected by deamidation (see Table 15). Amongst healthy subjects, there was no consistent induction of IFNy responses to specific gliadin peptide pools with gluten challenge (see Figure 30, and Table 16). IFN7 ELISpot responses were occasionally found, but these were weak, and not altered by deamidation. Many of the strongest responses to pools were also present on day 0 (see Table 17, subjects H2, H8 and H9). Four healthy subjects did show definite responses to pool 50,and the two with strongest responses on day 6 also had responses on day 0. In both subjects, the post-challenge responses to pool 50 responses were due to peptide 390 (QQTYPQRPQQPFPQTQQPQQ (SEQ ID NO:30)).
HLA-DQ2 coeliac subjects Following gluten challenge in HLA-DQ2+ coeliac subjects, median IFNy ELISpot responses to P04722 77-93 E85 rose from a median of 0 to 133 sfc/million (see Table 4). One of the six coeliac subjects (C06) did not respond to P04722 QE85 (2 sfc/million) and had only weak responses to gliadin peptide pools (maximum: Pool 50+tTG 27 sfc/million). Consistent with earlier work, bioactivity of wild-type P04722 increased 6.5 times with deamidation by tTG (see Table 15).
Interferon-gamma responses to gliadin-digests were present at baseline, but were substantially increased by gluten challenge from a median of 20 up to 92 sfc/million for chymotryptic-gliadin, and from 44 up to 176 sfc/million for peptide-gliadin.

Deamidation of gliadin increased bioactivity by a median of 3.2 times for chymotryptic-gliadin and 1.9 times for peptic-gliadin (see Table 15). (Note that the acidity required for, digestion by pepsin is likely to result in partial deamidation of gliadin.) In contrast to healthy subjects, gluten challenge induced IFNy ELISpot responses to 22 of the 83 tTG-treated pools including peptides from a-, y- and gliadins (see Figure 31, and Table 17). Bioactivity of pools was highly consistent between subjects (see Table 18). IFNy ELISpot responses elicited by peptide pools were almost always increased by-deamidation (see Table 17). But enhancement of io bioactivity of pools by deamidation was not as marked as for P04722 77-73 Q85, even for pools including hoinologues of A-gliadin 57-73. This suggests that Pepset peptides were partially deamidated during synthesis or in preparation, for example the Pepset peptides are delivered as salts of trifluoracetie acid (TFA) after lyophilisation from a TFA solution.
One hundred and seventy individual tTG-deamidated peptides from 21 of the most bioactive pools were separately assessed. Seventy-two deamidated peptides were greater than 10% as bioactive as P04722 77-93 QE85 at an equivalent concentration (20 [Tim (see Table 19). The five most potent peptides (85-94%
bioactivity of P04722 QE85) were previously identified a-gliadin homologues A-gliadin 57-73. Fifty of the bioactive peptides were not homologues of A-gliadin 57-73, but could be divided into six families of structurally related sequences (see Table 20). The most bioactive sequence of each of the peptide families were:
PQQPQQPQOPFPOPOOPFPW (SEQ ID NO:31) (peptide 626, median 72%
bioactivity of P04722 QE85), QQPQOPFPOPOOPQLPFPOQ (SEQ ID NO:32) (343, 34%), QAPPOPQQTFPHOPQQQFPQ (SEQ ID NO:33) (355, 27%), TQQPQQPFPOOPQOPFPQTQ (SEQ ID NO:34) (396, 23%), PIQPOOPFPQOPQQPQQPFP (SEQ ID NO:35) (625, 22%), PQQSFSYOQOPFPOOPYPQQ (SEQ ID NO:36) (618, 18%) (core sequences are underlined). All of these sequences include glutamine residues, predicted to be susceptible to deamidation by transglutaminase (e.g. QXP, QXPF (SEQ ID NO:37), QX_X[FY] (SEQ ID NO:38)) (see Vader et al 2002). Some bioactive peptides contain two core sequences from different families.

Consistent with the possibility that different T-cell populations respond to peptides with distinct core sequences, bioactivity of peptides from different families appear to be additive. For example, median bioactivity of tTG-treated Pool 81 was 141% of P04722 QE85, while bioactivity of individual peptides was in rank order:
Peptide 631 (homologue of A-gliadin 57-73) 61%, 636 (homologue of 626) 51%, and 635 19%, 629 16%, and 634 13% (all homologues of 396).
Although likely to be an oversimplification, the contribution of each "peptide family" to the summed IFN7 ELISpot response to gliadin peptides was compared in the HLA-DQ2+ coeliac subjects (see Figure 32). Accordingly, the contribution of P04722 77-73 E85 to the summed response to gliadin peptides is between 1/5 and 2/3.
Using the peptide homology search programme, PepPepSearch, and by direct comparison with Genbank sequences for rye secalins, exact matches were found for the core sequences QQPFPQPQQPFP (SEQ NO:39) in barley hordeins (HORS) and rye secalins (A23277, CAA26449, AA035598), QQPFPQQPQQPFP (SEQ ID NO:40) in barley hordeins (HOG1 and HOR8), and for PIQPQQPFPQQP (SEQ ID NO:41) also in barley hordeins (H0R8).
HLA-D08-associated coeliac disease Seven HLA-DQ8+ coeliac subjects were studied before and after gluten challenge. Five of these HLA-DQ8+ (HLA-DQA0*0301-3, HLA-DQB0*0302) subjects also carried one or both of the coeliac disease-associated HLA-DQ2 complex (DQA0*05, DQB0*02). Two of the three subjects with both coeliac-associated HLA-DQ complexes had potent responses to gliadin peptide pools (and individual peptides including P04722 77-93 E85) that were qualitatively and quantitatively identical to HLA-DQ2 coeliac subjects (see Figures 33 and 34, and Table 18). Deamidated peptide pool 74 was bioactive in both HLA-DQ2/8 subjects, but only in one of the 6 IILA-DQ2/X subjects. Pretreatment of pool 74 with tTG
enhances bioactivity between 3.8 and 22-times, and bioactivity of tTG-treated pool 74 in the three responders is equivalent to between 78% and 350% the bioactivity of P04722 77-93 E85. Currently, it is not known which peptides are bioactive in Pool 74 in subject CO2, C07, and C08.
Two of the four HLA-DQ8 coeliac subjects that lacked both or one of the HLA-DQ2 alleles associated with coeliac disease showed very weak IFN7 ELISpot responses to gliadin peptide pools, but the other two did respond to both protease-digested gliadin and specific peptide pools. Subject C12 (HLA-DQ7/8) responded vigorously to deamidated Pools 1-3 (see Figure 35). Assessment of individual peptides in these pools identified a series of closely related bioactive peptides including the core sequence LQPQNPSQQQPQ (SEQ ID NO:42) (see Table 20).
Previous work (by us) has demonstrated that three glutamine residues in this sequence are susceptible to tTG-mediated deamidation (underlined). Homology searches using WWW PepPepSearch have identified close matches to LQPQNPSQQQPQ (SEQ ID NO:43) only in wheat a-gliadins.
The fourth HLA-DQ8 subject (C11) had inducible TEN? ELISpot responses to tTG-treated Pool 33 (see Figure 36). Pools 32 and 33 include polymOrphisms of a previously defined HLA-DQ8 restricted gliadin epitope (QQYPSGQGSFQPSQQNPQ (SEQ ID NO:44)) active after deamidation by tTG
(underlined Gln are deamidated and convey bioactivity) (van der Wal et al 1998).
Currently, it is not known which peptides are bioactive in Pool 33 in subject Cll.
Comprehensive T cell epitope mapping in HLA-DQ2-associated coeliac disease using in vivo gluten challenge and a set of 652 peptides spanning all known 12 amino acid sequences in wheat gliadin has thus identified at least 72 peptides at 10% as bioactive as the known a-gliadin epitope, A-gliadin 57-73 E65. However, these bioactive peptides can be reduced to a set of perhaps as few as 5 distinct but closely related families of peptides. Almost all these peptides are rich in proline, glutamine, phenylalanine, and/or tyrosine and include the sequence PQ(QL)P(FY)P
(SEQ ID NO:45). This sequence facilitates deamidation of Q in position 2 by tTG.
By analogy with deamidation of A-gliadin 57-68 (Arentz-Hansen 2000), the enhanced bio activity of these peptides generally found with deamidation by tTG may be due to increased affinity of binding for HLA-DQ2.
Cross-reactivity amongst T cells in vivo recognizing more than one of these bioactive gliadin peptides is possible. However, if each set of related peptides does activate a distinct T cell population in vivo, the epitope corresponding to A-gliadin 57-73 E65 is the most potent and is generally recognized by at least 40% of the peripheral blood T cells that secrete IFNI, in response to gliadin after gluten challenge.
5. No gliadin-peptide specific responses were found in HLA-DQ2/8 coeliac disease that differed qualitatively from those in HLA-DQ2/X-associated coeliac disease. However, peripheral blood T cells in HLA-DQ8+ coeliac subjects without both HLA-DQ2 alleles did not recognize A-gliadin 57-73 E65 homologues. Two different epitopes were dominant in two HLA-DQ8+ coeliacs. The dominant epitope 10 in one of these HLA-DQ8+ individuals has not been identified previously (LQPQNPSQQQPQ (SEQ ID NO:46)).
Given the teaching herein, design of an immunotherapy for coeliac disease utilizing all the commonly recognised T cell epitopes is practical and may include fewer than six distinct peptides. Epitopes in wheat 7- and w-gliadins are also present 15 in barley hordeins and rye secalins.
Example 14 Several ELISpot assays were performed as previously described and yielded the following results and/or conclusions:
20 Examination of tnzdtiple a-gliadin polymorphisms with PQLPY
Potent agonists of A-gliadin 57-73QE (G01) include QLQPFPQPELPYPQPQS (G01), PQL-Y ---------------- P (G10), and PQPQPFL--------------- (G12). Less potent include ---- L -- P (G04), ------------------- P (G05), and -------- S -- P (G06). Less potent yet 25 include ----- L -- S -------- P (G07), -- S -- S ------ P (G08), -------- S--S -- P (G09), and PQPQPFP ----- (G13). Dashes indicate identity with the GO1 sequence in the particular position.
Gluten challenge induces A-gliadin 57-73 QE65 T cells only after two weeks of 30 gluten-free diet in newly diagnosed coeliac disease Additional analyses indicated that tTG-deamidated gliadin responses change after two weeks of gluten-free diet in newly diagnosed coeliac disease. Other analyses indicated that deamidated gliadin-specific T cells are CD4+a4f37+ HLA-restricted.
Optimal epitope (clones versus gluten challenge) A "dominant" epitope is defined by 7IFN ELISpot after gluten challenge.
QLQPFPQPELPYPQPQS (100% ELISpot response). Epitopes defined by intestinal T cell clones: QLQPFPQPELPY (27%), PQPELPYPQPELPY (52%), and QQLPQPEQPQQSFPEQERPF (9%).
o Dominance among individual peptide responses Dominance depends on wheat or rye. For wheat, dominant peptides include peptide numbers 89, 90 and 91 (referring to sequence numbers in Table 23). For rye, dominant peptides include peptide numbers 368, 369, 370, 371, and 372 (referring to sequence numbers in Table 23). Some peptides, including 635 and 636 (referring to sequence numbers in Table 23) showed activity in both rye and wheat.
In vivo gluten challenge allows T cell epitope hierarchy to be defined for coeliac disease The epitope hierarchy is consistent among HLA-DQ2+ coeliacs but different for HLA-DQ8+ coeliacs. The hierarchy depends on what cereal is consumed.
Deamidation generates almost all gliadin epitopes. HLA-DQ2, DQ8, and DR4 present deamidated peptides. HLA-DQ2/8-associated coeliac disease preferentially present DQ2-associated gliadin epitopes. Gliadin epitopes are sufficiently restricted to justify development of epitope-based therapeutics.
Other analyses indicated the following: HLA-DR3-DQ2 (85-95%) and HLA-DR4-DQ8 (5-15%).
Other analyses indicated the following:
HLA-DQ HLA-DQA1 HLA-DQB1 Duodenal Gluten EMA on allele allele histology free gluten (on GFD) C01 2,6 102/6, 501 201, 602 SVA 1 yr -1-(--) CO2 2,2 501 201 SVA 1 Yr +0 CO3 2,5 101/4/5, 501 201, 501 PVA 1 yr +(-) C04 2,5 101/415,501 201,501 SVA 7 yr +(-) C05 2,2 201, 501 201, 202 SVA 4 mo +(ND) C06 2,2 201,501 201,202 SVA 2 yr +(-) C07 2,8 301-3, 501 201, 302 SVA 1 yr -- +(-) C08 2,8 301-3,501 201,302/8 SVA llyr ND(-) C09 2,8 301-3,501 201,302 SVA 29 yr +(-) C10 2,8 201, 301-3 202, 302 TEL 1 yr +(-) C11 6,8 102/6,301-3 602/15,302/8 TEL 9 mo -(ND) C12 8,7 301-3, 505 302, 301/9-10 SVA 2 yr - (-) C13 8,8 301 302 SVA 1 yr +(+) Another analysis was carried out to determine the bioactivity of individual tTG-deamidated peptides in pools 1-3 in subject C12. The results are as follows (sequence numbers refer to the peptides listed in Table 23): Sequence 8 (100%), Sequence 5 (85%), Sequence 6 (82%), Sequence 3 (77%), Sequence 1 (67%), Sequence 2 (59%), Sequence 9 (49%), Sequence 7 (49%), Sequence 10 (33%), Sequence 4 (15%), Sequence 12 (8%), Sequence 11(0%), Sequence 23 (26%), Sequence 14 (18%), Sequence 15 (18%), Sequence 17 (18%), Sequence 16 (13%), Sequence 14 (8%), Sequence 22 (5%), Sequence 18 (3%), Sequence 19 (3%), Sequence 20 (0%), Sequence 21 (0%). The predicted deamidated sequence is LQPENPSQEQPE.
Individual ELISpot responses by PBMC (Spot forming cells determined by ELISpot Reader) Peptide (see Table 23) CO1 CO2 CO3 C04 C05 ' 68 =

71 ' 1 1 0 0 1 73 95 21 42 31 . 31 76 , 108 13 28 16 22 ' 82 3 3 5 2 2 83 14 2 0 0 1 .

Cross-reactivity To deal with data from 652 peptides in 29 subjects, or to determine when a .
particular response is a true positive peptide-specific T-cell response, or to determine when a response to a peptide is due to cross-reactivity with another structurally related peptide, expression of a particular peptide response can be as a percentage of a "dominant" peptide response. Alternately, the expression can be a "relatedness" as correlation coefficients between peptide responses, or via bioinfonnatics.
Additional epitopes A representative result is as follows:
Combination of peptides with P04722E (all 20mcg/m1) (n=4) . Alone P04722E+

Pep 626 60 135 HLAa 0 85 (expressed as percent P04722E) 626+tT: PQQPQQPQQPFPQPQQPFPW
P04724E: QLQPFPQPELPYPQPQL
TTG-dcamidation of peptide- 626 (n=12) No tTG = 100%
TTG = 170%
Substitution at particular positions Substitution of Peptide 626 PQQP[Q1}QP[Q21QPFPQP[Q31QPFPV (n=12) Glu = Arg (expressed as percent wild-type peptide) Bioactivity of tTG-treated 15mers spanning Peptide 626/627 (PQQPQQPQQPFPQPQQPFPWQP) (n=8) P1-.15 5 (expressed as percent of maximal 15mer response) Multiple epitopes:
P04724E: QLQPFPQPQLPYPQPQL
626+tTG: PQQPQQPQQPFPQPQQPFPW
Minimal epitope: QPQQPFPQPQQPFPW
5 Immunomagnetic depletion of PBMC by beads coated with anti-CD4 and by anti-integrin 137 depleted IFNy ELISpot responses, while immunomagnetic depletion of PIKVIC by beads coated with anti-CD8 or anti-alphaE integrin. Thus, the PBMC
secreting IFNy are CD4+ and c417+, associated with homing to the lamina propria in the gut.
10 Blocked by anti-DQ antibody but not by anti-DR antibody in heterozygotes and homozygotes for HLA-DQ2. This may imply multiple epitopes within one sequence.
7' cell epitopes in coeliac disease 15 Other investigators have characterized certain intestinal T cell clone epitopes. See, e.g., Vader et al., Gastroenterology 2002, 122:1729-37; Arentz-Hansen et al., Gastroenterology 2002, 123:803-809. These are examples of epitopes whose relevance is at best unclear because of the in vitro techniques used to clone T cells.
20 Intestinal versus peripheral blood clones Intestinal: 1) intestinal biopsies, 2) T cell clones raised against peptic-tryptic digest of gluten, 3) all ILA-DQ2 restricted, 4) clones respond to gliadin deamidated by transglutaminase.
Peripheral blood: I) T cell clones raised against gluten are HLA-DR, DQ and DP
25 restricted. Result Intestinal T cell clones can be exclusively used to map coeliac disease associated epitopes GDA _Wheat 307 aa Definition Alpha/Beta-Gliadin MM1 Precursor (Prolamin) Accession P18573 Genbank Intestinal T cell clone epitopes A definition of intestinal T cell clone epitopes can be found in, for example, Arentz-Hansen et al., J Exp Med. 2000, 191:603-12. Also disclosed therein are gliadin epitopes for intestinal T cell clones. Deamidated QLQPFPQPQLPY is an epitope, with a deamidated sequence of QLQPFPQPELPY. There is an HLA-DQ2 restriction. A homology search shows other bioactive rAlpha-gliadins include PQPQLPY singly or duplicated. A majority of T cell clones respond to either/or DQ2-al: QLQPFPQPELPY DQ2-aII: PQPELPYPQPELPY
Dominant gliadni T cell epitupes-0 All deamidated by transglutaminase.
Peripheral blood day 6 after gluten challenge: A-gliadin 57-73:
QLQPFPQPELPYPQPQS
Intestinal T cell clones: DQ2-cd: QLQPFPQPELPY DQ2-all: PQPELPYPQPELPY
Intestinal T-cell Clone Epitope Mapping a-Gliadins Al PFPQPQLPY

Glia-20 PQQPYPQPQPQ
F-Gliadins G1 PQQSFPQQQ

Glu-21 QSEQSQQPFPQQF
Glu-5 Q(IL)PQQPQQF
Glutenin Glt-156 PFSQQQQSPF
Glt-17 PFSQQQQQ

Gluten exposure and induction of IFNy-secreting A-Gliadin 5 7-73QE65-specific T
cells in peripheral blood Untreated coeliac disease, followed by gluten free diet for 1., 2, or 8 weeks, followed by gluten exposure (3 days bread 200g/day), followed by gluten free diet Result 1: Duration of gluten free diet and IFNy ELISpot responses on day 0 and day 6 of gluten challenge: A-gliadin 57-73 QE65 (results expressed as IFNy specific spots/million PPBMC) Day 0: none (5), 1 week (1), 2 weeks (2), 8 weeks (1) Day 6: none (0), 1 week (4), 2 weeks (28), 8 weeks (48) Result 2: Duration of gluten free diet and IFNy ELISpot responses on day 0 and day 6 of gluten challenge: tTG-gliadin (results expressed as IFNy specific spots/million PPBMC) Day 0: none (45), 1 week (62), 2 weeks (5), 8 weeks (5) Day 6: none (0), 1 week (67), 2 weeks (40), 8 weeks (60) Result 3: Duration of gluten free diet and IFNy ELISpot responses on day 0 and day .6 of gluten challenge: A-gliadin 57-73 P65 (results expressed as IFNy specific spots/million PPBMC) Day 0: none (1), 1 week (2), 2 weeks (1), 8 weeks (1) Day 6: none (0), 1 week (0), 2 weeks (0), 8 weeks (0) Result 4: Duration of gluten free diet and IFNy ELISpot responses on day 0 and day 6 of gluten challenge: PPD (results expressed as IFNy specific spots/million PPBMC) Day 0: none (90), 1 week (88), 2 weeks (210), 8 weeks (150) Day 6: none (0), 1 week (100), 2 weeks (210), 8 weeks (100) Result 5: Duration.of gluten free diet and IFNy ELISpot responses on day 0 and day 6 of gluten challenge: tTG (results expressed as IFNy specific spots/million PPBMC) Day 0: none (5), 1 week (4), 2 weeks (3), 8 weeks (2) Day 6: none (0), 1 week (4), 2 weeks (1), 8 weeks (2) Gluten challenge in HLA-DQ2 coeliac disease on long term gluten (SIODV) cladOdAdrIOddAdodbd (t7IDV) d0A0dA.CIOdciddbdod (EIDV) SbdOdAdlOcIdAdOdbd 0 (Z ov) sbabdAnOctlaaemba =
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OgrZ0/09-9/13d fLZt0I/0 OM
30-3T-t0OZ 8T388T730 YD

o / bubd utbs dbjs Abss dAbb to Tu-\/-0 / bubd ubbs dbjj 2bEs dAbb to Iu19r / bubd ubbj dbjj ONs dAbb to guy OC
z / bubd ubbs dbjj 2132s dAbb ioc lzv .1s /.(1)01EPIumP) Pod (z) adopda iitvzquiop 211?dchntr-al1ig 0 / 111 / A0aadOabsamadbaban sz o ail + IE1 9 / AbabababsdNaenban LI I aLl+OZOZ 60VGD / AbaadOOOsamadOlOan 8 / azoz 60VGD 18/ AOHUdOnScINIOdOlOdA

ado1ldaulEPIIII0 SOCI LZ / Abaubbbsambenban oz IL 1 AbaaaboOsambaMOJA
dA09lbaboOscrxbab ocI AbababObsaNORn /
AbaOdoubsambdoloa.
56 / bababbbsdNbablo .0011 Abababaosamodbaban 65 / ababbOsambabiba ci ott I OclOOOScINIORTINIA 9L /
A.OHOdOOOS(11\aleY1OdA
OEI / doOOScINORYTOdAd 1 AbababObsdmadOiodn.
06/ OboSdNodolodAd.M.
= 091 Obsamod1banam.11 9L / AbababObsambanOdA
ojg aouanbas ojs aouanbas o adopdo pagydossv ,90a lumillop zp 2wdelinu-2uLT
=smotiojge sasodind Butddetu io flo pat_uuo osre 010Ak SkUSSU 1.0dSfla gi aidurexa -odoltdo utpult2 g reuTdo ire Si gomm ,c9a6 EL-Ls ugreq2-17 01. paredtuoo IMLIRdO-CFES 0.1-0 SOITOTO
ITOOL LEI.IRSOTIII JO sadoltda `pootq reia-qdliad u sauoto -ago J, ruutlsaiut jo mug.
loatjal Tao' pootq rataqdpod jo samomoads `auaireqo uagpuu Fez Imp Atjapg t7L, OgrZ0/09-9/13d fLZt0I/0 OM
30-3T-t0OZ 8T388T730 YO

A3b2 301 qqyp ssqg sfqp sqqn pqaq /2 A4a 301 eqyp sgqv sfqs sqqn pqaq / 28 Albl 309 sfy sqqn plaq gsvq pqql / 2 Al al 309 sfrp sqqn pqaq gsvq pqql / 2 Example 16 Bioactiviiy of gliadin epitopes in IFNy-ELISpot (25 mcghnl, n=6) (expressed as % A-gliadin 57-73 QE65 response) DQ2-All: wild type (WI) (4), WT tTG (52), Gin-substituted (52) 10 DQ2-AI: wild type (WT) (2), WT + tTG (22), Glu-substituted (28) GDA09: wild type (WT) (1), WT + tTG (7), Glu-substituted (8) A-G31-49: wild type (WT) (2), WT + tTG (3), Glu-substituted (0) Dose response of A-Gliadin 57-73 QE65 (GOIE) (n=8) (expressed as %G01E
15 maximal response) 0.025 mcg/m1 (1), 0.05 mcg/ml (8), 0.1 meg/m1 (10), 0.25 mcg/ml (22), 0.5 mcg/ml (38), 1 mcg/ml (43), 2.5 mcg/m1 (52), 5 meg/m1 (70), 10 mcg/ml (81), 25 mcg/ml (95), 50 mcg/ml (90), 100 mcg/m1 (85).
IFNy ELISpot response to gliadin epitopes alone or mixed with A-gliadin 20 57-75 (GO1E) (all 50 mcg/ml, tTG-gliadin 100 meg/ml, PPD 5 mcg/ml; n=9) (expressed as % GO1E response) Alone: DQ2-A1 (20), DQ2-A2 (55), Omega G1 (50), tTG Gliadin (80), PPD
(220), DQ2 binder (0) GO1E+: DQ2-A1 (90), DQ2-A2 (95), Omega G1 (100), tTG Gliadin (120), 25 PPD (280), DQ2 binder (80) Effect of alanine and lysine substitution of A-gliadin 57-73 QE65 on IFNy ELISpot responses in individual coeliac subjects (ii=8) Epitope sequence: QLQPFPQPELPYPQPQS
30 Alanine substitution at positions 57-59 and 72-73 showed little to no decrease in % A-gliadin 57-73 QE65 response. Alanine substitution at positions 62 and 68-71 showed moderate decrease in % A-gliadin 57-73 QE65 response.

. 76 Alanine substitution at positions 63-67 showed most decrease in % A-gliadin 57-QE65 response.
Effect of lysine substitution of A-gliadin 57-73 QE65 on IFN'y ELISpot responses in individual coeliac subjects (n=8);
Epitope sequence: QLQPFPQPELPYPQPQS
Lysine substitution at positions 57-59 and 71-73 showed little to no decrease in % A-gliadin 57-73 QE65 response. Lysine substitution at positions 60-61 and 70 showed moderate decrease in % A-gliadin 57-73 QE65 response. Lysine substitution at positions 62-68 showed most decrease in % A-gliadin 57-73 QE65 response.
Example 17 Table 24 shows the results of analyses examining the 652 peptides with several patients challenged with wheat or rye.
References 1. Molberg 0, et al. Nature Med. 4, 713-717 (1998). =
2. Quarsten H, et al. Eur. J. Immunol. 29, 2506-2514 (1999).
3. Greenberg CS et al. FASEB 5, 3071-3077 (1991).
4. Mantzaris G, Jewell D. Scand. J. Gastroenterol. 26, 392-398 (1991).
5. Mauri L, et al. Scand. J. Gastroenterol. 31, 247-253 (1996).
6. Bunce M, et al. Tissue Antigens 46, 355-367 (1995).
7. Olerup 0, et al. Tissue antigens 41, 119-134 (1993).
8. Mullighan CG, et al. Tissue-Antigens. 50, 688-92 (1997).
9. Plebanski M et al. Eur. J. Immunol. 28, 4345-4355 (1998).
10. Anderson DO, Greene EC. The alpha-gliadin gene family. II. DNA and protein sequence variation, subfamily structure, and origins of pseudogenes. Theor Appl Genet (1997) 95:59-65.
11. Arentz-Hansen H, Korner R, Molberg 0, Quarsten H, Van der Wal Y, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM, McAdam SN. The intestinal T cell response to alpha¨gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med.
2000;
191:603-12.
12. Vader LW, de Ru A, van der Wal, Kooy YMC, Benckhuijsen W, Mearin ML, Drijfhout JW, van Veelen P, Koning F. Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 2002; 195:643-649.
13. van der Wal Y, Kooy Y, van Veelan P, Pena S, Mearin L, Papadopoulos G, Koning F. Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J. Immunol. 1998; 161:1585-8..
14. van der Wal Y, Kooy Y, van Veelan P, Pena S, Mearin L, Molberg 0, Lundin KEA, Sollid L, Mutis T, Benckhuijsen WE, Drijfhout JW, Koning F. Proc Natl Acad Sci USA. 1998; 95:10050-10054.
15. Vader W, Kooy Y, Van Veelen P et al. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. GastroenterolOgy 2002, 122:1729-37 . 20 16. Arentz-Hansen H, McAdam SN, Molberg 0, et al. Celiac lesion T
cells recognize epitopes that cluster in regions of gliadin rich in proline residues. Gastroenterology 2002, 123:803-809.

=
78 ' Table 1. A-Gliadin protein sequence (based on amino acid sequencing) VRVPVPQLQP QNPSQQQPQE QVPLVQQQQF PGQQQQFPPQ QPYPQPQPFP SQQPYLQLQP FPQPQLPYPQ

PQSFPPQQPY PQPQPQYSQP QQPISQQQAQ QQQQQQQQQQ QQQILQQILQ QQLIPCMDVV LQQI-INIAHAR

Table 2. Coeliac disease subjects studied Age Gluten HLA-DQ2, Bread Symptoms Sex free diet challenge with bread = I 64 f 14 yr Homozygote 3 days Abdominal pain, lethargy, mouth ulcers, diarrhoea 2 . 57 in 1 yr Heterozygote 10 days Lethargy, nausea 3 35 f 7 yr Heterozygote 3 days Nausea 4 36 in 6 wk Homozygote 3 days Abdominal pain, mouth ulcers, diarrhoea 5 26 in 19 yr Heterozygote 3 days None .
6 58 in 35 yr Heterozygote 3 days None 7 55 in 1 yr Heterozygote 3 days Diarrhoea 8 48 f 15 yr Homozygote 3 days Abdominal pain, diarrhoea =

Aminoacid at position 65 Range Mean Glutamate (100) 100%
Asparagine (50-84) 70%
Aspartate (50-94) 65%
Alardne (44-76) 64%
Cysteine (45-83) 62%
Serine (45-75) 62%
Valine (24-79) 56%
Threonine (46-66) 55%
Glyeine (34-47) 40%
Leucine (8-46) 33 A
Glutamine (16-21) 19%
Lsoleucine (3-25) 14%
Methionine (3-32) 14%
Phenylalanine (0-33) 12%
Histidine (0-13) 8%
Tyrosine (0-17) 8%
Tryptophan (0-17) 8%
Lysine (0-11) 4%
Proline (0-4) 2%
Arginine (0-2) 1%
Table 3 Elisopt response Peptide sequence Corresponding residues in glindin protein sequences (Accession no.) No TG TG
8(143) QLQPFPQPQLPYPQPQS 57-73 rz-Gliadin (T.
aestivum) Q41545 00(100) QLQPFPQPELPYPQPQS 57-73 cs-Gliadin (T.
aestivum) Q41545 5(1-7) 53(44-67) QLQPFPQPQLPYSQPQP 77-93 cap-Gliadin precursor (Tricetum. aestivum) P02863 76-92 ct-Gliadin (T.
aestivum) Q41528 77-93 cr-GIiarlin storage protein (T.
aestivurn) Q41531 57-73 cc-Gliadin Mature peptide (T.
aestivurn) Q41533 77-93 cc-Gliadin precursor (T.
spelta) Q922.09 12(0-20) 83(61-113) QLQPFPQPQLPYPQPQP 77-93 cdp-GI iadin A-1:1 precursor (T. aestivurn) P0472 19 (0-33) 83 (74-97) QLQPFPQPQLPYPQPQL 77-93 a./11-011adia A-IV
precursor (T, aestivum) P04724 77-93 ct/13-Gliarlin Will precursor (T. aestivum) P18573 3 (0-7) 109 (41-152) PQLPYPQPQLPYPQPQP 84-100 a./13-01.indin A-IV precursor (T. aestivum) P04724 ND PQLPYPQPQLPYPQPQL 84-100 ctip-Gliadin MMI
precursor (T. aestivum) P18573 0 (0-1) 3 (0.7) QLQPFLQPQLPYSQPQP 77-93 ct43-Gliadin A.-I
precursor (T. aestivurn) P04721 77-93 ca-Gliadin (T.
aestivum) Q41509 0 (0-0) 2 (0-7) QLQPFSQPQLPYSQPQP 77-93 cc-Gliadin storage protein (T. acstivutn) Q41530 ND PQPQPFPPQLPYPQTQP 77-93 cc/111-Gliadin A-ITI
precursor (T. aestivum) P04723 17 (0-40) 24 (11-43) PQPQPFPPQLPYPQPQS 82-98 cdf3-Gliadin A-V
precursor (T. aestivum) P04725 (0-30) 19 (11-33) PQPQPFPPQLPYPQPPP 82-98 cc/13-G1iadin clone PV/1215 precursor (T. aestivum) P04726 82.98 a/E-Gliadin (T.
urartu) Q41632 10(0-30) 21(11-33) PQPQPFLPQLPYPQPQS 79-95 aip-GUadin clone PW8142 precursor (T. aestiv-urn) P04726 79-95 a-Gliadin (T.
aestiv-urn) Q41529 79-95 ctill-Gliadin precursor (T.
aestivum) Q4I546 Table 4 Table 5. T cell epitopes described in coeliac disease Source Restriction Frequency Sequence*
Gamma -gliadin DQ2 3/NS (iTCC) QQLPQPEQPQQSFPEQERPF
Alpha-gliadin DQ2 12/17 (iTCL) QLQPFPQPELPY
Alpha-gliadin DQ2 11/17 (iTCL) PQPELPYPQPELPY
Alpha-gliadin DQ2 1/23 (bTCC) LGQQQPFPPQQPYPQPQPF
Alpha-gliadin DQ8 3/NS (iTCC) QQYPSGEGSFQPSQENPQ
Glutenin DQ8 1/1 (iTCC) GQQGYYPTSPQQSGQ
Alpha-gliadin DQ2 11/12 in vivo QLQPFPQPELPYPQPQS
NS not stated in original publication, iTCC intestinal T cell clone, iTCL
intestinal polyclonal T cell line, bTCC peripheral blood T cell clone *All peptides are the products of transglutaminase modifying wild type gluten peptides except the fourth and sixth peptides Table 6. Relative bioactivity of gliadin T cell epitopes in coeliac PBMC after gluten challenge Sequence* ELISpot response as % A-gliadin 57-73 QE65 (all 25mcg/m1) Wild type Wildtype+tTG E-substituted QQLPQPEQPQQSFPEQERPF 9(3) 18(7) 10(5) QLQPFPQPELPY 6(2) 19 (1) 8(3) PQPELPYPQPELPY 13(6) 53 (8) 48 (9) QQYPSGEGSFQPSQENPQ 10 (3) 9 (3) 14 (8) QLQPFPQPELPYPQPQS 18(7) 87(7) 100 PQLPYPQPELPYPQPQP 14(4) 80(17) 69(20) * sequence refers that of transglutaminase (tTG) modified peptide and the T
cell epitope. Wild type is the unmodified gliadin peptide. Data from 4 subjects. Blank was 5 (I) %.

=
TIOdbaclibcibdadblb (3) 6-LL Rods tunoOn NICIVI1D-VHcITI 60dZ6b TictdbdAd'Ibdr1.3d0d0d (d) 86-Z8 11-1=1E-111 m11011111 'EdAl-ViaE{/VHCIV Z91tb Sbcibd.AdlbdIdclbdOci (IA) SG-6L 1\11(IVIID-VIES/VHCIV
9trgItb TtbabDc.aiOdbciadOlb (a) EL-LS Sit7b -dbabKikcnbabaaablb (a) 6-LL 1\TIGVFID-VHFIV 1E51176 abclbS.A.ctlOdbgadblb = (H) 6-LL NIGVFID-VHdqV
0E5.11,0 SbabdAd'Ibd:IddOdOd 56-6L NICENTID-VHEMY 6ZgitO
JbabRA.albabaadbab (a) 6-9LZ NIGNTID-VHdl-V sctb abablkaibabladblO (D) 6-LL NIGNTID-VHCIV 60g Itb lEfiAlEILL
"abc1bdzictlbdbcgc1T071 ()I) L01-16 L Id ivaam 6VGD
qbdoclAcilbc1bcadTOTI (7) 00 I-tr8 EL58Id IVEHM-6V00 qbabdAdrIbdbc1.3clbqb b) 6-LL ELC8Id ivaHm-6VGD
Sbabdikd'IbctEd0d0d (it) g6-6L LZLtOd IVEHM¨LVGD
Pcibc1AdlociciddOciOd (d) 86-Z8 9ZLt0d .LVal-li\A-9VGD
Sbctbd.AdlodclAclOdOd (14) 86-Z8 SZLVOd IVEHM¨SVGD
dOclbdAd1Oclbcadlb7 01) 001178 trZLI70c1 1,VEHAA-17VGD
7Thdoditcnbaba1ab-lb (3) 6-LL 17ZL170d IVEHM¨trYGD
abibdAtiTbaciadodod (o) 6-LL ZLI7Od IVEHM¨VGD
TiOdbdA.cflOciOdthiblb (E)E6-LL ZZLP0c1 IvaHm-zvuo ababgAaabaMadb-ib (D) 6-LL IZL.170d IVEHAA¨IVGD =
abaOlkalbabaaabab (g) 6-LL 980d Ivalim-OVCD
:1011c1SSIMS
sbabaAcr1babaadb7b (v) L-Lc (uplold paouanbas nio1J) o!Pt112-y c1751-17b =
Inspp:liougrod Jaquinn uoIssaoot uweqa (palms sun umAnson tunom.u) intim xxxxxxaloaxxxxxxx :aauartbas aII tuTum.uoa sulpullt JOJ ( .6671'010 11U3s UM/Valli Puu IOIMSSIMS
SOcIOIA.diOdOdadolO . (G) (L-Lc 01.9EE :1131AIE) :01 aldruuo bdbigAd'IbclodAdolo (A) ( 7EINE) :II '6 'S BI-IdIu jodoclikd'IocRIEIdclolo (U) (E-LS tr09I 1-161 !ID
cloclUgARIbcIocIdclolo (I) (EL-LS 809EIfY `L,09IfV `909EIfd :IEHAIS) :CVE
mIcilt !ID
dbibaxcnbaciacioab (r) (tt-ss ZO9EEI1V g09I f V :-IfiNg) sbabdiTicribcemadblb (v) EL-Lc (uploid paouanbas luau) ulptip-y ciqgito insIgdzotukiod (sopgdad j.21,4 oT =jai apoo Japai apup) uiojcI ugre112-aidiy Joupolyi uFells pawn uumpiu alpaoN poApap saouanbas .17 =
L-LS ultgell2-y Jo smsIttdioluzilod .L aiqui, OStZO/COR3/I341 ELZ170I/0 OAA
30-3T-t003 8T3813D30 YD

Table 8. Bioactivity of substituted variants of A-gliadin 57-73 QE65 (Subst) compared to unmodified A-gliadin 57-73 QE65 (G) (mean 100%, 95% CI 97-104) and blank (no peptide, bl) (mean 7.1%, 95%
CI: 5.7-8.5) Subst ' % P vs G Subst % P vs G Subst % P vs G Subst %
PvsG Pvsbl Super-agonis ts F62 71 0.001 1162 47 <0.0001 N66 24 <0.0001 Y61 129 <0.000 V63 70 <0.0001 G69 47 <0.0001 R64 24 <0.0001 Y70 129 0.0006 S69 70 <0.0001 N63 47 <0.0001 K63 23 <0.0001 Agonists R63 70 <0.0001 1168 47 <0.0001 V65 23 <0.0001 W70 119 0.017 F63 70 0.008 M68 46 <0.0001 1166 23 <0.0001 K57 118 0.02 P70 69 <0.0001 D68 46 <0.0001 H67 22 <0.0001 Y59 117 0.04 T62 69 <0.0001 V69 46 <0.0001 L64 22 <0.0001 A57 116 0.046 L61 69 <0.0001 G63 45 <0.0001 S66 22 <0.0001 S70 116 " 0.045 S61 69 <0.0001 V64 45 <0.0001 F67 21 <0.0001 1{58 114 0.08 T61 69 .<0.0001 61 45 <0.0001 W66 21 <0.0001 W59 110 0.21 T63 69 <0.0001 A69 43 <0.0001 G64 21 <0.0001 A73 109 0.24 1166 68 <0.0001 R62 42 <0.0001 G65 21 <0.0001 159 108 0.37 T69 67 <0.0001 G68 42 <0.0001 D64 21 <0.0001 G59 108 0.34 1{60 66 <0.0001 A64 42 <0.0001 165 21 <0.0001 A58 108 0.35 S62 66 <0.0001 C65 42 <0.0001 M64 20 <0.0001 <0.0001 W60 105 0.62 1161 66 <0.0001 N67 41 <0.0001 G67 19 <0.0001 <0.0001 A59 104 0.61 P61 65 <0.0001 W63 41 <0.0001 T65 19 <0.0001 0.003 1(72 104 0.65 1162 64 <0.0001 F69 41 <0.0001 A66 19 <0.0001 <0.0001 S59 103 0.76 Q61 64 <0.0001 N68 40 <0.0001 164 19 <0.0001 0.0003 1C73 102 0.8 G61 64 <0.0001 V66 40 <0.0001 R63 19 <0.0001 <0.0001 A70 102 0.81 A63 64 <0.0001 1169 40 <0.0001 W67 19 <0.0001 <0.0001 Y60 101 0.96 L62 60 <0.0001 M69 40 <0.0001 K68 18 <0.0001 <0.0001 A72 100 0.94 168 60 <0.0001 R69 40 <0.0001 H64 18 <0.0001 <0.0001 S63 98 0.67 S67 59 <0.0001 W69 40 <0.0001 W64 18 <0.0001 0.0001 1(59 96 0.46 N61 59 <0.0001 Q69 39 <0.0001 Q65 18 <0.0001 0.0002 160 96 0.5 169 59 <0.0001 L67 38 <0.0001 F64 16 <0.0001 0.0008 G70 95 0.41 V61 58 <0.0001 K69 38 <0.0001 L65 16 <0.0001 0.0022 D65 95 0.44 D61 58 <0.0001 K62 38 <0.0001 N64 16 <0.0001 <0.0001 E70 93 0.27 60 57 <0.0001 E67 37 <0.0001 F65 16 <0.0001 0.12 163 92 0.19 A61 57 <0.0001 L69 37 <0.0001 Q67 15 <0.0001 0.0012 S60 92 0.23 Q62 56 <0.0001 S64 36 <0.0001 M65 14 <0.0001 0.015 P59 88 0.08 F68 56 <0.0001 G62 36 <0.0001 D66 14 <0.0001 0.013 M63 87 0.03 N65 56 <0.0001 E69 36 <0.0001 1167 14 <0.0001 0.002 1(71 85 0.047 A62 56 <0.0001 68 36 <0.0001 .. Non-agonists V62 84 0.04 A68 53 <0.0001 V67 35 <0.0001 P63 13 <0.0001 0.002 170 84 0.04 P66 53 <0.0001 D62 35 <0.0001 E64 12 <0.0001 0.053 161 83 0.01 R61 53 <0.0001 1168 34 <0.0001 W65 11 <0.0001 0.24 V68 82 0.0045 S68 53 <0.0001 Q66 34 <0.0001 Q64 .. 11 <0.0001 0.15 E59 81 0.01 Y63 52 <0.0001 A67 33 <0.0001 1366 11 <0.0001 0.07 Partial agonists N69 51 <0.0001 N62 32 <0.0001 1165 11 <0.0001 0.26 W61 79 0.002 63 51 <0.0001 F66 31 <0.0001 Y67 10 <0.0001 0.13 A60 78 0.002 T64 51 <0.0001 62 31 <0.0001 E66 10 <0.0001 0.17 Y62 78 0.006 T67 51 <0.0001 D69 31 <0.0001 K66 10 <0.0001 0.21 G60 77 0.003 Y69 50 <0.0001 D67 30 <0.0001 R66 10 <0.0001 0.23 A71 77 0.003 D63 50 <0.0001 M67 29 <0.0001 K67 10 <0.0001 0.11 W62 76 , 0.0009 A65 49 <0.0001 Y66 28 <0.0001 P65 8 <0.0001 0.57 Q60 76 0.001 1161 49 <0.0001 167 28 <0.0001 1(64 8 <0.0001 0.82 L63 74 0.0002 166 49 <0.0001 H65 26 <0.0001 K65 8 <0.0001 0.63 162 74 0.0005 168 48 <0.0001 P68 26 <0.0001 Y65 7 <0.0001 0.9 1170 74 0.001 365 43 <0.0001 Y64 25 <0.0001 1-161 72 <0.0001 L68 48 <0.0001 EK65 25 <0.0001 W68 72 <0.0001 Q68 48 <0.0001 T66 25 <0.0001 Table 9. Antagonism of A-gliadin 57-73 QE65 interferon gamma ELISPOT
response by substituted variants of A-gliadin 57-73 QE65 (Subst) (P is significance level in unpaired t-test). Agonist activity (13/0 agonist) of peptides compared to A-gliadin 57-73 QE65 is also shown.
Subst 'V0 Inhibit. P % agonist. Subst % Inhibit. P
% agonist.
Antagonists 65R 13 0.18 11 65T 28 0.004 19 65M 13 0.16 14 67M 27 0.0052 _ 29 68P 13 0.16 26 64W 26 0.007 18 63R 13 ' 0.19 19 67W 25 0.0088 19 66G 12 0.19 11 Potential antagonists 65Q 12 0.2 18 671 24 0.013 10 65Y 12 0.22 7 67Y 24 0.013 21 66S 12 0.22 22 64G 21 0.03 21 67F 11 0.25 21 64D 21 0.029 16 66R 10 0.29 10 65L 20 0.046 26 67K 10 0.29 10 66N 20 0.037 24 64F 10 0.29 16 6511 20 0.038 16 65F 9 0.41 16 64N 19 0.05 16 63P 8 0.42 13 64Y 19 0.06 25 65EK 8 0.39 25 66Y 19 0.048 28 64Q 7 0.49 11 64E 19 0.049 12 641 5 0.6 21 67A 18 0.058 30 68K 5 0.56 19 6711 18 0.052 22 67Q 5 0.61 18 Non-antagonists 65G 5 0.62 15 65V 17 0.07 23 64M 4 0.7 20 651 17 0.086 21 66H 4 0.66 23 66T 17 0.069 25 66 E 3 0.76 10 65W 15 0.11 11 66D 1 0.9 14 67R 15 0.13 14 63K 1 0.88 23 65P 15 0.13 8 64H 1 0.93 18 .
65K 15 0.11 8 66K 0 0.98 10 66W 15 0.12 21 64K -2 0.88 8 67G 14 0.14 19 64L -11 0.26 22 66A 14 0.14 19 ' Table 10. Inhibition of A-gliadin 57-73 QE65 interferon gamma ELISPOT
response by peptides known to bind IlLA-DQ2 (P is significance level in unpaired t-test).
Peptide % Inhibit. P
TP 31 <0.0001 HLAla 0 0.95 Table 11. Antagonism of A-gliadin 57-73 QE65 interferon gamma ELISpot response by naturally occurring polymorphisms of A-gliadin 57-73 QE65 (P is significance level in unpaired t-test).
A-gliadin 57-73 QE65 polymorphism % Inhibit.
P04725 82-98 QE90 PQPQPFPPELPYPQPQS 19 0.009 Q41509 77-93 QE85 QLQPFLQPELPYSQPQP 11 0.15 Gli a 1,6 58-74 QE66 QPQPFPPPELPYPQTQP 11 0.11 P04723 77-93 QE85 PQPQPFPPELPYPQTQP 10 0.14 Gli a 3-5 57-73 QE65 QLQPFPQPELSYSQPQP 7 0.34 Q41509 77-93 QE85 QLQPFLQPELPYSQPQP 6 0.41 P04727 79-95 QE65 PQPQPIiLPELPYPQPQS 6 0.39 P04726 82-98 QE90 PQPQPFPPELPYPQPPP 5 0.43 Table 12. Prolamin homologues of A-gliadin 57-73 (excluding alpha/beta-gliadins) Prolamin Accession number Sequence %
Bioactivity*
Wheat: ct-gliadin A-gliadin (57-73) QLQPFPQPQLPYPQPQS 100 (0) Wheat: ce-gliadin AAG17702 (141-157) ..... PQ .. F QSE 32 (6.4) Barley: C-hordein Q40055 (166-182) ..... . .... ...QPFPL F -- Q -- 2.3 (2.0) Wheat y-gliadin P21292 (96-112) ....... ...QTFPQ F . QPQ
2,1 (4.2) Rye: secalin Q43639 (335-351) ...... ...QPSPQ F Q 1.6 (1.4) Barley: y-hordein P80198 (52-68) ....... ...QPFPQ HQHQFP -1.0 (1.8) Wheat: LMW glutenin P16315 (67-83) _. .. LQ .QPIL FS. Q...Q -0.9 (1.0) Wheat: HMW glutenin P08489 (718-734) .. HGYYPTS SGQGQRP 6.4 (4.0) Wheat y-gl iadin ' P04730 (120-136) ...QCCQQL ......
I...QQSRYQ 0.7 (0.9) Wheat: LMW glutenin : P10386 (183-199) ...QCCQQL ......
I...QQSRYE -0.7 (0.5) Wheat: LMW glutenin 049958 (214-230) ...QCCRQL ......
I...EQSRYD -1.1 (0.3) Barley: BI-hordein P06470 (176-192) ...QCCQQL ......
I...EQFRHE 1.8 (1.4) ' Barley: 137hordein Q40026 (176-192) ...QCCQQL
ISEQFRHE 0.5 (0.9) *Bioactivity is expressed as 100x(spot forming cells with peptide 25mcg/m1 plus tTG Smog/nil minus blank)/(spot forming cells with A-gliadin 57-73 25mog/m1 plus tTG 8mog/m1 minus blank) (mean (SEM), n=5).
Peptides were preincubated with tTG for 21137 C. Note, Q is deamidated in A-gliadin 57-73 by tTG.

' Table 13. Clinical details of coeliac subjects.
HLA-DQ ITLA-DQA1 HLA-DQB1 Duodenal Gluten free EMA on gluten alleles . alleles histology (on GFD) C01 2,6 102/6,501 - 201,602 SVA 1 yr -- 4- 0 CO2 2, 2 501 201 SVA 1 yr + 0 CO3 2,5 101/4(5,501 201,501 PVA 1 yr +(-) C04 2,5 101/4-5, 501 201., 501 SVA 7 yr -- + 0 COS 2,2 201,501 201,202 SVA 4 mo + (ND) COG 2,2 201,501 201,202 SVA 2 yr + 0 C07 2, 8 301-3, 501 201, 302 ' SVA 1 yr + 0 _ _ C08 2, 8 301-3, 501 201, 302/8 SVA 11 yr ND (-) = COP 2, 8 301-3, 501 201,302 SVA 29 yr -- + 0 --=
_ C10 2, 8 201, 301-3 202, 302 IEL 1 yr -- + (-) C11 6,8 102/6,301-3 602/15,302/8 TEL 9 mo - (ND) C12 8,7 301-3, 505 302, 301/9-10 SVA 2 yr - 0 C13 8,8 301 302 SVA 1 yr +(+) SVA subtotal villous atrophy, PVA partial villous atrophy, TEL increased intra-epithelial atrophy, GFD gluten-free diet, ND not done. .

, Table 14. HLA-DQ2+ Coeliac (C01-6) and healthy control (1101-10) IFNI, ELISpot responses to control peptides (20 pg/m1) and gliadin (500 g/m1) before and after gluten challenge (sfc/million PBMC minus response to PBS alone) Peptide Healthy Day 0 Healthy Day 6 Coeliac Day 0 Coeliac Day 6 P04722 77-93 0 (-4 to 17) 0 (-5 to 9) -2 (-3 to 0) 27 (0-100)*
P04722 77-93 + LTG 0 (-5 to 4) 0 (-9 to 3) 0 (-4 to 11) 141 (8 to 290)**
P04722 77-93 QE85 0 (-5 to 5) 0 (-3 to 4) 0 (-6 to 14) 133 (10 to 297)*
P02863 77-93 0 (-4 to 13) 2 (-3 to 5) -2 (-3 to 2) 8 (-2 to 42)**
P02863 77-93 + tTG -1 (-5 to 4) -1 (-4 to 11) 1 (-4 to 6) 65 (8-164)**
P02863 77-93 QE85 0 (-4 to 13) 0 (-4 to 14) -1 (-4 to 6) 42 (-2 to 176)* ' Gliadin chymotrypsin 2 (-5 to 20) 18 (0 to 185)* 20 (11 to 145) 92 (50 to 154) Gliadin chymotrypsin + tTG 0 (-1 to 28) 16 (-9 to 171)* 55 (29 to 248) 269 (206 to 384)**
Chymotrypsin 0 (-4 to 5) 1 (-4 to 11) -2 (-5 to 5) 1 (-4 to 8) Chymotrypsin + tTG 0 (-5 to 8) 6 (0 to 29) -2 (-3 to 11) 2 (-3 to 18)*
Gliadin pepsin 4 (-4 to 28) 29(0 to 189)*** 44(10 to 221) 176 (54 to 265)**
Gliadin pepsin +tTG 2 (-3 to 80) 27 (-4 to 241)*** 61(8 to 172) 280 (207 to 406)**
Pepsin 0 (-4 to 10) 0(-3 to 12) 0 (-2 to 3) 2 (-2 to 8) Pepsin + tTG 0 (-3 to 8) 0 (-5 to 9) 1 (-6 to 3) 0 (-3 to 14) PBS alone 4 (0 to 6) 2 (0 to 6) 4 (Ito 12) 4 (0 to 4) PBS + tTG 3 (0 to 8) 3 (0 to 11) 4 (2 to 10) 4 (2 to 11) Day 6 vs. Day 0: *P<0.05 **P,0.02, ***P<0.01 by one-tailed Wilcoxon Matched-Pairs Signed-Ranks test Table 15. Effect of deamidation by tTG to gliadin (0.5 mg/ml) and A-gliadin 57-73 homologues on IFN7 ELISpot responses in HLA-DQ2+ coeliac (C01-6) and healthy control subjects (1101-10) (median ratio tTG:no tTG pretreatment, range) Peptide Healthy Day 6 Coeliac Day 0 Coeliac Day 6 Gliadin chymotrypsin 0.94 (0.4-9.0) 2.1 (0.8-6.8)* 3.2 (1.8 -4.2)**
Gliadin pepsin 1.4 (0.5-1.4) 1.4 (0.8-4.0)* 1.9 (1.1-4.4)**
P04722 77-93 Q85 6.5 (2.3-1'2)**
P04722 77-93 E85 0.7 (0.6-1.1) P02863 77-93 Q85 7.5 (3.9-19.9)**
P02863 77-93 E85 1.0 (0.8-1.2) TTG>no tTG: *P<0.05 "11,0.02, ***P<0.01 by one-tailed Wilcoxon Matched-Pairs Signed-Ranks test Table 16. Healthy subjects: LUNT ELISpot Responses (>10 sfc/million PBMC
and >4 x buffer only) to tTG-treated gliadin peptide Pools on Day 6 of gluten challenge (sfe/million PBMC) (italic: response also present on Day 0):
Group 1 ¨ HLA-DQ2 (DQA1*0501-5, DQB1*0201) Group 2 ¨ 1[LA-DQ8 (DQA1*0301, DQB1*0302) and absent or "incomplete"
DQ2 (only DQA1*0501-5 or DQB1*0201) Group 1 Group 2 Subject H01 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 HLA-DQ 2,6 2, 7 2, 8 2, 5 2, 6 2, 6 2, 6 2, 7 2, 5 2, 5 8, 8 Pool 1 .
2 .
3 . . . . . . . . . .
4 . 13 . . . , ' = 5 . 24 . =
31 . .
7 . . . . .
-8 . . . . . . . . . .
9 . . . . . . . . . 10 . . . . . .
. . . . 11 . . = = = . . . . . 12 .
. . . . . . . . . .
13 . . . . . . . .
=
14 . . . . . . . . . . 15 . . . .
. . . . . . . .
=
16 . . . . . . .
17 . . . . . . .
18 . . . . . 20 . . . .
19 . . . . . . . .
=
20 _. 11 . . . . . .
21 . 11 . . . . . . 27 . 22 . . . .
23 . 43 . . . .
. , -= 24 . . . . . . . . 25 . 11 .
. . ' ' . . . . 26 . . . . . . . 27 . . . . . . . . . , 28 . . . .
. . _ 29 . . . . . . . , 30 . . . . . 23 . .
31 . . . .
=
. 32 . . . . . . . . 33 . 20 . . . .
. .
. , 34 . . . . . . . .

35 . 11 . . . . . . . '

36 . . 0 0 . . . . . . 37 . .. . .
18 . , .
38 14 . 12 . . . . . . . .
39 . . 11 . . . .
40 . 14 . . . . . 17 . . . 41 . . . . .
42 . . . . _ . . . .

CA 0248 8218 20 0 4 ¨12 ¨02 43 . . . = = = 11 . =
= =
44 . 14 . . . . . .
. . 45 . 11 . . . = = = . =
.
46 . = . . = = = . = .
.
47 . . . = = = = = = = =
48 . = = = =
= = = = = =
49 . = = = = . = = = =
=
50 . 14 .
. 12 . 22 . 14 . .
51 . . . . = = = = =
= = 52 . 14 . . . . . . .
. 53 - . 26 . . . .
. = = .
54 . = . . . 12 . . .
= .
55 . . . . . . =
.
56 . = = = =
= = - 57 . . 23 . . . 12 . . . .
58 . . . . . =
59 . . . . . . . . =
. 60 . = . = . . = = = = = = 61 . 23 . . . . //
// . .
62 . = = = = = = = = = . 63 . = = = =
= = = =
= = 64 . 20 . . . . . . = .
.
65 . = = = = = = = = = =
66 . 14 . . . . . = . . 67 = 11 . . =
= = = = = = 68 = 20 = . = . 20 . = =
=
69 . 20 . . . . . = = = =
70 . = = = = = = =
= = .
71 = = = = = . . . 16 = . 72 . 11 . . = = = = = = =
73 . 14 . . . . . = = = =
74 . = = = = = = . = =
. 75 . = . . . . = . =
76 . 14 . . . . . . . = 77 . . . .
. - - = =
. , 78 . 11 . = = = = = = = = 79 . ' 11 . .
. . 19 = . = =
80 . . . . = . = = .
81 . ' . . . . . ' . . . 82 = =
= = = = = . = =
. 83 . . . . ' . . . . P0472277-93 . . .
. . . = ' .
P04722 77-93 E . . . . '. . . . . . =
P04722 77-93 E . . . . . . . . . P02863 77-93 .
. . . . 11 . = = .
. P02863 77-93 E . . . = . . . . . . .
Gliadin+C 171 40 25 16 10 18 14 . 17 90 Chymntrypsin 29 26 18 . . . . . 22Gliadin+Pepsin 24/ 151 29 24 48 . 16 45 . 19 35 Pepsin Table 17: tTG-deamidated gliadin peptide pools showing significant increase in IFN gamma responses between Day O. and Day 6 of gluten challenge in HLA-.
DQ2 coeliac subjects C01-6 (Day 6 ¨Day 0 response, and ratio of responses to tTG-deamidated pool and same pool without tTG treatment) .
IFNg ELISpot tTG: no tTG IFNg ELISpot tTG: no tTG
Pool (Median sfc/million) . (Median) Pool (Median sfc/million) (Median) 9 59*** 1.0 49 . 46*** ______ 1.4 116** , 1.7 50 50*** 4.6 . 11 24*** 2.5 51 40*** 1.7 12 133*** 1.1 52 , 30*** 3.1 13 26** 2.1 53 27** 1.4 , 42 33** 1.2 76 17*** 1.1 43 32*** 1.3 79 20*** 0.9 44 24*** 1.5 80 83*** 1 45 10*** 1.1 81 141*** 1.1 46 12*** 2.1 82 22*** 1.5 48 17*** 1.4 83 16** 1.8 Day 6 vs. Day 0 **P<3.02, "*P<0.01 by one-tailed Wilcoxon Matched-Pairs Signed-Ranks test ..
, Table 18. Coeliac subjects: IFNy ELISpot Responses >10 sfc/million PBMC and >4 x buffer only to tTG-treated Pepset Pools on Day 6 of gluten challenge (sfc/million PBMC) (italic: response also present on Day 0):
Group 1 ¨ HLA-DQ2 (DQA1*0501-5, DQB1*0201/2), Group 2 ¨ HLA-DQ2/8 (DQA1*0501-5, *0301, and DQB1*0201/2, *0302), and Group 3 ¨ HLA-DQ8 (DQA1*0301, DQB1*0302) and absent or "incomplete"
DQ2 (only DQA1*0501-5 or DOB1*0201/2) Group 1: Group 2: Group 3 Subject CO1 CO2 CO3 C04 COS C06 C07 COS C09 HLA-DQ 2, 6 2,2 2, 5 2,5 2, 2 2, 2 2, 8 2, 8 2, 8 2, 8 6,8 7, 8 8, 8 Pool 1 . 23 223 _ 2 . 155 3 . 41 4 11 22 .
5 .

7 . 353 = =
8 11 64 14 20 480 . 13 .

. .

11 32 118 33 14 26 27 12 .
12 204 379 54 225 61 129 587 12 .

14 . 45 21 17 .
18 30 = 38 43 . .
16 . 37 . .
17 . .
18 .
. . 19 .. 11 21 . 11 .
22 . 21 =
23 . 18 21 12 .
24 . 15 10 . 15 12 _ .
26 . 18 ' 13 12 .
27 . 15 28 . 11 29 . 11 .
Ii II . .
= 31 . 70 32 . 18 20 33 11 10 14 11 . 40 11 34 . 11 . .
.
36 .

37 . 23_ 14 . .

38 . 24 19 20 ,

39 . 49 15 // . .
. 14 . 93 41 . 21 . .
42 39 42 44 21 _ _ 11 63 12 . .

. .
44 32 97 17 _ 96 13 87 , 107 . .
45 . 21 10 _ 100 11 38 110 . .
46 14 55 102 _ 18 63 163 . - .

. .
_ 48 21 106 60 14 144 , 353 . 57 .
49 75 170_ 17 142 30 202 293 39 . .

. .

= .
52 43 121 79 13 16 175 180 . .
53 36 94 92 29 69 _ 53 . .
_ 54 36 _ 35 11 166 27 . . _ 19 13 55 . . .
56 29 . ...
11 . .
57 . 36 20 13 . .
58 _ 59 . 10 53 . . . ..
60 . 18 15 11 53 .
= 61 . 20 . . _ 62 14 18 13 _ _ 60 . .
63 . 10 14 28 .
, 64 . 15 18 65 36 25 23 _ 35 27 . 11 66 31 11 10 17 . .
67 . 17 17 = .
68 . 19 127 14 . .
69 . 15 10 20 20 .
70 . 12 31 - 13 10 .
71 11 21 13 _ 14 . _ = 18 72 . , 16 . .
- 73 . 13 14 11 . .
_ 74 . 239 254 447 . .
_ 75 . . .

. . _ = 77 . 88 10 13 . .
78 . 18 17 r 69 _ . .
_ 79 11 85 44 29 12 44 43 . .
80 132 133 33 240 39 12 208 467 12 . 70 81 171 318 113 367 104 12 211 530 74 . .
82 18 300 17 125 32 16 241 _ 723 83 14 164 31 21 163 277 15 . _ P0472277-93 211 291 75 _ 281 66 78 740 . .
P04722 77-93 E 164 297 108 _ 221 64 10 84 _ 653 .
.
P04722 77-93 E 161 182 98 256 73 16 63 500 .
. .
P0286377-93 139 164 35 94 36 29 603 . .
902863 77-93 E 46 176 19 88 41 23 520 . .
Gliadin+C 214 273 265 360 384 206 278 543 17 25 527 71 Chymotrypsin 18 . . _ Gliadin+Pepsin 239 315 269 406 207 292 357 _ 557 42 89 335 87 _ _ Pepsin , . 14 _ S.
, (gA),:nod nuartbas woo otr4 timm EL-is ullnlig-y Jo ongotoutog saluo!pui *
(c) It baldbbabblodAdbodood LGE CL (9) at tIdbbcil6l6dobbc11t\bnoNd 600 99 (9) I 1 dbcIaIndbocIttlibbbdbb 60E IL (9) 01 bbdS8dddOdAd1OddAnd 96 SE..
' (9) II thlAdlOdladOdOdadtiltil 60 at. (s) a 1 NasONO080d.4.100.860s c i t, re (0 it AdoodUkIddOOLlb1.4662M OLC 69 (9) 61 06,16c1166bclOadObc166-1 111' CE
(a) II O6A,1106143,1bev6dtrI9II LL 89. (0)6!
babOd.gclObabbabagooba Lci: Ee (s) at babaAabbatmsbaaboab sot, 09 (5) 61 bbr111.3dode/dAditiddgdbd 96 IC.
(9) Z1 bba-aadbabsA.nbacuaba 88 99. (9) 6 r 6,161.1c100,300cLIdoba1d 999 DE
(9)91 olaasMatmaibbabiindu 809 59 (8) OZ
6bbc/d<lbt/dOld1cl6t)dt)bd 099 60 (V) 91 adnekkmtibdbb,r.I.LOO LSE 69 (OI) 00 ,u0OctObag,I.DbabIAMb3 ILE AZ
(a) at dOd-tbdgclobdObibdadOO 66E 9 (01) ZZ
Obaba.gObOabbaabbabb ,ELE LZ
IL) CI X)74.4dababbarcsbOlsa6 199 Z9 (6) ZZ , dAibbdbbdttoldobeEld 50.9 9Z
(61)9! 66.366.01.4abOabbamob sac 19 = (8) ZZ
OddiadOOdObibaaabbab 909 SZ
(9) CI aboaabbab-wabb-ibba 909 09 (01) EZ
bbaNgabbabbabEdbOd 9/.9 Pt .
. (9) 91 11,11bribbaidbbdbct6eibb LEE 65 (L) 99 OZ)d6)brIddlbdt)Odbd.4dbb SSC CZ
, (9) 91 babigabbabbsbaktbOdb LOh Its (al) EZ
sbOacriatOOmalialostos 609 ZZ
abbaadObabaubbabia 2,911 Ls (6) a O.LN6dObabba400babtu.
969 it OcurtmagabektaabiOi OS as. . (9) 90 boausbabamabduaba 56 09, (9) 91 NAntmagabablaamba ZS Ss. (I 1) sz Oaddaabbabbabaddobbb SEE 61 (L) 91 bbbabbEabbabbdodabb 890 95 (0)9c liabbabsArribabdadbibl L9 at, (5) 91 NIAdib6r13dbdodgdb7b11 90. CS. (SOU
OdabbbabuddlObabagvb 95E Li (9) SI daalbibaAcribaagababa IS ZS', (8) L0 Ob6bba0)3gabbabba.daib 969 91 (11) SI saddbosasbbdalabbbma 019 IS (9) SZ
OrmaJsoaba,kaibariaabd . 96 ' St.
(oldi 6600abHaubbabagibo 69C OS Woo luab6b5AdlbdorWerlol 99 PI.
(9) 91 Jababctaababaxabbsaaa 99 . 69 (01) EC
113,10<lblAdlbdbd6do161 09 94 . .
(8) 51 d.NbOdblIagIbbdbd&ob 050 82, (11) Pr bbdad1OdOOdol46ObabO t:ri Et (9)91 bbabbi6abbab610adabb 689 Li' (61) 1,9 bbidadOONI6id6dOtidObS alt' . II
. (9) 91 .dboada6babbadabbabld 91,9 91, (11)6a AdlodOam-10<1066entn CZ GI.
(9) 91 Obc1.4c1b6dOl13.30010d6d 699 St, - (01) If bdbMILIbbdts/thlt/babbd 999 6 (9)91 ObabOld,tgabOJOOcrgan ZSC Ph (Z1)19 SZ)bd:ILrlbdtLOJbodbbd.3 1E9 8, (0) 91 Oemminabb-rOamobad . 601, 09 (00) 99 tagabbablaidObabavb 09 L
(01) 91 . .1:19t6batiklalbbeMalbb SPE ZP (EZ) CL ,.
Mct0ctObdbc16dbt)6bbdnd 999 9 (6)zi bbanitxtadobalbdAibb 060 IS, (Si)ca mgdbakuartbatmaablb-i 90 S.
(o) Li 21.44bdbdAdlbL12143,10-101 SL Ob. -(91) LS .
ctiRdOdOdAdloakLIZIN . 06 V
' (L) si bbaAabbkiribbbAsEZZa 019 69 ' ()'1) 813 Aai1dbcua1b1ba69ftbv4 90 E.
_ (9) 81 ,13,410dObab6anbabbab OSC RC (01) 68 dodAdibdbdAcrIbcId.idtid 16 Z.
(9) al brusobabbabcustobabm C14 LE (Ii) 96 dAillt)dbdAdlbdOdAdlOd 68 (was) (iAlas) Inrolv Dauonbas, .0N numli ucam! ananbas =ohl Nueu s-103 spofqns zurpoo tOci-v-TH u! (iuOiri OZ) ssa zzLvoa jo %c4-! < AjlApaeoici uuoux twitet sappdad pa4-upiumou '61 giclui OgrZ0/09-9/13c1 fLZt0I/0 OM
30-3T-t0OZ 8T388T730 YD

Table 20. Peptides >10% as bioactiye as P04722 QE65 grouped by structure.
Rank Peptide no. Sequence IFNg ELISpot (Pool) response Gliadin-subtype compared to QE85: mean (SEM) Group 1: Homologues of A-gliadin 57-73 1 89(12) a PQL...Y LPYP 94 (18) 2 91(12) a PQPFPPQL...Y 89 (12) 3 74(10) a M LPY 88(14) 4 90(12) a PQL...Y PFRP 87(16) 5 76(10) a L PER 85(15) 8 631 (81) o) - FPQQPQ F QS 61(12) 10 73(10) a L LPY 49(11) 13 68 (9) a L L PFR 33 (10) 14 66 (9) a L S PFR 32 (7) 18 67 (9) a L S QFR 26(6) 20 95 (13) a PQPFL FPPQQ 24 (6) 31 93(12) a PQPFP PFRPQQ 19(5) 35 94(12) a PQPFP PPFSPQQ 18(3)

40 78 (10) a L R PFR 17(8) 52 81(11) a PQPQPFP T...PFPP 15(5) 53 75 (10) a MQLQPFPQPQPF 14 (5) 55 82(11) a PQPQPFPQPQPF
14(3) 56 80 (10) a LQLQPFPQPQPF
14(4) 66 88(11) a PQPFP S PFRPQQ 12(3) 68 77(10) a LQLQPFPQPQPFP
11(4) ' 70 79 (10) a LQLQPFPQPQPFL 11(5) Group 2: Homologues of peptide 626 QQPFPQPQQPFP
6 626(80) o) PQQPQQP W 72 (23) 7 627(80) co WQPQQPFPQ 66 (30) 9 636(81) co PQQP I VQPQ 51(10) 11 412(53) y sQQP Q PQQ 34 (19) 33 411(53) y LQQP Q PQQ 19(4) 36 329(42) y PSGQVQWPQ 18 (4)

41 390(50) y _ QQTYPQRP T QQ 17 (9) 59 337(43) y Q CQQPQRTI 13 (4) 61 388(50) I QQPYPQQP T QQ 13 (3) Group 3: Homologues of peptide 355 , . FPQPQQTFPHQPQQQFP
17 355(46) 7 QA Q 27 (15)

42 348(45) 1 QQT 16 (10) 48 353(45)y QQ1 A 15(8) -50 349(45) y QQI 15 (9) Group 4: Homologues of Peptide 396 QQPFPQQPQQPFP
21 396(51) y TQQP QTQ 23 (9) 27 378(49)-y QQP QPQQ 22(10) 28 371(48) y PQQQFIQP TY 22 10) 29 642(82) co PQQP L QQP 20 (8) 30 635(81) to PLQP QPQ 19(5) 44 332(49) y QTQQPQQ 16(6) 45 629(81) co PFPQT S L QQ 16 (5) 46 643(82) co PLQP QQP 16(6) 60 634(81) o) PQQL L QQP 13(3) 64 387(50) y T L QQPQQPP 13 (4) _ 62 641(82) co FPEL I LQP 13 (7) Group 5: Homologues of Peptide 343 (overlap_Groups 2 and 4) _ QQPFPQPQQPQLPFPQ , 12 343(44) 7 QQP Q 34 (11) 16 393(51) 7 QLPFPQQP 27(8) 19 335(43) y _ QQ Q PQ __ 25(11) 23 385(50) 7 QPQQ 23 (7) , 24 375(48) 7 P Q PQQ 23 (10) , 25 406(52) 7 QP L Q PQ _ 22 (8) . _ 32 377(49) 7 P Q Q QPQ 19(9) = 34 415(53) 7 _ SQQP QS 18 (5) _.
37 413(53) 7 SKQP QS 18(4) 38 380(49) 7 QPQQP 18 (6)

43 409(53) 7 _ QP L Q...L PQ 16(2) 47 389(50) 7 _ T Q QPQQ 16(6) 58 407(52) 7 . , QP S Q PQ 14(5) 63 ._ 399(51) 7 T Q_ LQQP . 13 (5) 67 408(52) If QP SK Q PQ 12 (5) _ 71 379(49) 7 _ QQP Q Q P 11(5) 72 397(51) y PQQP T Q 11(3) Group 6: Peptide 625 .
PIQPQQPFPQQP
26 625(80) (I) QQPQQPFP 22 (9) , 57 624(80) a) FTQPQQPT 14 (6) , 65 628(80) (.0 , PF...W TQQSFPLQ 12 (4) ' Group 7: Peptide 618 39 ' 618(79) a) _ PQQSFSYQQQPFPQQPYPQQ 18 (7) Table 21. Bioactivity of individual tTG-deamidated Pools 1-3 peptides in Subject C12:
No. Sequence % No. Sequence 8 AVRWPVPQLQPQNPSOQOPO 100 23 LQPQNPSQQQpQEQVPLMQQ 26 3 M'VRVPVPQ H 17 KQVPLVQQ

,59 8 1VIVRVPVPQ' L 19 L EQVPLVQE

Core sequence of epitope is underlined. Predicted deamidated sequence is:
LQPENPSQEQPE

Table 22: Phylo genetic groupings of wheat (Triticum aestiyum) gliadins Alphaibeta-gliadins (n=61) Al al AAA96525, EEWTA, P02863 A1b13 B22364, P04271 A1a2 CAB76963 A2a1 AA1323109, CAA35238, P18573, A1a3 AAA96276 A2a2 CA1376964 A1a4 0AA26384, S07923 A2b1 P04724, T06500, AAA348282 Al a5 AAA34280 A2b2 D22364 A I a6 P04728 A2b3 P04722, T06498, AAA34276 Al bl CAB76962 _ A2b4 C22364 A I b2 CAB76961 A2b5 0AB76956 A1b3 BAA12318 A3a1 AAA34277, CAA26383, PO4'726, A1b4 CAB76960 A3a2 1307187B, A27319, S13333 Al b5 CAB76958 A3b1 AAA96522 A 1b6 CAB76959 A3b2i AAA34279, P04727, A1b7 CAB76955 A3b2ii 0AA26385, S07924 A1b8 AAA96524 A3b3 A22364, AAA34278, AAB23108, 061218, P04725 Al b9 CAA10257 A4a P04723, AAA34283, T06504 Alb10 AAA96523, TO6282 A4b E22364 Albl 1 AAA17741, S52124 A4c CA1376957 A1b12 AAA34281 A.4d CAB76954 Gamma-gliadins (n=47) Gamma-gliadins GI 1 a P08079, AAA34288, PS0094, CACI 1079, GI5a AAK84774, AAK84772 AAD30556, CA011057, CACI1065, CAC11056 Glib CAC11089, CACI1064, CAC11080, CAC11078, GI5b AAK84773 GIlc CAC11087 GI5c AAK84776 Gild CAC11088 GI6a JA0153, P21292, AAA34272, 1507333A

Clic CACI 1055 GI6b AAK84777 GI2a JS0402, P08453, AAA34289 GI6c 1802407A, AAK84775, AAK84780 GI21 AAF42989, AAK84779, AAK84779 GI7 AAB31090 GI3a AAK84778 _ GI1a AAA34287, P04730, 807398 GI3b CAB75404 G1113 1209306A
G13c BAA11251 01111a P04729 GI4 EEWTG, P06659, AAA34274 GIII1b AAA34286 Omega-gliadins (n=3) 0Ia AAGI 7702 Olb P02865 01 c A59156 , I69 Oddd bbdb bbd0 Oddd 70db 1791 V519 19 dabd bddd MOJA' Adbb Sddd 69 ZUZV
069 bbdb bibd ddbb dlibd Ad,bb179I 'KID 09 dlbd Odic! blblk Adbb Sddd 69 IEIZV
689 ONO OcLad bbdb 0.Lbd Adbb PEI VZ1D 65 &loci O&M
trIbl Adbb Stidd 69 IVZV
889 bodo b..1Lbd 3d00 dbbd Adbb PEI VI1D 85 dclOd bldd biO-I Adbb Sddd 69 Zia 1 V
L89 delb (IOW ddlb dbbd din 9Z1 V9I0 LS d-Tbd bridd blb-1 Adbb Sddd 69 I IHIV
989 bbab Oadd IbVb bdbd adbb 9Z1 ORD ' 8 '100d 589 oOdb Oddd lbdb Odbd Adbb 9Z I VSID 95 dlOd bSdcl b10-1Adbb SdAd 69 01E11 V
= OS -100d 55 dibd bddd bibl Adbb Sddd 69 IEI1V
1789 ddbb dbbd Adbb dbbd adbll 9Z I 1710 Pc d-lbd Oddd b-lbl Adlb SIM 69 I VI V
989 ddbb albd Albb dbbd dclbb 9Z 1 VC1D ES baba.
Adbb &al bbHd Adbb 19 VW
Z8E bbab tuba ,aabb dbbd ad00 9Z I VZ10 ZS biblkl Adbb &IR obbd Adbb 19 I EIZV
189 aabb dbbd Adbb dbbd ddbb 9Z1 V I ID 15 blbl AdOO Sddd bdbd Adbb 19 I Eft V
089 dddl bdOO dOdd (ION bbdb 11 YSID 05 dblb lAdl bSd.4 bdbd Adbb 19 EV1V
6LE clOdd dbbd bbdb aibb bdbb El I V51D 617 blbl AdlO &TM. bdbd Adbb 19 IYIV
8L9 bbab dabb babb dddb belb gal vgrn = L '100J
LIE bdbb fiddb bobb dbc13 000a L6 V5ID 817 Sadd bbHd Adbb dddll R000 5 (WV
' 61,100d Lt7 ScIdd bbHd Adbb dcIdO

9LE gorlb clbbd dibb dbda dbbd 68 V911) 91' &Ed Odbd Adbb dcldb 0000 ES IVEY
5LE obdb tide) 0.100 dbdd clbbd 68 3510 St Sdal bbba Adbb ddAd 0000 5 10ZY
tLE bdbb dbaa 000a Obal (IOW 68 V5I0 PP Sddd bdbd A.:TOO &HS 0000 95 r HIV
EL E Aabb dbbd Adbli (IOU 000c1 68 PID 917 bSd.3 bdbd Adob &III bbbo ES EV IV
ZLE Ode) dAIO Odbb dbid bbbd 68 Ef CID 917 SdAd bdbd xabb ddAd 0000 ES UV I V
1L9 AIbb dbbd &Ebb end bbbd 68 VEID It dbbd &PG
0094 abbb bbiAn tt atv ' 86100d ' 9'100d OLE MOO dbbd dabb dna bbba 68 VIID 017 dbbd ddba 000d Abbb oblAil tt VtV
69E glob ;Todd nod. bodo &lob IR V9ID 69 Adbb &TA) 0600 dAbb 00/11 PP 1Y9V
899 000d bbda dbbd bbdb ddb?) 18 VETO SE Adbb ddld bbbD babb abAl tt EEIZV
L99 dem end bbod obab aab0 Is mo LE Aabb dad 0000 adbb bbni tt IVZV
99E Al?)?) (lbw 000a Obab sib?) 18 VEID 99 Adbb dddd Obbo idbb bbn-1 tt zis iv 59E aabb end abod bbab a/06 IR Van SE Adbb ddaS
bbbo ldbb bbnl tt, 191I V
1799 aabb dna bbOd Obab clabb Is vim 1,2 Adb0 &fad bbbo -Hob bbn7 tt iv iv 99 dbbd bbib dabb bee dAib EL V910 EC 000d 6000 obi/51a./09 bdbb 99 VPV
= L17 100d = .. 5 '100d 999 (Ind bbdb dabb OdbH (13.1.6 EL 3910 99 0000 dab0 00na dAblIbdbd 99 ZV9V
199 000a bbab sabb Odbli dub EL VEID IC 000o dab0 00/17 dAb0 0<10<199 IVCV
099 dbbd bbdb dnbb bdbx coal) EL VZID 09 000o WOO
abni dAOH (MOO 99 CEIZV
659 000.d bbdb dabb babx da,L0 EL V IID 6Z 0000 Ebb bbKI dAbM bdbb 96 ISZV
85E baab bbab Hal badb adab 99 3910 89 000D dab?) bbNI dAbH WOO 99 IVZ=V
L59 baab bbab HdAI 00ab &ILO 99 V910 LZ bbbo labb bbnl dAba bdOb 92 FA Ef I V
959 ddbb OdbH dalb bdbd LLIdo 993910 99 MOD 'Hob boN1 clAbg bdbb 99 ZHIV
559 baab bbab Hal, bbab davb 99 VSID sz bbbo labb Obn-ianba bdub 99. 1911V
' 917 100.1 ' t 100.1 1759 HdAI badb ddlb bdbH HALO Sc 3910 179 bbOD'Idbb 00/1.1 dAbH Mb 99 1V IV
5 MINI ONO mit) babll HALO SS V910 EZ b0IA1-1 dAbU OcIbb bSdN OdOl 86 VtV
659 MIL Obdb d3Vb Odbld ddLO Sc V5ID ZZ Obnl anba WOO bSdN Md0-1 8? 1 CIEV
ISE &ILO bdbH 11410 lidbd 1E210 05 V9I0 1Z ObAl dAbll bdbd bSdN. bdbd 86 ZV9V
OS daV0 OdbH ddib babd dIbb 05 V510 09 00/11 dA00 OdOcIOSEIN odbd 86 IVEY
61179 Ebb bdbfl alLb baba 100 05 1710 61 gbA1 dAbH
0(00 bsaN bibi 89 589V
8179 d300 OdbH dab bdbd ILbb 05 VIID 81 30A1dAba bdbb bSdPI_Olol 89 ESZV
L'179 HALO Hdbd Illfb dbaD delb 917 3910 LI boAl dAbM
babb bsd/1/4/ ben sz I EIZV
= 517 =1[00c1 = '100d 9179 HALO lidbd Jib?) (1003 MOO 917 H910 91 ObAldAba bdbb OSdill Odba 8? MTV
gve = HALO Hdod II210 dObJ ddbb Ztr V9ID 5 I - DOKI
cIA6U0,1110 OS(11\1 OdOl SZ 1131 V
trt daLb bdOd d.LHO dObA adOO Z17 3510 171 00A1 dAb3 0h100 O&M odbl 8? ZV IV
9179 bbdd d-iod Obab clad() babb a, Eisto El bbAl dAbH bdlb bSdN 0d01 8Z [VI V
9179 dlLb bdbd 4166 doOS41.160 917V510 91 brabb bsdN
men bdAd ANAV OZ I HEV
IVE dal?) Oa:hi dIbb dbOS 3c1bH 917 VID II bdbd bSdN bdod bdAd A.21AV 09 I VEV
OPC &lib bdbd aibb dbbs '1<10091g WED 01 bdbO bScIN
blO1bdAd A.I1AI4I 09 506V
6E6 garb bdbd 3100 dbbS Ad:41617 VIID 6 WU) bScIN
bibl bdAd ANAV 09 99IZV
rt 'mod - 1100d 899 11110 dbg3 3,300 dbaa abbb 6173910 8 babb bsdN
ben band M.WAV 06 01E11 V
LEE 11110 d003 Adbb dbd3 dObb EL V910 L 0d00 OSdNI
OdblOdAd MAY OZ Sal V
999 dIllo dboA ddbb dbIld a000 EC DOD 9 Odob bSdN NM
INAS ATIAY OZ 8a1V "
SCE bd0,1 Adbb dbbd Oak! 6060 EE 1510 5 babb bSdN
berl bdA.I. AlIAIN 06 LEI I V
17E9 100<100s<1 abba Odaa 0000 cs Y5ID t bdbb bsda ben bd1Ald AllAIAI OZ 6E11 V
999 abb dot's dem dm abbb 29 1710 9 balb bSdN bdo7 bclAd KIIAIN 09 1911 V
ZEE abb abbs aabH abdd (1000 Et YEID 1 bdbb bSdN bdb-1 bdAd AlIAIAI OZ ZV IV
ICC Ebb dbbS Id?)?) abdA a000 EC YZIO 1 Odlb OSdNI
Odbl OdAd .411AV 09 IYI V
= Et '100.1 ' 1100<1 =olq aauanbas tuoppod ulawid =ON aouanbas ,uop!sod up;old sialuzy ulpe0 lump& umatui im &i1uuds sappdad 3pa1puSs -cz aiqui OgrZ0/090/13c1 LZt0I/0 OM

5517 21Vlb 0000 nIA/A MOOS 11d1.1 05Z WO SZI bbu bbbb bbbb bbOb bbOb sct t tow 175t OVlb 000b 0)11A1A boas wit osz No tzi . bbli bbab bbbb bbb WO TEl 9V I V
1St OVIO boob MINA boas uni OSZ V OD EZ I bb-ll bbbb bbbb nbb 0000 so Iv iv Est, Ova?) boob bum boas banns OSZ V110 NI 0000 0000 v000 me) baba SE! tvzv ' 15t boINIA boas 21d11 ISA1 SSA1 Z17Z Y910 1Z I
1000 Obbb 0000 staa bibs NT arty ost bbinin boas Ildll IAISA1 55A1 Zi7Z OSID NI
0000 0000 0000 slab babs sz I tviv 617t 01.11AIA )10(IS TICII INSA1 551(1 Z17Z V519 611 v000 stab baba Abe) ObdA ozi EEO/
= 89100d =
91100,1 ' 817t ' 01.11A1A
bYIS ddlI 1SN1.1 SSA1 Z17Z WO 811 Vbbb Sldb ben A111d21 00dA OZ I al EV
. LW' blIIAIA ODCIS I1d1.1 IAISM.1 SSA Zt7Z
VE1D LI 1 V000 SIdb ben AMdll ObdA OZ I I 8 EV
91717 OTIVIA 00CIS OdM1 ISAVI SSA1 Zi7Z VZI 9 911 NI060 Slab bdbd Abdo dbdA OZ I IVEY
WV blivv, boas bamr INSM1 SSA1 Zi7Z Y 1 ID SI I
Obbb stab Nibs Abe) sbaA Et tYZV
171717 1.1d11 I51(1 SSA SAI-11=1 0001 tEZ V9I0 tl I
0000 SIda bd6S Abdb dbdA OZ I 101V
It'!' 11d11 IAISA1 SSA1 SAdN 0001 ta Y5ID Eli 0000 Slab NOS Abdb dbdA OZ I WI V
Ett7 Ildll IAISM1 SSA1 SAd")I Dool ta YEID ZI 1 bdbd Abdb abaA abba dddb Z I I VtV
Ill' O&M ISM1 SSA SVO1 S001 VIZ Vt10 III bdbl Abdb OW abba aasb El I suv ' LS 100J = 51 100d .
01717 OdPAI IAISM1 SSA1 SAd>1 0001 tZ VIII) 011 baba AMU ObaA dbbd dISO El I MEV
6117 551(1 SAM 0001 IdI\DI DdNIAI 9EE V9I0 601 baba Afld11 ObdA dbbd aasb z t t lacy sct, SSA1 SAdN0001 "I.A.N=51 041=11AI 9ZZ V5ID ' 801 6dbd Abdb dbdA dbbd Said Z 11 IVEY
at SSA SAD! D001 1.31,D1 Od1,11 9ZZ VETO LOT
babs Abdb abaA sbba uaab El I lazy 9E17 SSA1 SVOI Sbbl aux 0d1\11 9EE VZIO 901 bibs Abe) sbaA abba liaab Et I ivzv cot' SSA1 SA.DI 3061 131,DI DdNA 9ZZ VI ID 501 bibs Abdb abaA.abba _T.actb Eli 98 I V
171=17 0001 -HINDI DdNIN 0001 aka 8IZ V9I0 1701 NOS
Abdb dbdA dbbd lddb ELI 1V1V
117 0061 -TANN OdNIAI bbba AaN 8IZ V510 01 bdAd bOdd Ad01 bdAd lbdd 901 Vt7V
= 99100d ' VI
100d . Eli' 00011.11µ11. 0d1\11 bbba sbN TIE VIM
ZOI bdAd Obaa asba baAa abaa 901 I GEV , !Et SOZYI moi oama 0001 saN 8IZ VZID 101 dbdb dAdb OdSd dddb dAc11 901 IVEY
- Kt 0001 latix DdNA Obbi saN TIE vuo oot abab aAsb baud abab dAdl 901 I UV
at DdNIAI 0001 .350I va00 Obaa cut V9I0 66 dbSb dAdb bald dbdb dAdel 901 IVZV
SZt DdNIAI bbbi AdbI Idb0 Obad 01Z V5ID 86 Mad ddbd. bdAd -Ibdd dabd S8 ow LZ17 0d1\11 0001 sON 'Nab 00dd OTZ V10 L6 Odbd Aa00 dada bibd Adlb 58 VtV
9Z17 DEINT0001 &IN &tub bbaa otZ VETO 96 Obaa asba OaAa abaa AdOd ss MEV
5Zt DdNIA bObl SdbI 3ddb bbad OIZ VI1D 56 Obda 3S0:1 OdAdlbal Adbd S8 10V
= 55100d . tZt 500IrlScib bOdd Sood 00db ZOZ VIM 176 bbdS addd bdAd1bdd ddbd S8 I VEV
at, saN Laub bbaa sbba bbab zoz VETO 16 obd21 ddbd bdAd rIbtdd adbd 58 1741ZY
ZZi7 sabi ArTab bbaa sOba bbab zoz vuo Z6 dbdA
abba ulab abaA alba 58 stazv let abra 1000 Odds Obab OdOd 1761 MD 16 dbdA dlbd bdAd lbdd ddOd 58 zazv Ott abn abbb ball bbab baba t6I YOU 06 dlIdd baba Adlb dbdA alba 58 IEIZV
6I1' bbIl Sdbb WAS nab baba 1761 tID 68 6161 Odbd 1610 dbdA 6106 58 1VZV
8117 obda Sbbd bbab 6100 baba t61 VETO = El 1006 Lit bbc1.3 sbba bbab aabb Odbd t61 VETO 88 606>1 adbd 051610.m AdOd 58 Z10 IV . =
' 175 100d L8 IOTA ibbd xaab absA
slOa 58 901V
9117 ()Oda sOba 00:10 a.abb bibs 1761 VI ID 98 dbdA dbbd Ilddb dbSA S1bd 58 PO IV
911' OdIS 00db bdbd aabb abbs 981 D510 58 dbdA
dbOdlIddb dbll dlbd 58 111V
t It Nod OM 06011600 6001 9BI VS1D 1/8 dbdA dbbd 21.40b dOSA d1bd 58 9V1V
Lit baas 0060 baba 6600 ems 981 MO 8 dbdA (Ind Ildab dbSA dlbd 98 IV IV
Zit bbab aabb baba aabb abbs 981 VETO ZS bald 10dd adbd Od.dd bdbd LL OW
II!' bbab a100 baba aabb abba 981 VZID 18 dad(' tuba Aaab &rad baba LL \ivy oil' bbab a.abb bads 6000 abbs 981 Y110 = 11100d 6017 bdbd labb db01 badd bbab 8LI VSID 08 bald lbddadba badd 0101 LL 1V
= 5100.1 6L
bdAd lbdr1 ddbd badd 0101 LL tav 8017 baba 6600 dbMS bald bbab 8L1 17ID 8L ' 113db clod/ 6106 Ilddd 0101 LL SUZY
LOP baba aabb abbs bald bbab SL! vao LL baAa abaa LIN bddd 0101 LL tilZY
901/ baba aabb abba Ociaa bbab sLI VZID 9L Ilddb dbdA dlbd Nal blbl LL 0ZY
. 5017 babs a000 abbs baaa nab 8L1 V [ID Si.
baAa -1066606 Nal bibini LL zazv trolg abbs Wad bbab baba aabb OL1 V910 PL Adlb dbdA
drIbd bad blbIAI LL I FIZV
1017 dbOS bad 0060 Odbd1(100 OL I 0510 EL AdlO
dbdA dlOd Oddd 0161 Li I VZV
ZOt . dbb1 bad bbab baba aabb OL I V510 ' OI 100d 1017 dONIS bard Obdb Olbd mob OL I VID EL 0516 ibaa aaba Oldd 0101 LL ZIEI 1 V
151006 IL 11660 doSA drIbd Mid 0101 LL 1101V
0017 6005 badd bbab 01066100 OLI VIII) OL )1.3db dOSA dlbd bSId blb1 LL 010 TV
66E dObl badd 00db WM 3,30b OL I VETO 69 )daab absx saba baaa baba LL 1'H IV
86E dboS Oddl 00d0 bibd ad00 OL I VI ID 89 21.3d0 dbrIA alba bald blbi LL 10 IV
L61 bald bbab biba aabb abOa 991 VETO L9 xa 00 absx alba baaa baba LL 1'V IV
961 Olbd ddbb dbbd aabb abb.', ss I VZID 99 113db JOS.& d1bd bd.dd blbrl LL IVIV .
, 561 dbbd aabb abbi bald bbab HI VZID 59 ddbd 0,1,4d 0.106 MOO ScIdd 69 EItIV
t61 abb.'. bagri ONO bald bbab Et! vzio = 6 '100d . 161 bbab baba aabb JON nab tEl V9I0 179 1610 &Ida Nod Ad00 Sdad 69 V1'V
= 15100d 9 dibd lidad blorl Adbb Slid 69 5EZV
Za and bald 00d0 OdddlOV017 I 0510 E9 Adbd bdad bibel Adbb &lad 69 t1ZV

Ogra/09-9/13d fLZt0I/0 OM
30-31-t0OZ 8T388T730 'VD

L9100,1 ' SZ 100a , 815 dAND IA1141 LDIA "ISIII A310 LEE VSID 881 HIV() 311SO MO 1100 D310 0E lad LIS' AAND IALIN1101A Sail nalb LEE Dab L8I Hrvb 0-850 3,30 M100 Da30 0Z I EIZV
91S AAND TALLY-ILO-IA ism naib LEE VED 981 111V0 31150 adib in100 3310 az Ivry sts AAND TAISal .LOIA -MR V3100 LEE YOU 98I HIV0 30-1,1 0410 M1110 3310 COE 01111V

DbSb 0110 AVIHO D310 OK 901V
EIS .1311A ISIII 0010 vabb 400 6ZE V9I0 81 Fint0 0bsb 3410 isAlHO 3313 COE I VI V
tic 1111A 16/31 A2A0 Vd00 400 6ZE 3910 ' I7Z 100d 115 101A Sall A310 V400 doll 6ZE DOD Z8 I 3410 11100 3310 bibb Ass() 561 V17V
' 991004 181 2410 '1100 D310 0110 ALSO S61 1 El ITV ' 019 .1.01A ISIII Aalb Vd00 d011 GEE VEID 081 all0 Ak100 3010 01 JO ALSO 561 I VEV
609 ..1,01A "1ST VE10 Vd00 401! 6ZE VI ID 6/1 3410 805 0310 Vd00 40II 0190 VIOd TEE V91D IL! 11410 m100 3310 0A10 ALSO S6I I VEV
- LOS A310 Vd00 (10I1 DODO A10.3 TEE VSID Li! 11410 4111-Ib 331a 11110 ALSO 56I 801V
909 VE10 WOO d0II 0000 AlSO t ZE VEID 9/1 3410 MHO

SOS V310 WOO dill DODO MID lZE V HO 5L I 0d10 tA1H0 3310 0110 ALSO S6I I VI V
1709 - 40I1 DODO A110 ODA0 0005 El E MD " CZ

EH 40II DODO AII0 00A0 bbbs LIE VEID tLI 3310 = 59 1004 ELI

ZOS 4011 DOD0 NISD 00A0 000S El E EIZIO ELI 3310 0140 ALSO 01AO 511 VII LSI 1.VV
109 d011 DODO AlSO bantratths LIE 'RID ILL 3310 0A10 xisa 01A0 SSDH LSI ESZV
005 dOII DODO AILD 0DA0 000A El E VIID OL I 3340 0.A10 ALSO blisb 55011 L8 I I SZV
661' d0II D100 V-10d1d-111IdAD SQL VOID 691 3310 0A-8617 doll DODO A=1071 Ian 1010 SOL 3510 891 331a 6110 ALSO 01A0 S)IDH L81 118IV
L617 40II DODO A103 1.11.11/11010 SU VSID L91 DD1N
11110 xisb MAO SIIDH L8 I 881V
9617 A1I0 ODA?) 0005 1dAl IOAD SOE 1710 ' ZZ 1004 5617 AlID ODA0 0005 IdAl 10A0 SOL VETO 991 ' 1791004 591 3310 0110 ALSO 01A0 SIIDH LSI IV! V
17617 AlSO 00A0 bbbs 1414 0111/0 SU EIZID 1791 ASSO
01A0 SSVH VINH Obin 6L1 V17V
E617 AlSO banb 31-105 14113KII9 50E VZ1D 1)!! ALSO

Z61' Al,LD ODA?) 000A 1411111ND SOS VIID Z91 ALSO

1617 0A10 31411 vuOr o000 0000 86Z VSID 191 Aisa 0-1.A0 SSDH VINE 001A 6/1 ECIEV
0617 loby /On (111114A00 0030 06E V9ID 091 ALSO MAO

680' 000A 10E1 &III blob 0030 06E 3510 651 ALSO

8817 mbi 0000 0000 0000 00H0 06Z VSID - LE '1004 L8t 000s 14A1 TOAD buba 0030 06Z 1710 851 ALSO
01A0 511011MN.11 001A 6/1 1181V
= 91004 LSI
ALSO 01A0 SIIDIIYINTI 001A 611 OISIV , 9817 Obbs 'IdA1 TOAD 0):103 0030 06Z VEID 951 ALSO

5817 00S1d1d1HIAIDO 0000 0000 06Z um 551 ALSO 01AO

, 17817 HOS1 dial mob 0000 0000 06Z VEID 1791 ALSO
01A0 STIDH V}11,111 001A 6/1 EV I V
E8t 000A 1d11 limo 000a 00ab 06Z V110 ES! ALSO
01A0 SIIDH 'VINE 001A 6/1 IV IV
Est' ant dA00 0030 011III SOYA Z8Z V9I0 Z5 I , SSVH
VINE 001A AGED drib ILI !UV

STIVH VINH 001A AMID drib ILI IVEY
0817 0000 0000 00ab OIAIII SHA1 ESE VSID ' ' , VINH 00-IA AG113 d110 IL! EERY
19 'mod 6171 6500 VISH 001A AMID 4110 IL I IEZV
8Lt. 'Ono baba 0030 OY\III MAI Z8Z VEID PI SSDA
VISH 00-in AMID 1110 ILI IVEY .
LLt Glob 0000 0000 bmi SHAA Z81 VZID Lt I 631011 S11011 VINH 001A AMAID dI10 ILL 01111V
9/17 IHIAID 0003 0030 bvoi SHIT Zia V I ID 3171 .
51100AINE 001A AGIND alb IL! LEW

ELI' 0030 OIAIII slim pm vDOita YSID 171 SIIDH
V}INH 001A AGIAID drib ILI EV I Y
EL17 0030 01-SIT slam. sinv v00-1 viz I710 = 611004 =
I Lt bbab OIAIII SHAI SHIV V30117LE VEID E171 -511011 VINE 001A AGIAID 4110 IL! IV IV .
.
191004 1171 0,01A AMID 41100011 0014. 91 VI7V
0017 0000 01A111 SHAA SHIV v301tra VEID 0171 001A
AMID 4110 0011 Odell E9 I EVEV
. - 6917 0030 011III SHIA IHIV V301tLE E 1 ID 6E1 8907 Oloa0 01AIII SHIT .LHIV V301 tLE Y110 8E I
001A ACIIAID d110 0011 0011 91 OIEIV
L917 SOYA SHIV vobl 0041 0v-10 99Z Y910 LEI 001A

9917 SHAA SHIV V301 bun 0V10 99E 3910 9E1 001A
AGIND 4110 001I 0011 (91 IYI V
9917 SHAT 9111V V301 00dI IIVIO 99Z VSID SE I drib 0011 0011 0000 0000 9171 VtY
.
091004 = 811004 17917 SEAT SHIV v301 0041 OV-10 99Z VEID 17E1 4110 0011 0411 0000 0000 9171 ZVEY .
917 SHAA SHIV VDU' 0041 beib 99Z VETO LEI
000110011000 n00 0)100 9171 IYZY ' Z917 SHIA IHIV V30100,11 bvib 99Z 0110 ELI 4110 I917 SHII IHIV V301 004I 0V10 99Z V HD ILI drib 0011001! bbab 00009171 OIEIV
0917 V301 0041 OVIO ODDO 001A1A. 85E VOID 0E1 dI10 0011A1 0011 003.0 0000 9171 90119r 6917 V301 011.41 0V-100330 001,AIA 85Z DOD 60 drib 00=II 0011 0030 0000 9171 9V1V
8907 v001 0041 Irvib 0030 031NA 8SZ VSID 8E1 4110 LS17 v001 0041 Ovlb 0330 buyin 89Z VIID LZI
00110000 0000 0000 000-v 6E1 017V
= 651004 = LT 1004 9517 bvib 0330 bblAin 03CIS1141I OSE 33I0 90 1000 0000 03100 0000 0000 SE! ivzv IOI
OgrZ0/09-9/1.3c1 fLZt0I/f 0 OM
30-31-t0OZ 8T388T730 VD

Z85' cISOO dlAd 0000 Sddd 0000. L6 VI HID ' 1100d , I2S 0000 Sadd 0000 SAdd 0000 68 VI HID 151 OV0d KOOS db3S DODS dAbb 10 IVZV
' SL '100.1 I SZ OVOS NObs d113S
D601 dA00 10 ZEI1 V
ass 0000 S3dd 0000 INNI IldIN II VI HID OE
OvOd mObs d213S 0001 dA00 10 !VW

Sb3S AODS dAba Odb0 ASA0 61 VW
BLS Hd11\1 MINS &LIE A03N NNdS 59 VI IUD 8I7Z
dbdS DOSS dAbb ?Mob dSAO 61 ZEIEV
La dIIIH Abdtµl NNMS NINI-01 NS,IS LS VI IUD
Lt7Z dbdS AbSS dAbo babb dSAO 61 111EY
9L5 NI\MS NNHH NSdS NINNd SININN 617 VI 1110 9171 dOld 0005 dAbb odbb 1SAO 61 'WV
SLS NSdS NNNd SNNININIGIN NHOd IV VI 1110 51,Z
doll DODS JAN) bdb0 ASA0 61 SSIV
17LS SNNIN1 N131\1 NHOd SNNN SAdb EE VI 1110 ' 1100d ELS NHOd SINNN ?IAA?) NNNS HdliFI 51 VI 1110 1717Z dOdd DODS dA00 Odb0 dSAO 161 111V
' pL 700d 117Z dOAS DODS dA00 ZLg 1IA3b NNNS HdHH NSNS 1141114 LI VI 1110 1.171 . dOAS DODS dA00 0360 dSAO 61 I VZV
I LS HAHH NSNSIEHHININI,IS Slid 6 VI IIID 1.17Z
3213S 0001 JAZ/6 bd00 dSAO 6Z ZS IV
OLS 1131111NNNS Slid Ida ULM I VI IIID OK
altIS 0001 dA00 rldbb 3 Sdb 61 9V I V
695 AVDA OIDA DMA SILL -21Ald SE HD 61 dILIS
DO01 dAbb -AO dSAO 161 IV1V
891 93dA SILL 11Ald ANAS atAuci LIZ 110 811 AbSS dAbb babb asAb SSdb 681 IS EV
L91 YAld ANAS Kid 1011 VIS.1 601110 LE DODS
dAbb bSbb 30A0 Sldb 681 IRV' 995 JINJA 11111 VISI IAIATI bYlb 101110 ' If 100,I
591 VISA IAIATI bviO IldbIlL0b 61 110 9E1 DODS dAbb 0,100 dsA0 Slab 681 1VZV
' EL 100d SE Odbb dsA0 ssab 1795 0V10 Hd01 IMO v-TOO 00db 581 110 VIZ 33A0 51cIb 0000 0000 baimd 091 ESZV
95 AIDO VIbb bbdb 0010 00S0 LL1 110 11 0s00 dan0 slab 0000 0000 091 I E1ZV
195 bbdb bol) Obs0 00d0 SADO 691110 1E1 0,100 dsA0 slab 0000 0000 09Z I V1V
195 00s0 bOdb sA00 0-100 d000 191110 IEZ .-SAOS Sd00 00)10 0000 1006 Ill FISINI
09S SAD0 Dibb d000 OS01 SOOA 51 IID OE 60)10 6SS d000 0S01 SDOA 000a 010 St7I 00 611 dAba odOb ASAO SSdb 0000 scz vv = Zi. 100d = OC 100d 815 SDOA 0006 b-al am VIIIV LE I II0 811 0000 LSS 0111 SAII 'PITY ONUS 00d1 611 HO LIZ SdAO
Obab 0'15A 0S5d obblt SE IVEV
955 IISA IIVIIIvaAlis00 diOd az' I1D 911 1000 555 vlav bAus bOdi Odib boob 111110 szz 1660 0000 0016 0)1011 0000 scz c t e IV
1755 bbdI 0310 0000 bolAIA MSS 11110 till 'MAO
00d0 03SA bssa 0OZDI 1E1 OI E I V
155 0330 00wA HDSS ObilAl 0SIIV SOT II0 EN
dA00 OdOZ) ASAO SS'10.006)1 SE ZEIIV
Zgg 14355 bblIAI 05211µ1110dIAIVAd L6 IID ZZZ
dA00 id00 asiO scab 00011511 9VI V
ISS 0SIIVIII0d IAIVAd 530b 0l3A 68110 I ZZ
dAbb 1100 LISAO Scab 000)I SEZ IVIV
= IL '100d = 6Z 100d 055 . IAIVAd 5300 OITA )13(11\11bbel 18 110 OE
MAO S5d0 0000011116 11IV LZZ VI7V
6171 01.3A SINN 7001 1Sdb AAdI IL IID 611 0010 Oxba 0000 bOtad wav LZZ 1111V
8175 1001 1Sdb AAdl 000v ibbH 59 110 8IZ 0006 060-i 0000 RbOH MOW LZZ ISEV
L175 AAdI 000v -100x 0000 badd Lg IID LIZ 015A
bSSd 60011 000H 111V LZZ IVEY
9175 "ibbIl 0000 Od3d VSid .)10-VS 617110 911 0010 b)102 01-1116 HOOH111V LZZ 17111V
" 5171 bd.dd VS..14 )IDVS dIII dllid 117110 SI Z 0000 0000 OHHH H001-I 'TIIV LZZ ESZV
I7171 31DVS din diVd sub nAbb Is 110 I'll slab 0000 00000001-1 lav LZZ tvzv Etc diva sub nAbb dOds dbba sz IID I Z 0010 0)10a 6006 000HTIV LZZ 161 V
= 01.100d =
81100d ZI75 AA06 dbdS dbbc1 0013 dbOS LI IID lit OASA
OSSd 000x 0006 II1V LE 01E1 I V
It'5 dbOd 001d dbbs 0m-id sbOd 6 IID Ill ' .3SA0 SS1b 000)1600H111V LE ZEIIV
0-17S dOZ)S 0.8i1d SbOd 01dd cIbbd 1110 011.
S3OS SdOb 00)10 0001-111TV LZZ. 9V1V
619 OD DIDV Q1NV AdANIISD 191 V9ID 601 3SA0 SSdb 000>I 000H 11IV L11 IV I V
815 60 SIOV, AISV AdVN IISD 191 DSID 801 0000 LES OD DIQV ANY ddV)IIISD 191 VSID LOZ ' . 915 00 DISV AISV .3dVIIII53 19 'KO 901 0000 11001-I wily HAAN MVO 611 I 01V
SE g 11100 OIDV AISV .3dVII 1153 191 VEID 50Z .
- ObOu 000a 'Ind HAAN HIVO 611 IVEY
' 69100d = a 100.1 I755 OD DIOV AISV ddVII IAIISD 191 VZID 1701 Z)HHH 14006111V HAAN HIVO 611 ESZV
Etc 00 DIOV AASS AdV>1 1153 191 VI 10 COZ 0066 006H ariv HAAN HIVb 611 1ISIV
115 QINV AdAN ',LSD laddA AAND ESE V9I0 ZOZ
000x 000H111V RAIN HIVO 611 6/3 IV
III AISV AdVIN USD Q/121A AAND 51 3110 101 000)100011-my HAAN bivb 611 EE1 I V
0E5 AISV adVX LLS3 HddA 3AND ESE VSID '00Z

615 AISV &PM IISD AddA AAND ESE YOU 661 000)1000H 1IIV HAAN HIV() 611 I.VIV
El, AISV 3JY-a BlISD addA AAND ES1 VZ1D 861 111V HAAN MVO also ac.110 Ill vt,k, LZS AASS 3dVX IISD addA IAN) ESE VIM L61 IAMV
RYAN Hivb o0s0 aai0 Ill CIICV
= 89 100d = 91 '100d 91c USD (IddA IAN) WITT I)I1A 9171 VIII) 961 -ary HAAN favb ousb sin Itz [WV
szs USD CMITA AAN0 YiNdl III1A 5=17 C DSID 561 111V HAAN HIVZ) 3013I 3dI0 Ill DI 11 V
17ZE 1150 HcidA dAND JAILdliOlA 5171 YOU 1761 II1V HAA3Imv0 obis?) 6r110 Ill 6ER V
ES USD AddA AAND PUN/ iolA 5171 VIII) 61 1IIV HAAN MVO obsb glib 11Z 9E11 V
NE LUSO addA IAN) IA1Sdl IblA 517 VZID 161' 111V HAAN 01Vb DOSO Ralb Ill 1131V
IZ5 ITS) HddA IANDIAILdliolA 5171 VIID 161 'IIIV HAAN atv0 00150 ad10 Ill Pill v 015 IAN) TALId11011A 1SV 0610 LEE VIII) 061 619 IAN) IAINdl 1217.1A15111A6A0 LE/ DgID 681 Hiv0 3050 gaib -1-100 0)10 COZ COZY
ZO I
OStZ0/c0113/13.1 ELZ170I/0 OAA

9179 sris saw bOba Adbb <IOW LSZ VI 0 91 '1000 imb.A. ODsa cti\br NVID Li VZID
S179 Obbd AcIO0 dbOd AS00 dbOd 6179 VI 0 91 aObb div,bA bssd CAOIAI NV1D L I VI ID
8 700(1 It lood 171'9 abba asbb abba 4dbb and itz vi o 1'1 NANI DEO ddVI IISD AddI OS Ut7V
179 dood Adob (ION id?)?) doll EEZ VI 0 Elf MID SID3 dVII ISDN &HA 617 MEV
91,9 dna aabb db-ta gabb dbba szz Vi o 91 c ' RID AIDA ad-U.1SM ddlA 617E !BEV
It9 and aabb abbd tabb Tada Liz Vi 0 1H
NID AIDA dYLL ISDA ddIA 6ti ZVEV
0179 µ100d Iibb lad -100d Ovad 609 VI 0 OIE
NW AIDA dVII ISDA ddIA 617E ZE1ZV
. 6E9 -odd IOW blidd 0050 OS?)?) 109 VI 0 60E
IAN 1DAI DAdV LOA dill 617 IVZV
8E9 OVII bOSO bsbb .14s0 ORA E6I VI 0 80E
NI IDA! Dail [IDA ddIA 617E 601V
L9 OSOO attsO babn oat) baba 591 VI 0 LOE
NAN IDA! DAdY IAILDA ddIA 617 SGIV
98 MEI 017109d 9E9 odbA did?) bdOd ddOb dbOd LLI Vi 0 90E
NAN 10d1 DAdV IAIVDA ddll 617 19E11 V
' 5E9 Nod Ain db?)d Ado?) tIold 691 VI 0 SOE
NAN IDAI 03dA IIDA ail/ 617E 10I6 179 dbba aabb dbid adbb ibbd 191 VI 0 tO
NAN IDAI 03,3V 'IDA dill 617 IV I V
9 dbad adbb ibba tiba 900d Est Vi o coc dV11. ISDH ddIA ANDIAIVI/L It7E lad 9E9 -IOW llba sbba adib abda stt vt o zoc &VILMA ddIA. ANDIAI Nall 117 ZVEV
1t9 900a adib abda db0a ?)Odd LEI vi o 10 dVIIISDA ddIA ANON 11d1J, 117 IVEY
0E9 dOda d0bd 00da dbod olcILI 6ZI VI 0 00 iVII ISDA MA ANDIAIWIL 117E ZEIZV
6Z9 bodd dbbi Old3 5001, Wad 191 vi o 669 0/A.:WILDA ddIA ANDIAI Vd1.1. I t7E IVZV
18 100d = 6 Ptmd 899 0-Idd SOW. Oddi Obdb /Mad 11 VI 0 869 Dddl IIDA MA ANDIAI Vc.111 117E 601V
L19 O&M bbdb Mddd bbdb did) 901 VI 0 L61 DdiV IANDA ddIA ANDIAI Vd-11 117 1701V
999 AkddcI Obab die/ Odbb JON L6 Vi 0 969 Jail. IIDA cldIA AND111 \rill 117 101 V
S19 ditto bdbb dbbd ddbb dbId 68 VI 0 569 DidVIIDA ddIA ANDIA1Sdli It7E EV I V
1799 dbbd adbb dna id00 dbia 18 Vi 0 1,69 DAdV LIDA ddIA ANDIA1 Vd-11 117 IVI V
99 JOIN MOO dbid dbOS bactd EL VI 0 6Z
ddIA ANDIAI 1.1d71 01V1 N2112 E I VEV
999 ' a:13 dOOS bald Obod IdAd 59 VI 0 969 ddIA ANDIAI Vd11 TIV1 N2113 EEC 1 VZV
199 bada 0001 IcIdd 00Sd Ad?)?) LS VI 0 16Z
dill ANDIAI Sd11 0-1V1 N2113 EEC (Vi V
08 100(1 = = 8E100d 099 UR bbsa .kabb SdAd bodA 617 VI 0 069 &HA ANDIAI WM 07V-I N2113 EEL I VI V
619 Adob ScIA.d obdA dbOd ddob It VI 0 689 VcrIL O-TV1NITTR babd 'ION SZE VbV
819 ONA dbbd gabb bASa sOba El VI o ssz vd-a bivi t\nuu vabd and SZE I REV
LI9 ddbb USA sbOd 90191 )11.15a sz .vi o Lsz Vali 01V-I NNW Udbd Ibbd SZE 5EIZV
919 SOW Sb-In )1I\ISd NrIalIVVIV LI VI 0 989 vdii TIV-1 NNE adbd 100d SZE I VZV
919 Ad00 bOSA ant) Sild bbOS ti. Ell HID 589 MIIVI INIIIIa=adOd IOW SZE ZIEtI V
1'19 din Slid 0009 Add0 ObS.3 99 111 1199 189 TAIIdl 101Y 1N211 adba -100d sZS 9V IV
19 000s Jae) Obsa sa00 dub sc al MD 89 lidlI 01V1 NNE 3,30d 1bod SZE IV I V
6L lood LE pod , 9I9 boal sdbb a-tab bbbb sada os HI 1119 zsz N.Illa babd -100d 0199 bv0d Lit Vt7V
119 die) nob SAdd bbsa sbba 917 Hi 1119 I 89 INNIF vabd ibbd bAso Ovbd Lit IEIEV
019 SAdd obS3 Sbod &Mb 00/Yk.d it HI IIID
089 MEV adbd lbod 0..ISD bvbd LIE 5EIZV
609 SbOd aidb bbmdlIE-10 9135 99 HI MD 6LZ
NNIR Edbd -100d bASD bV0d LIE 149V =
809 AID ADID ADAd ASIA VSKI COE VI MD SLZ
/µ12112 3A0d -100d OASD OVIld LIE t/EtI V
L09 Aadd ASIA VSK-1 dANA SDKI. 969111119 LLZ
Rum Raba -100d Ons9 000d LIE altv 909 VSKI JANA SMALL d11-21 TVIS L89 VI MD ' 9E 100d 509 SDIAII d11217VIS inva liivi 6L9 VI HID
9L9 1,11.113 adbd ibbd bASD bVld LIE 101V
8L 100d. SLZ INIII adbd Ibbd bAso bvOct L I E 9VI V
1709 1VIS IAI9111-1V1 oHdo 'HID I LZ VI IIID
tLZ - NAM Raba ibba bAso bibd LIE EV I V
09 'MI blidb IUD MAO 0006 99 VI IIID ELZ
NUM aibd lobd bASD bV0d LIE I VI V
909 1.310 MAO 0000 bbab 09-1b L99 VI MD ZLZ
100d 01110 0vbd Nbbs SOAS 60E Vt7V
109 0000 00a0 09-10 bbab OASD Ott 11 1119 1LZ lbOd OASD OVOci NObs ems 60 -MEV
009 0o-10 00d0 bap 0910 0050 119 vt mo az 100d 0159 Ovba Nibs ems 60E lEIEV
665 OdSD 0010 OOSO 0090 9/90 9E9 vl HID 699 - 100d OAS 0v0d Nibs dbas 60E !GEV
869 bOSO bon SAD?) DSOO dbOb SZZ VI HID ' sE ma LOS SAD?) Os?)?) dbbb bab/ A900 LIZ VI HID
899 ibbd bASD bVbd NObS dbid 60E 11EV
= LL 100d L99 Ibbc1 O.ISD bYbd Nbb..4 dbda 60E sazv 965 dbbb babn aobb babi IISA 609 VI IIID 999 100cT thaso bvbd Nbbs dbaa 60E MEV
565 30Ob 0301 IISA. MI IVRA. 109 VI 1110 599 ibbd 6159 bv0d NbOs dOES 60E IVEY
'MS - IISA. IIVN IV91A1111091 d100 E61 VI
HID 1791 100d OASD 0V1Ici MOOS aliS 60E bill V
65 11911 115091 dibb Ina DObb 581 VI IIID
99 '100d OASD ba0d MOOS dlliS 60 111 969 at00 1003 3?)?)?) rAt/H9 990b at VI IIID
999 100d 0/99 0v05 MOOS dlIAS 601 9E11 V
169 DObb IAIA.HD 9500 mnbs Irrni 691 VI HID
I9Z lOOd OASD OLOd MOOS dlIZS 60E EVI V
069 9900 mintbsuv-9.1 OdYµIV AdSD 191 VI HID
' FE 100(1 685 = ITTRI OdIAIV Ad50 0001 AAND 91 VI HID
099 Ibbd bASD bIbd NbbS (11.13S 60E I VII
9L 100d 659 lbod bASO WE NObS dlIES 60E IS I V
885 AdS0 00011AM dN10 blAS 5171 VI HID ssz bvbd Nbbs sbas Abos 11091 roc v6'v L85 AAND dN10 blAS dbAI alb?) LEI VI HID L5Z
bvba Nbbs adds obss dAbb toc 9EIEV
98c 01AS d0A1 dibb ON-10 0006 691 11 1110 999 OVOd N'IOS dOIS AOSS dA00 10E 11EV
S8S MOO bAlb 0600 badi A-Ibb 191 11 1110 SSZ
OVOd MOOS d0.33 DODS 1100 I OC IVEY
HS 0000 baa11100 bbsa d900 El I 111110 VSZ
0101 NOW glad obDs dA00 tot cant HS 1100 OOSA dS00 crIAd 0000 901 11 1110 Esz bvba Nbbs dbaa obos dAbb toc Env COI
, OgrZ0/09-9/13d fLZt0I/0 OM
30-3T-t0OZ 8T388T730 ID

Pool 42 GI2A 25 PSGQ VQWL QQQL 'VPQL QQPL 324 014 25 PSGQ VQWP QQQP FLQP HQPF 326 =

*Position of N-terminal residue in a-, yl-, 72-, y3-, or co consensus sequence nsdaodOaxabOddadOOODEv 6666onex6M6636000609' abOaaauaMpaa6000014,19 doOdaa0a009,13o0000mor, xd0DaazOMODda0000m6 xaOndaanDODOzOnOinec . II ______________________________ 11111 - ' 1111 1111 Adaoaaaa000bda0000AaLc xaboadadOODDriADOODArlec 111 ____________________________________________________________ ___________________________________________________________ xdbOdaasObaouONDAqs0 AaDbaaaaWouoDOODAripe 000636ODOOmaA00,160cs bOODasa500AazADOdCau DOODea000DAuA0D0anau DOODO3bOX0A7,3A02C3CCOC
000962bDWA72A0XCd0C60 ObOal.a0000AaaA0.0aMez 00 DriabOODAuA02baMu, 000D71MDDAlaACOa0Dez DOODriaDOWArmA020aROgz ' . ".i. I . .
' I I 1111 00C07,0000AugnOdaDvz DON7dAbsbeMsaNDasricz 00.ividgManbOsammaDrIz0 00indA0u0d0ansdNoaDaTz 66manzendadosaNnabaoz 2bArld.02C,abOosaN0a016-c mmdA0s6ann0sanD10r1e1 ODArlaAbOaMbsdN0d0aLI
06K1mO60a000saa0nI91 DONLIAO303HMS,IN0a0701 60A73A03C30006361030761 , 00.A7dA03Cd700630,507E1 DabbpSdNN.30703/13A6TAVZT
0.3080SENC30,30,3A3AENVTT .
; 11111111 1111 DdODOSdN07D7C2A6MAW01 66000saND10106AaA6A,ff6 ba0W6allOa0lOandmi3m3 1111111111111111111111111111 , 0,6000saNannandAuAliz DaDDCsdNaicribansAliAv9 OaNOsaNbernaAINdAws Oa=sandOlOaNanump Cd1100030,16703A2AAWC
111111111ffil 0,61006660ffOnamAuAwz nd76063ND3C770dAdaUAVT
. . apuanbas avpdaa , . .
ZTZLZE9T-TES'OPZT
. . DSS /036273 600 Zg 91 OI Dg 600 601 LS 60 901 610 561 96 96 COZ
DaS I066I72 ECl/d3d INV6126I0C1 'i0E-10-0'0CCEC9C90 -C (SWG) n61377'00 30 610II10900 2E6 1.11296 20N2TIVED

, - XZ XZ xiez ez -- xi xe- xi xZZZ -ZZ zz zz zz zz 00-16au 230 Lb D 900 000 900 EZO ZZO 100 000 610 810 LTD 910 SID VID .10310E06 01617200 - _______________________________________________________________ Aualluffa ualni2 pile swaged Ippek stsifiguv lodsna pug sappdad agarpuAs zsg .pz aiqui sO I
OgrZ0/09-9/13d fLZt0I/0 OM
30-3T-t00Z 8T388T730 YD

. 106 . .,..

44 GQQQSFPPQQPYPQPQPFPS

45 GQQQPFPPQQPYPQQQPFPS

48 GQQERFPPQQPYPHQQPFPS . .
49 QQPYPQPQPFPSQLPYLQLQ ..
50 QQPYPQPQFPSQLPYLQLQP _ 53 QQPY PI-1QQ PFPSQQPYPQPQ - .
_ , 54 PFPSQLPYLQLQPFPQPQLP _ _ 56 PFE'SQQPYLQLQPFSQPQLP _ . 58 PFPSQQPYLQLQPFLQPQPF .
59 PEPS QQPYLQLQPFPQPQLP .
_ _ _ , 60 PEPS QQPYMQLQPFPQPQLP , 61 PETSQQPYMQLQPFPQPQPF .

64 PFPSQQPYPQPQPFPPQLPY .

66LQLQ9FQ9QLYSQPQPFR lir-i: = =. . . . = .= =
. .
. .
67 LQLQPETQPQLPYSQPQQFP = =. . = 1 , . ..
.
. .
. .
68 LQLQP FPQPQLPYLQPQP FP ..
= ________________________________ . . = 1,...:74- . . . .
.=
..
.
69 LQLQE'FPQPQLSYSQPQPFR = :::' :, i, :--*:
71 LQLQPFLQPQLPYSQPQPFR ' , 72 LQLQPFLQPQPFPPQLPYSQ . _______________________________ .
73 LQLQPFPQPQLPYPQPQLPY offli= = ,. ..,...,..k,õ: .. ==
,,........:.õ..õ,õ
, = ________________________________________________________________ =

75 MQLQPFPOPQPFPPQLPYPQ = ' ' ','" ' ., 11111 " =
7 6 LQLQPFPQPQL PYPQPQP FP i .; .ii =.: .1 78 LQLQPFPRPQLPYPQPQPFR . = ' .
, : .. , . = .
79 LOLQPFPQPQPFLPQLPYPQ ' ' '. ' = . .... = ' :
30 LQLQPFPQPQPFPPQLPYPQ = ' , 1 ' .. = .; ',--,:. ' :, ',:.
_ .
81 PQPQPFP PQLPY PQTQP FP P .
82 PQPQPFPQPQP FP PQLPY PQ , :
34 PQLPYSQPQQFRPQQPYPQP .

87 PQLSYSQPQPFRPQQLYPQP ' 89 PQLPYPQPQLPYPQPQLPYP , _ I. I = - ... . .
. .
90 PQLPYPOPQLPYPQPQPFRP re =, 4 91 PQPFPPQLPYPQPQLPYPQP M.1 92 PQLPYPQPQPFRPQQPYPQP .
93 PQPFPPQLPYPQPQPFRPQQ - = - ' 111 ,-.= ='' . .
;14. III ________________________ 94 PQPFPPQLPYPQPPPFSPQQ -., "
r,^
95 EQPFLPQLPYPQPQS FP PQQ = , = .- .
96 PQPFPPQLPYPQPQSFPPQQ ' .. ' = :- ' . .

_ 100,1,PYPQPQPFRPQQSYPQPQP

102LPQLPY9QPQSFPPQOPYPQ _ 103PPQLPYPQTQPPPP0Q9Y9Q _ _ 104QPERPQQPYPQPQPQYSQPQ _ 1050P8'P.PQQLYPQPQPQYSQPQ

107QPFRPQQSYPQ8'QPQYSQPQ

109QS8PPQQPYP0002KYLQPQ _ _ 112QPFPPQQPYPQ9QEQYPQRQ _ _ 114YPQ9QPQYSQPQEPISQQQQ _ _ _ 117YPQQAPKYLQPQQPISQQQA _ _ 118YPQOP4YLQ9QQPISQQQA _ _ 120 SQPQQP I SQQQQQQQQQQQQ .

126QQQ000QQQQKQQQ000QQI.

128QQQ000QOILQQILQQQLIP _ _ 13240.0000QQIIQQILQQQLIP

135 4QC2440QINIMIL0QQLI _ _ 13/ILQQMLQQQLIPCMDVVLQQ _ 138ILQQILQQQLTPC14DVVLQQ _ 140IL90IL000LIPCRDVVLQQ _ , _ _ _ 153VLQQHNIAHGRSQVLQQSTY =

155VLQQHNLAHGRSQVLMSTY - _ 167HGRSQVLQQSTYQLLRELCC _ 171HGSSQVLQESTYQLVQQLCC =

175QSTYQLLQELCCQHLWQIPE . .

= =

=

188QLCCQQLLQIFEQSACQAIH =

190QLCCQQLFQIPEQSRCQAIH .

209 Al ILHQQQKQQQQPS SQVS F

218 AI IMHQQEQQQQLQQQQQQQ õ
219 Al IMHQQQQQQQEQKQQLQQ

229 QQQQQPsSQVSYQQPQEQYP

. .

248 QVS FQQPQQQYPSSQGSPQP , 253,QQYPSGQGFFQPSQQNPQAQ = =
254 QQY PSGQGFFQPFQQNPQAQ =

=

266 FFQPSQQNPQAQGSFQPQQL _ 268 FFQE'SQQNPQAQGSVQPQQL ' I

I I

297 TLPAMCNVY I PPYyCcANAPFG
, TIT

. .

306 Y I PPYCANIAP FG I FGTNYR .

308 Y I PPYCTITPCGIFGTN .
-312 YI E'PHCSTTIAPEGIFGTN -324PSGQVQWLQQQLV9QLQQPL =
325PSCQVP99PQMP9'PQPHQPF

329PSCQVQWPQQQPFPQPQQ2F =

= 333QQQPFLQPHQPFSQQPQQIF
334000QPFPCPQCPFSQQPQQI = . .

EE I..

341 HQPFSQQPQQIFPQPQQTFP 1.111..1111.11111111111.111111.111111.* =
= 342QQPFSCAIPWIFPQPQQTFP MIME= fF''MTM

347QQPFCEQPQRTI9Q9HQTFH .

349QQI9'PQPQQTFPHQPQQQFP

353QTT4IHUMTFPQPQQTYP9 =

,==
. , .=

369QQF9QTQQPQQPFPQPQQTF . . .
=
370PQQQFL09009F900PQQPY =
=

= :

=
112 , 374 PQQPFPQQPQQQFPQPQQPQ , 375 PQQPIPOPS)Q8Q4PFPQ940 ' . _ ' _ , ,, .
376 PQQPFPQPQQTFPQQPQLPF _ -, 377 PQQQFPQPQQPQQPFPQQPQ ' . ' , , - ¨ , - - , , . , ' _L ' ' =
381 QQPFPQQPQOPYPQQFQQPF _ _ - _ _ . . ¨

- ¨ - . .
384 RQPFPQQPQQPYPQQPQQPF' 385 QQPFPQPQQPQLPFPQQP.Q0 ' ..,. . , 386 0QPFPOPQOAQLPFPOQPQQ , , .
387 QQTFPQQPQLFFPQQPQQPF .
..:. .. . µ 388 QQPIPQQP4(20r0QT4Q0QQ - ' ' .
389,QQPFPQTQQPQQPF800PQQ , ' ..dS.L./
390 QQTYPQRPQQPFPQTQQPQQ _ _ 391 QPQL8FPQQPQQQ800PFPQ .
PEO - _ _ 392 QAQLPFPQQPQQPLPQPQQP .µ . - _ _ ¨
393 QLP FPQQ PQOP ETQPQQP(X) ¨

¨ ¨
395 QPQQPFPQTQQPQQPFPQQP r .:,..-,41N
396 TQQPQQPFPQQPQQPFPQTQ , . , . _ , , 391 PQQPOQPFPQTQQPQQPFPQ , . .
398 QQPFPQTQQPQQLFPQSQQP _ -399 QQPFPQTQQPQQPFPQLQQP - .
400 QUP ¨ - PQTQQPQ4PFPQSQ4P _ , 402 QQPFPQPQQPQQPFPQLQQP ¨11111111-7", _ .. "-404 QQPFPQPQQPQQPFPQSQQP _ ' '..._ _ .

, 407 Q0,14PFPQ64.424Q0EPQ0Q __ --11.-, 408 Q PQQP ETU KMQPFPQPQ __________________________________ , 409 QPQQPFPQLQQPQQPLPQPQ ¨ , - .
410 SQQPQQQFSQPQQQFPQPQQ IMI , _____________________________________________________________ . _ 411 LQQPQQPFPOPQQQLPQPQO = , _ _ .
, 412 S0QPQQP EPOPQQ4FP(RO _ : , _ .
i .
413 SKQPQQPFPQPQQPQQSFPQ ...., . _ 1- r;
415 SQQPQQPFPQPQQPQQSFPQ .
'L
-416 SQPQQQFPQPQQPQQSFPQQ , 417 8Q8QQQLPQ8Q0P003ETZ2 - .
.
, II L
418 PQPQQQFPQPQQPQQSFP4S2 _ _ - .
- .
419 PQPQQPQQSENGQPSLISTIQ _ _ . _ õ
, 423 QPQQFQQS FPQQQRPFIQE'S

- _ , _ 425 80QQQ0PVIUSLQQQV9P0 . _ _ _ -- --=

. =
432 IQQSLQQQLNPCKNFLLQQC .

=
.õ
.= .

õ.

447 LVSSLWSMILPRSDCQVMRQ .
= .=

. 451 LVSSLVSIILPRSDCQVMQQ

=
458 VMRQQCCQQLARIPQQLQCA , =

=. . .

461 QLAQI PQQLQCAAIHTI I HS . .
=

. .

. 472 LQCAAI HSVVHS I IMQQEQQ

=

=

485 QQQQQQQQQGMHIFLPLSQQ _ 486 QEQQEQRQGVQILVPLSQQQ _ _ , 491 00QQQQQG I Q:MRPLFQLVQ _ _ _ 498 GIQTLRPLFQINQGQGIIQP _ 499 GVPILRPLFQLAQGLGIIQP _ _ 502 SQQOQVGQGSLVQGQG I IQP _ - -_ 518 QLEVIRSLVLGTLPTMCNVF _ 521 VLQTLPTMCNVYVPPECSII _ 523 VLQTLATMCNVYVPPYCSTI _ 525 VLRTLPNMCNVYVRPECSTI _ 528 CNVYVPPECSIMRAPFAS IV _ _ 529 CNVYVPPYCSTIRAPFASIV _ _ 530 CliV FVP PECSTTKAPFAS IV
531 CNVYVRPECSTINAPFAS I V _ 532 CNVYVPPDCSTINVPYAN ID , ___________________________________ = 535 CST IRAPPAS IVAGIGGQYR
536 CSTIRP.PFASIVASIGGQ
_ 541 PQQSELWQSQQPFLQQPQQP õ..

546 SAGRPTSAPEPQQQQQHQQL = = =
.=

554 S SC HVMQQQCCQQLPQI PQQ ' 55,6 PQI PQQSRYEAIRAIIYSI I _ 559 I ILQEQ(24VQCSIQSCH.XIQP

561 QQQPQQLGQCVSQPQQQSQQ .
. .

566 QIAQLEVMTSIALRILPTINC _ , . . .
582 000OPPFSQQQQPVLPQQSP . - -==

599 QGVSQSQQQSQQQLGQCSFQ =

606 SIALRTLPTMCSVNVPLYSA =

612 PPFSQQQQQPL?QQPSFSQQ

615 SQQQPILSQQP?FSQQQQPV
616 ATAARELNPSNKELQSPQQS 11111111.1111111111.11.1111111111,111111.111.1.111110 617 PSNKELQSPQQSFSYQQQPP Mal 111 MIMI

623 FPEQSQQPFTMQPTPIQP =
111111111mien.
624 FTQPQQPTPIQPQQPFPOOP .
625 PIOPQQPFPQQ?QQPQQPFP .
626 PQQPQQPQQPFPQPQQPFPW ?A=1 1111 =

NEM I NEN ===
1.11 631 FPQQPQQPFPQPQLPFPOQS __________ MIEN MEW NINE
632 F POPQLPFPQQSEQI 'PQM
633 PQQSEQI I PQQLQQP FPLQP L Ell 111E11 635 PLQPQQPFPQQPQQPFPQPQ 11111". '''''111111=11011V;

=

642 PQQPQQPFPLQPQQPFPQQP I =

6-19 Qa.Q5 KVA PGWYPOQFY
L .

6 51. ISELHTPCM.CIEVWFPCIPQ
652;9E.Polqprpqe9wQ
____________________________________________ -I--70,1 to 100 ngsW: 40.1 to 70 2s,i In An .1:10.1 to 25 5.1 to 10 <F, >3 .<
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description contains a sequence listing in electronic form in ASCII text format (file: 67674-83 Seq 07-05-2018 v5.txt).
A copy of the sequence listing in electronic form is available from the Canadian Intellectual Property Office.

Claims

CLAIMS:

1. A peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19.

2. The peptide of claim 1, which is 10-50 amino acids in length.

3. The peptide of claim 1 or 2, which is 15-30 amino acids in length.

4. The peptide of any one of claims 1 to 3, which comprises the sequence of transglutaminase-deamidated SEQ ID NO: 39.

5. The peptide of claim 1, which consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID NO:779).

6. The peptide of any one of claims 1 to 5, which is HLA-DQ2-restricted.

7. The peptide of any one of claims 1 to 6, which comprises a modification that corresponds to a natural post translational modification present on the N or C
terminus.

8. The peptide of any one of claims 1 to 7, which is bound to an HLA
molecule or a fragment of an HLA molecule which is capable of binding the peptide.

9. Use of at least one agent for the preparation of a medicament for treatment or prevention of coeliac disease, wherein the agent is:
(a) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19: or (b) the peptide of (a) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (c) optionally, in addition to the peptide of (a) or (b), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2.

10. The use of claim 9, wherein the peptide of (a) or (b) is 10-50 amino acids in length.

11. The use of claim 9 or 10, wherein the peptide of (a) or (b) is 15-30 amino acids in length.

12. The use of any one of claims 9 to 11, wherein the peptide of (a) comprises the sequence of transglutaminase-deamidated SEQ ID NO: 39.

13. The use of any one of claims 9 to 12, wherein the peptide of (a) consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID NO:779).

14. The use of any one of claims 9 to 13, wherein the peptide of (a) or (b) is HLA-DQ2-restricted.

15. The use of any one of claims 9 to 12, wherein the peptide of (a) or (b) is HLA-DQ2-restricted and the medicament comprises a second agent that is HLA-DQ8-restricted.

16. The use of any one of claims 9 to 12, wherein:
the peptide of (a) or (b) further comprises another wheat epitope and the medicament comprises a second agent that comprises a rye epitope: or the peptide of (a) or (b) further comprises a rye epitope and the medicament comprises a second agent that comprises a wheat epitope.

17. The use of any one of claims 9 to 12, wherein:
the peptide of (a) or (b) further comprises another wheat epitope and the medicament comprises a second agent that comprises a barley epitope; or the peptide of (a) or (b) further comprises a barley epitope and the medicament comprises a second agent that comprises a wheat epitope.

18. The use of any one of claims 9 to 12, wherein:
the peptide of (a) or (b) further comprises a rye epitope and the medicament comprises a second agent that comprises a barley epitope; or the peptide of (a) or (b) further comprises a barley epitope and the medicament comprises a second agent that comprises a rye epitope.

19. The use of any one of claims 16 to 18, wherein the second agent is a peptide.

20. The use of any one of claims 9 to 12, wherein:
the peptide of (a) or (b) further comprises another wheat epitope and the medicament comprises a second agent that comprises a barley epitope and a third agent that comprises a rye epitope; or the peptide of (a) or (b) further comprises a barley epitope and the medicament comprises a second agent that comprises a wheat epitope and a third agent that comprises a rye epitope; or the peptide of (a) or (b) further comprises a rye epitope and the medicament comprises a second agent that comprises a barley epitope and a third agent that comprises a wheat epitope.

21. The use of claim 20, wherein the second agent is a peptide and the third agent is a peptide.

22. The use of any one of claims 9 to 12, wherein the peptide of (a) or (b) further comprises another wheat epitope, a barley epitope, and a rye epitope.

23. The use of any one of claims 9 to 22, wherein the agent is present within a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

24. The use of any one of claims 9 to 23, wherein the medicament tolerises T-cells which recognise the agent.

25. A pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier or diluent, wherein the agent is:
(a) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (b) the peptide of (a) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (c) optionally, in addition to the peptide of (a) or (b), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2.

26. The pharmaceutical composition of claim 25, wherein the agent is a peptide which is 10-50 amino acids in length.

27. The pharmaceutical composition of claim 25 or 26, wherein the agent is a peptide which is 15-30 amino acids in length.

28. The pharmaceutical composition of any one of claims 25 to 27, wherein the agent is a peptide which comprises the sequence of transglutaminase-deamidated SEQ ID
NO: 39.

29. The pharmaceutical composition of any one of claims 25 to 28, wherein the agent is a peptide which consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID NO:779).

30. The pharmaceutical composition of any one of claims 25 to 29, wherein the agent is a peptide which is HLA-DQ2-restricted.

31. The pharmaceutical composition of any one of claims 25 to 30, wherein the agent is a peptide which is bound to an HLA molecule or a fragment of an HLA
molecule which is capable of binding the peptide.

32. The pharmaceutical composition of any one of claims 25 to 31, for use in tolerising an individual to a gliadin protein to suppress the production of a T-cell or antibody response to the agent.

33. A method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising:
(a) contacting a sample from the individual with at least one agent that is:
(i) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (ii) the peptide of (i) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (iii) optionally, in addition to the peptide of (i) or (ii), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
and (b) determining in vitro whether T-cells in the sample recognise the agent;
whereby recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease.

34. The method of claim 33. wherein the peptide of (i) or (ii) is 10-50 amino acids in length.

35. The method of claim 33 or 34, wherein the peptide of (i) or (ii) is 15-30 amino acids in length.

36. The method of any one of claims 33 to 35, wherein the peptide of (i) comprises the sequence of transglutaminase-deamidated SEQ lD NO: 39.

37. The method of any one of claims 33 to 36, wherein the peptide of (i) consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID
NO:779).

38. The method according to any one of claims 33 to 37, wherein the agent comprises the peptide of (i) or (ii) bound to (A) an HLA molecule. or (B) a fragment of an HLA molecule capable of binding the peptide of (i) or (ii).

39. The method according to claim 38, wherein the HLA molecule or fragment is in a complex comprising four HLA molecules or fragments of HLA molecules.

40. The method according to any one of claims 33 to 39, wherein the sample is a blood sample.

41. The method according to any one of claims 33 to 40, wherein the T-cells are not restimulated in an antigen specific manner in vitro before said determining.

42. The method according to any one of claims 33 to 41, wherein the recognition of the agent by the T-cells is determined by detecting the secretion of a cytokine from the T-cells.

43. The method according to claim 42, wherein the cytokine is IFN-y.

44. The method according to claim 42 or claim 43, wherein the cytokine is detected by allowing the cytokine to bind to an immobilised antibody specific to the cytokine and then detecting the presence of the antibody/cytokine complex.

45. The method according to any one of claims 33 to 41, wherein said determining is done by measuring whether the agent binds a T-cell receptor of the T-cells.

46. Use of at least one agent which is:

(i) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (ii) the peptide of (i) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (iii) optionally, in addition to the peptide (i) or (ii), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
for the preparation of a diagnostic means for use in a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual, said method comprising determining whether T-cells of the individual recognise the agent, wherein recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease.

47. The use of claim 46, wherein the peptide of (i) or (ii) is 10-50 amino acids in length.

48. The use of claim 46 or 47, wherein the peptide of (i) or (ii) is 15-30 amino acids in length.

49. The use of any one of claims 46 to 48, wherein the peptide of (i) comprises the sequence of transglutaminase-deamidated SEQ ID NO: 39.

50. The use of any one of claims 46 to 49, wherein the peptide of (i) consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID NO:779).

51. The use according to any one of claims 46 to 50, wherein the agent comprises the peptide of (i) or (ii) bound to (a) an HLA molecule, or (b) a fragment of an HLA molecule capable of binding the peptide of (i) or (ii).

52. The use according to claim 51, wherein the HLA molecule or fragment is in a complex comprising four HLA molecules or fragments of HLA molecules.

53. The use according to any one of claims 46 to 52, wherein the recognition of the agent by the T-cells is determined by detecting the secretion of a cytokine from the T-cells.

54. The use according to claim 53, wherein the cytokine is IFN-.gamma..

55. The use according to claim 53 or 54, wherein the cytokine is detected by allowing the cytokine to bind to an immobilised antibody specific to the cytokine and then detecting the presence of the antibody/cytokine complex.

56. The use according to any one of claims 46 to 52, wherein said determining is done by measuring whether the agent binds a T-cell receptor of the T-cells.

57. A method of determining whether a composition is capable of causing coeliac disease comprising determining whether a protein comprising the sequence set forth in SEQ ID NO: 19 is present in the composition, the presence of the protein indicating that the composition is capable of causing coeliac disease.

58. The method according to claim 57, wherein said determining is done by contacting the composition with an antibody specific for the protein, binding of the antibody to said protein in the composition indicating the composition is capable of causing coeliac disease.

59. A kit for carrying out the method according to claim 33 comprising:
(a) at least one agent that is:
(i) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (ii) the peptide of (i) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (iii) optionally, in addition to the peptide of (i) or (ii), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
and (b) a means to detect the recognition of the peptide by T-cells.

60. The kit of claim 59, wherein the peptide of (i) or (ii) is 10-50 amino acids in length.

61. The kit of claim 59 or 60, wherein the peptide of (i) or (ii) is 15-30 amino acids in length.

62. The kit of any one of claims 59 to 61, wherein the peptide of (i) comprises the sequence of transglutaminase-deamidated SEQ ID NO: 39.

63. The kit of any one of claims 59 to 62, wherein the peptide of (i) consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID NO:779).

64. The kit according to any one of claims 59 to 63 wherein the means to detect recognition comprises an antibody to IFN-.gamma..

65. The kit according to claim 64, wherein the antibody is immobilised on a solid support and optionally the kit also comprises a means to detect the antibody/IFN-y complex.

66. Use of a peptide comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19, to produce an antibody specific to the peptide.

67. Use of at least one agent for the treatment or prevention of coeliac disease, wherein the agent is:
(a) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (b) the peptide of (a) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (c) optionally, in addition to the peptide of (a) and (b), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2.

68. The use of claim 67, wherein the peptide of (a) or (b) is 10-50 amino acids in length.

69. The use of claim 67 or 68, wherein the peptide of (a) or (b) is 15-30 amino acids in length.

70. The use of any one of claims 67 to 69, wherein the peptide (a) comprises the sequence of transglutaminase-deamidated SEQ ID NO: 39.

71. The use of any one of claims 67 to 70, wherein the peptide (a) consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID NO:779).

72. The use of any one of claims 67 to 71, wherein the agent is HLA-DQ2-restricted.

73. The use of any one of claims 67 to 71, wherein the peptide of (a) or (b) is HLA-DQ2-restricted and the at least one agent comprises a second agent that is HLA-DQ8-restricted.

74. The use of any one of claims 67 to 70, wherein:
the peptide of (a) or (b) further comprises another wheat epitope and the at least one agent comprises a second agent that comprises a rye epitope; or the peptide of (a) or (b) further comprises a rye epitope and the at least one agent comprises a second agent that comprises a wheat epitope.

75. The use of any one of claims 67 to 70, wherein:

the peptide of (a) or (b) further comprises another wheat epitope and the at least one agent comprises a second agent that comprises a barley epitope; or the peptide of (a) or (b) further comprises a barley epitope and the at least one agent comprises a second agent that comprises a wheat epitope.

76. The use of any one of claims 67 to 70, wherein:
the peptide of (a) or (b) further comprises a rye epitope and the at least one agent comprises a second agent that comprises a barley epitope; or the peptide of (a) or (b) further comprises a barley epitope and the at least one agent comprises a second agent that comprises a rye epitope.

77. The use of any one of claims 73 to 76, wherein the second agent is a peptide.

78. The use of any one of claims 67 to 70, wherein:
the peptide of (a) or (b) further comprises another wheat epitope and the at least one agent comprises a second agent that comprises a barley epitope and a third agent that comprises a rye epitope; or the peptide of (a) or (b) further comprises a barley epitope and the at least one agent comprises a second agent that comprises a wheat epitope and a third agent that comprises a rye epitope; or the peptide of (a) or (b) further comprises a rye epitope and the at least one agent comprises a second agent that comprises a barley epitope and a third agent that comprises a wheat epitope.

79. The use of claim 78, wherein the second agent is a peptide and the third agent is a peptide.

80. The use of any one of claims 67 to 70, wherein the peptide of (a) or (b) further comprises another wheat epitope, a barley epitope, and a rye epitope.

81. A kit for carrying out the use according to claim 46 comprising:
(a) at least one agent that is:
(i) a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence of transglutaminase-deamidated SEQ ID NO: 19; or (ii) the peptide of (i) comprising a modification that corresponds to a natural post translational modification present on the N or C terminus; and (iii) optionally, in addition to the peptide of (i) or (ii), a peptide comprising at least one epitope comprising the sequence selected from SEQ ID NO: 1 and SEQ
ID NO: 2;
and (b) a means to detect the recognition of the peptide by T-cells.

82. The kit of claim 81, wherein the peptide of (i) or (ii) is 10-50 amino acids in length.

83. The kit of claim 81 or 82, wherein the peptide of (i) or (ii) is 15-30 amino acids in length.

84. The kit of any one of claims 81 to 83, wherein the peptide of (i) comprises the sequence of transglutaminase-deamidated SEQ ID NO: 39.

85. The kit of any one of claims 81 to 84, wherein the peptide of (i) consists of the sequence of transglutaminase-deamidated QQPFPQPQQPFPWQP (SEQ ID NO:779).

86. The kit according to any one of claims 81 to 85, wherein the means to detect recognition comprises an antibody to IFN-.gamma..

87. The kit according to claim 86, wherein the antibody is immobilised on a solid support and optionally the kit also comprises a means to detect the antibody/IFN-.gamma. complex.

88. A pcptide of not morc than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID
NO:796).

89. The peptide of claim 88, which comprises the sequence EQPFPQPEQPFP.

90. The peptide of claim 88 or 89, which comprises the sequence EQPFPQPEQPFPWQP (SEQ ID NO:795).

91. The peptide of any one of claims 88 to 90, which is 15 amino acids in length.

92. The peptide of any one of claims 88 to 91, wherein a modification that corresponds to a natural post translational modification is present on the N
terminus.

93. The peptide of any one of claims 88 to 92, wherein a modification that corresponds to a natural post translational modification is present on the C
terminus.

94. Use of a peptide for the treatment or prevention of coeliac disease, wherein the peptide is a peptide of not more than 50 amino acids in length comprising at least one T-cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID
NO:796).

95. Use of claim 94, wherein the peptide comprises the sequence EQPFPQPEQPFP (SEQ ID NO:797).

96. Use of claim 94 or 95, wherein the peptide comprises the sequence EQPFPQPEQPFPWQP (SEQ ID NO:795).

97. Use of any one of claims 94 to 96, wherein the peptide is 15 amino acids in length.

98. Use of any one of claims 94 to 97, wherein a modification that corresponds to a natural post translational modification is present on the N terminus of the peptide.

99. Use of any one of claims 94 to 98, wherein a modification that corresponds to a natural post translational modification is present on the C terminus of the peptide.

100. A pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier or diluent, wherein the agent is a peptide of not more than 50 amino acids in length comprising at least one T cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796).

101. The pharmaceutical composition of claim 100, wherein the peptide comprises the sequence EQPFPQPEQPFP.

102. The pharmaceutical composition of claim 100 or 101, wherein the peptide comprises the sequence EQPFPQPEQPFPWQP (SEQ ID NO:795).

103. The pharmaceutical composition of any one of claims 100 to 102, wherein the peptide is 15 amino acids in length.

104. The pharmaceutical composition of any one of claims 100 to 103, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the N terminus.

105. The pharmaceutical composition of any one of claims 100 to 104, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the C terminus.

106. The pharmaceutical composition of any one of claims 100 to 105, for use in tolerising an individual to a gliadin protein to suppress the production of a T-cell or antibody response to the agent.

107. A method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising:
(a) contacting a sample from the individual with at least one agent that is a peptide of not more than 50 amino acids in length comprising at least one T
cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796); and (b) determining in vitro whether T-cells in the sample recognise the agent;

whereby recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease.

108. The method of claim 107, wherein the peptide comprises the sequence EQPFPQPEQPFP.

109. The method of claim 107 or 108, wherein the peptide comprises the sequence EQPFPQPEQPFPWQP (SEQ ID NO:795).

110. The method of any one of claims 107 to 109, wherein the peptide is 15 amino acids in length.

111. The method of any one of claims 107 to 110, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the N terminus.

112. The method of any one of claims 107 to 111, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the C terminus.

113. The method of any one of claims 107 to 112, wherein the sample is a blood sample.

114. The method of any one of claims 107 to 113, wherein the T-cells are not restimulated in an antigen specific manner in vitro before said determining.

115. The method of any one of claims 107 to 114, wherein the recognition of the agent by the T-cells is determined by detecting the secretion of a cytokine from the T-cells.

116. The method of claim 115, wherein the cytokine is IFN-.gamma..

117. The method of claim 115 or claim 116, wherein the cytokine is detected by allowing the cytokine to bind to an immobilised antibody specific to the cytokine and then detecting the presence of the antibody/cytokine complex.

118. The method of any one of claims 107 to 114, wherein said determining is done by measuring whether the agent binds a T-cell receptor of the T-cells.

119. Use of at least one agent which is a peptide of not more than 50 amino acids in length comprising at least one T cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796) for the preparation of a diagnostic means for use in a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual, said method comprising determining whether T-cells of the individual recognise the agent, wherein recognition by the T-cells indicates that the individual has, or is susceptible to, coeliac disease.

120. The use of claim 119, wherein the peptide comprises the sequence EQPFPQPEQPFP.

121. The use of claim 119 or 120, wherein the peptide comprises the sequence EQPFPQPEQPFPWQP (SEQ ID NO:795).

122. The use of any one of claims 119 to 121, wherein the peptide is 15 amino acids in length.

123. The usc of any one of claims 119 to 122, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the N terminus.

124. The use of any one of claims 119 to 123, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the C terminus.

125. The use of any one of claims 119 to 124, wherein the recognition of the agent by the T-cells is determined by detecting the secretion of a cytokine from the T-cells.

126. The use of claim 125, wherein the cytokine is IFN-.gamma..

127. The use of claim 125 or 126, wherein the cytokine is detected by allowing the cytokine to bind to an immobilised antibody specific to the cytokine and then detecting the presence of the antibody/cytokine complex.

128. The use of any one of claims 119 to 124, wherein said determining is done by measuring whether the agent binds a T-cell receptor of the T-cells.

129. A kit for carrying out the method of claim 107 comprising:
(a) at least one agent that is a peptide of not more than 50 amino acids in length comprising at least one T cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796); and (b) a means to detect the recognition of the peptide by T-cells.

130. The kit of claim 129, wherein the peptide comprises the sequence EQPFPQPEQPFP.

131. The kit of claim 129 or 130, wherein the peptide comprises the sequence EQPFPQPEQPFPWQP (SEQ ID NO:795).

132. The kit of any one of claims 129 to 131, wherein the peptide is 15 amino acids in length.

133. The kit of any one of claims 129 to 132, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the N terminus.

134. The kit of any one of claims 129 to 133, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the C terminus.

135. The kit of any one of claims 129 to 134, wherein the means to detect recognition comprises an antibody to IFN-.gamma..

136. A kit for carrying out the use of claim 119 comprising:
(a) at least one agent that is a peptide of not more than 50 amino acids in length comprising at least one T cell epitope, said T-cell epitope comprising the sequence FPQPEQPFP (SEQ ID NO:796); and (b) a means to detect the recognition of the peptide by T-cells.

137. The kit of claim 136, wherein the peptide comprises the sequence EQPFPQPEQPFP.

138. The kit of claim 136 or 137, wherein the peptide comprises the sequence EQPFPQPEQPFPWQP (SEQ ID NO:795).

139. The kit of any one of claims 136 to 138, wherein the peptide is 15 amino acids in length.

140. The kit of any one of claims 136 to 139, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the N terminus.

141. The kit of any one of claims 136 to 140, wherein the peptide comprises a modification that corresponds to a natural post translational modification on the C terminus.

142. The kit of any one of claims 136 to 141, wherein the means to detect recognition comprises an antibody to IFN-.gamma..

143. The kit of claim 142, wherein the antibody is immobilised on a solid support and optionally the kit also comprises a means to detect the antibody/IFN-.gamma. complex.