Title of Invention

AGENT TYPICALLY IN THE FROM A PEPTIDE

Abstract A compound 1, 2, 3, 4 tetrahydroquinoline of formula (I) (R)m or a salt, or metabolically labile ester thereof wherein R represents a group selected from halogen, alkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, trifluoromethyl, trifluoromethoxy, nitro, cyano, SO2R2 or COR2 wherein R2 represents hydroxy, methoxy, amino, alkylamino or dialkylamino; m is zero or an integer 1 or 2.
Full Text THERAPEUTIC EHTOPES AND USES THEREOF
The invention relates to epitopes useft]] 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 file 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 tins 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 feat only intestinal T cell responses provide an accurate indication of the immune response against gtiadins. Therefore they have concentoated on me investigation of T cell responses in intestinal tissue1. Gliadin epitopes which require tcansglutaminase 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 mat this is
recognised by T cells in foe peripheral blood of individuals wjfli coeliac disease {see
WO 01/25793). Such T cells were found to be present at high enough frequencies to
be detectable without restimu!ation{Le. a 'fresh response' detection system could be
used). The epitope was identified using & 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 merefore an intestinal biopsy is not required. The test is more sensitive man 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:
(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 N0:3, (ii) an epitope comprising sequence comprising: SEQ ID NO: I, or an equivalent sequence from a naturally occurring hotnologue of the gliadin represented by SEQ ID N0:3 (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 ' arnino acids in length, or (iv) a product comprising two or more agents as defined in (i), (ii) or (in), and (b) detennhring in vitro whether T cells in the sample recognise the agent, recognition by the T cells indicating that me 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-DQ24- 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 NOS31-36), as well as in rye secalins and badey hordeins (e.g. SEQ ID NOS39-41); 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 Ihus provides the dominant epitopes recognized by T cells • in coeliac patients. This, fee above-escribed method and omer methods of the invention descnbed herein may be performed using any of these addnional identified epitopes, and analogues and equivalents thereof (i) and (u) 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 «se in a method of diagnosing coeliac disease, or suspeptibility 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.
The finding of an immunodominanl epitope which is modified by transglutamraase (as well as the additional other epitopes defined herein) also allows diagnosis of coeliac disease based on determining whether other types of immune response lo 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 me antibody indicating mat 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 me 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 mat binds an antibody (mat binds the agent) for use in a method of treating or preventing coeliac disease in an individual by tolerising the individual to prevent me production of such an antibody.
The invention provides a method of determining whether a composition is capable of cansing coeliac disease comprising determining whe&er a protein capable of being modified by a transglutaminase to an oligopepti.de sequence as defined above is present in me composition, the presence of the protein indicating mat 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 mat comprises an epitope comprising sequence as defined above, but which mutant griadm protein has been modified in such a way that it does not contain sequence which can be modified by a transglutaminase to a sequence mat 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 me agent, and which sequence is able to cause antagonism of a T cell that carries such a T cell receptor.
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 ammo acidf in length; and c) optionally, in addition to the agent selected from a) and b), a peptic s comprising at least one epitope comprising a sequence selected from SEQ ID NO.1 and SEQ ID NO2. In some embodiments, the agent is HLA-DQ2-res1ricted, ELA-DQ8-restricted or one agent is HLA-DQ2-restricted and a second agent is HLA-DQS-restrieted, In some embodiments, the agent comprises a wheat epitope, axye 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 pharmaceutically 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 lhan 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 detemaining 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; or b) adtainistering an agent as defined above and determining in vivo whether T cells in me individual recognise me agent, recognition of the agent indicating that the individual has or is susceptible to 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 earner, 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.
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 transgluiarninase 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-alse provides cells comprising a porynucleotide as defined above or which has been transformed wife such a porynucleotide.
The present invention also provides mammals mat 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 o£ SEQ ID NOS: 18-22,31-36,39-44, and 46, and equivalents thereof; and u) an analogue of 0 which is capable of being recognised by a T cell receptor mat recognises i) and which is not more man 50 amino acids in length; and in) optionally, in addition to the agent selected from i) and if), a peptide comprising at feast one epitope comprising a sequence selected from SEQ ID NOS:I and 2; and b) determining in vitro whether T cells in the sample recognise the agent; recognition by me T cells indicating feat fee 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 me composition, the presence of the protein indicating mat 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
ueiecnng 01 any sucn decrease in sata anility indicating tnai ine suoscrace 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 delect the recognition of the peptide by the T cell.
Hie 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 determining whether substance prevents or treats coeliac disease in the mammal, fee prevention or treatment of coeliac disease indicating that the substance is a therapeutic product
Hie present invention also provides processes for me 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 ceD 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.
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 fee harvested product
The present invention also provides food that comprises a protein as defined above.
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) BFN ELISPOT responses (vertical axis shows spot forming cells per 106 PBMC) to transglutaininase (tTG)-treated and untreated peptide pool 3 (each peptide 10 µg/ml) including five overlapping 1 Smers spanning A-gnadin 51-85 (see Table 1) and a-chymotrypsin-digested gliadin (40 µg/mi) 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 EFNy ELISPOT responses by Subject 2 to tTG-4reated A-gliadin peptide pools 1-10 spanning the complete A-gHadin protein during ten day bread challenge (b). The horizontal axis shows days after commencing bread-Figure 2 shows PBMC BFNy ELISPOT responses to tTG-toeated peptide pool 3 (spanning A-gliadin 51-85) in 7 individual coeliac disease subjects (vertical axis shows spot 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 IFN Elispot responses to tTG-treated overlapping 15mer peptides included in pool 3; bars represent the mean (± SEM) response to individual peptides (10 µg/ml) 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.)
Figure 3 shows PBMC IFNy EL1SPOT responses to tTG-trealed truncations of A-gliadin. 56-75 (0.1 µM). Bars represent the mean (± SEM) in 5 Coeliac disease subjects. (!B individual subjects, responses were calculated as the % of five maximal response elicited by any of fee peptides tested.)
Figure A shows how the rnminigl structure of the dominant A-gliadin epitope was mapped using tTG-treated 7-17mer A-gliadin peptides (0.1 µM) including the sequence, PQPQLPY (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 aftetbread 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 ammo acids that were deamidated by tTG. A-gliadin 56-75 LQLQPFPQPQLPYPQPQSFP (SEQ ID NO:5) (0.1 µM) was incubated with fTG (50 µg/ml) at 37°C for 2 hours. A single product was identified and purified by reverse phase BPLC. 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 ITmers: QLQPFPQPELPYPQP1S (SEQ ID NO:6) (E57,65), QLQPFPQPELPYPQPES (SEQ ID NO:7) (E65.72), ELQPFPQPS.LPYPQPES (SEQ ID NO:8) (E57,65,72), and QLQPFPQPgLPYPQPQS (SEQ ID NO^) (E65) in ftree Coeliac disease subjects on day 6 or 7 after bread was ingested on days 1-3. Vertical axis shows % of flie E65 response.
Figure 7 shows that tTG treated A-gliadin 56-75 (0.1 uM) elicited IFN-g ELJSPOT responses in (a) CD4 and CDS magnetic bead depleted PBMC. (Bars represent CD4 depleted PBMC responses as a % of CDS depleted PBMC responses; spot forming cells per million €D8 depleted PBMC were: Subject 4:29, and Subject 6:535). (b) PBMC JFNy ELISPOT responses (spot forming cells/million PBMC)
, •«
after incubation wiH. monoclonal antibodies to HLA-DR (L243), -DQ (LZ) and -DP (B7.21) (10 jig/ml) Ih prior to tTG-toeated 56-75 (0.1 pM) in two coeliac disease subjects homozr/gous for HLA-DQ al*0501, bl*0201.
igure 8 sbows tbe elieet of substituting Glu at position 65 for ofter aroino acids in the immuBodorninant epiiope. The vertical axis shows the % response in the 3 subjects in relation to the immunodomioant epitope.
Figure 9 shows the immimoreactivity of naturally occurring gliadin peptides (measuring responses from 3 subjects) which contain the sequence PQLPY (SEQ ED NO: 12) with (shaded) and without (clear) transglutaminase treatment
Figure 10 shows CDS, CD4, p7, and exE -specific immunomagnelic bead depletion of peripheral blood mononuelear cells ficom two coeliac subjects 6 days after commencing gluten challenge followed by interferon gamma ELISpot A-gliadin 57-73 QE65 (25mcg/ml), tTG-treated chymotrypsin-digested gliadin (100 meg/ml) or PPD (10 meg/ml) were used as antigen,
Figure 11 shows fee optional T cell epitope length. '
Figure 12 shows a comparison of A-gUadin 57-73 QE65 wife 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 bioactiyity of gliadin polymorphisms in coeliac subjects.
Figures 15 and 16 show the defining of fee 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 bioactiviry of prolamkhomologues of A-gliadin 57-73.
Figure 30 shows, for heaMry HLA-DQ2 subjects, fee change in IFN-gamma EUSpot responses to tTG-deanridated gliadm peptide pools. '
Figure 31 shows, for coeliac HLA-DQ2 subjects, fee change in IBN-gamma ELISpot responses to fTG-deaniidated gliadin peptide pools.
Figure 32 shows individual peptide contributions to "summed" gliadin peptide response.
Figure 33 shows, for coeliac BLA-DQ2/8 subject COS, gluten challenge induced IFNy ELISpot responses to tTG-deamidated gliadin peptide pools.
Figure 34 shows, for coeliac HLA-DQ2/8 subject C07, gluten challenge induced IFNy ELISpot responses to tTG-deamidated gliadin peptide pools'.

Figure 35 shows, for coeliac HLA-DQ8/7 subject C12, gluten challenge induced IFN-y ELISpot responses to tTG-deamidated gliadin peptide pools.
Figure 36 shows, for coeliac HLA-DQ6/8 subject CM, gluten challenge induced IFN^ ELISpot responses to tTG-deamidated gliadin peplide 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 nmcosa (hyperplastic vfllous 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 asyrcptomaiic) 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 ammo acids, such as 10 to 40, or 15 to 30 ammo acids in length.
SEQ ID N0:l is PQPELPY. SEQ ID NO2 is QLQPFPQPELPYPQPQS. S6Q ID NO3 is shown in Table 1 and is the sequence of a whole A-guadin. The glutamate at position 4 of SEQ ID NOil (equivalent to position 9 of SEQ ID NO:2) is generated by transglutammase treatment of A-gliadin.
The agent may be the peptide represented by SBQ 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 NO3. Thus the epitope may be a derivative of the protein represented by SEQ ID NO:3. Such a derivative is
typically a fragment of the gliadin. or a mutated derivative of fee 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-gh"adin (e.g. SEQ ID NO:3), which comprises the" sequence of SEQ ED NO:1, obtainable by treating (fiilly or partially) with transglutaininase, le. with 1,2,3 or more gluiamines substituted to glutamates (including fixe substitution wifliin SEQ ID NO:1).
Such fragments may be or may include me sequences represented by 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 me same extent that me . 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 me gliadin has been treated wim transgratarninase). Bioactive fragments of such sequences axe also agents of me invention. Sequences equivalent to any of SEQ ID NOS:18-22,31-36,39-44, and 46 or analogues of these sequences axe 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 gliatfrns which cause coeliac disease), such equivalent sequences win correspond to a fragment of a gliadin protein typically treated (partially or fully) with txansglutaminase. Such equivalent peptides can be determined by aligning the sequences of other gliadin proteins wim 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.
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 me 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 £he 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 the method of the invention). Demonstration of foe binding of the analogue to the TCRs can flien shown by determining-whether the TCRs inhibit the binding of foe analogue to a substance mat binds me analogue, e.g. ari antibody to the analogue. Typically the analogue is bound to a class n 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 fiie amount of (i) or (ii) which can bind the TCR in me presence of the analogue is decreased. This is because me analogue is able to bind die TCR and therefore competes with (i) or (ii) for binding to me 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.
Omer binding characteristics of me analogue may also be the same as 0) or (ii), and thus typically me analogue binds to the same MHC class H molecule to which the peptide binds (HLA-DQ2 or -DQ8). The analogue typically binds to antibodies specific fox (i) or (u), and finis inhibits binding of (i) or (ii) to such antibodies.
The analogue is typically a peptide. It may have bomology with (i) or (ii), typically at least 70% homology, preferably at least 80,90%, 95%, 97% or 99% bomology wife (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 MHC molecule) contiguous amino acids. Methods of measuring protein homology are well known in the art and it wfll be understood by those of doll
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 UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux 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 Iheir default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
Software for performing BLAST analyses is publicly available through fhe National Center for Biotechnology Information on the world wide web through flue internet at, for example, "wwwjncbLnbnja3i.gov/". This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence mat either match or satisfy some positive-valued threshold score T when aligned wim a word of the same lengfc in a database sequence. Tis referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood won! hits act as seeds for initiating searches to find HSPs containing them. The word bits ate extended in bom directions along each sequence for as far as the cumulative alignment score can be increased- Extensions for the wozd hits in each direction are halted when: me cumulative alignment score falls off by me quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to ^ accumulation of one or more negative-scoring residue' alignments; or fhe end of either sequence is reached. The BLAST algorithm parameters W, T and X determine ihe sensitivity and speed of the alignment The BLAST program uses as defaults a word length (W) of 11, me BLOSUM62 scoring matrix (see Henflcoff and Henikoff (1992) Proc. NatL Acad. ScL USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=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. Nad. Acad. ScL USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)X which provides an indication of tine 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 ten about 0.15 more preferably less than about 0.01, and most preferably less than about 0.001.
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 me 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 in me second column and preferably in me same line in the third column may be substituted for each other
(T able Removed)
Typically the amino acids in the analogue at me equivalent positions to amino acids in (i) or (ii) that contribute to binding the MEC 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.

Generally., the non-natural amino acid will have an N terminus 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 that is 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 Jhe 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 (0 or (ii) bound to a MHC class E[ molecule. 2,3,4 or more of such complexes may be associated or bound to each other, for example using a biotm/streptavidin based system, in which lypically 2,3 or 4 biotin labelled MHC molecules bind to a streptavidin moiety. This analogue typically inhibits foe binding of die (i) or (fi)/MHC Class E complex to a TCR or antibody which is specific for me complex.
The analogue is typically an antibody or a fragment of an antibody, such as a Fab or (Fab)z fragment The analogue may be immobilised on a solid support, particularly an analogue mat 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 fiie form of being bound to a MHC class n molecule, such as HLA-DQ2 or -DQ8. Analogues are generally selected from the library based on their ability to mimic the bbding characteristics© or (S). Thus they may be selected based on ability to bind a TCR or antibody which recognises © or(ii).
Typically analogues wfll be recognised by T cells to at least the same extent as any of the agents (i) or (ii), for example at least to me 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
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 mat 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 mis 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 BO the method may be used to identify analogues which are
-*
recognised to mis extent
In one embodiment the method comprises deternrining whether a candidate substance is able to inhibit me recognition of an epitope of the invention, inhibition of recognition indicating that the substance is an analogue.
Tlieagentmaybeapr6ductcomprismgatleast2,5,10 or 20 agents as defined by (i), (2) or (iii). Typically the composition comprises epitopes of me invention (or equivalent analogues) from different gliadins, such as any of me speciesorvarietyofortypesofglkdmmentionedherem. Preferred compositions comprise at least one epitope of me 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 me panel of species consisting of wheat, rye, barley, oats and triticale). Thus, the agent may be monovalent or muMvalent
Diagnosis
As mentioned above the method of diagnosis of the invention may be based on fee detection of T cells that bind the.agent or on me detection of antibodies that recognise the agent
The T cells that recognise the agent in the method (which includes the use
i 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.
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 m 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 flue APCs discussed below). The term 'contacting' as used herein includes adding the particular substance to fee solution.
Determination of whether fee T cells recognise me agent is generally accomplished by detecting a change in the state of fee T cells in fee 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 fee T cell after fee TCR binds the agent The change of state may be measured inside (e.g. change in intraceDular expression of proteins) or outside (e.g. detection of secreted substances) me T cells.
The change in state of me T cell may be fee start of or increase in secretion of a substance from fee T ceu, such as a cytoldne, especially IFN-^, EL/-2 or TNF-a. Determination of IFN^r secretion is particularly preferred. The substance can typically be detected by allowing it to bind to a specific binding agent and men measuring me presence of fee 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 me specific binding agent is immobilised on a solid support After fee substance is allowed to bind fee solid support can optionally be washed to remove material which is not specifically bound to me agent The agent/substance complex may be detected by using a second binding agent mat wfll bind the complex. Typically the second agent binds fee substance at a site which is different from fee site which binds fee first agent The second agent is preferably an antibody and is labelled directly or indirectly by a detectable label
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 biotia 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 WO 98/23960. In that assay IFN-7 secreted from the T cell is bound by a first IFN-y specific antibody that is immobilised on a solid support. The bound IEN-7 is then detected using a second IFN-y 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 feat can be measured may be the increase in the uptake of substances by the T cell, such as the uptake of rhymidine. 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 surfece markers on the T cell
In one embodiment the change of state is detected by measuring the change in the inttacellular expression of proteins, for example the increase in intracellular expression of any of the cytokines mentioned above. Such inlracellular changes may be detected by contacting the inside of the T cell with a moiety mat 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 anMHC class n molecule (typically HLA-PQ2 or -DQ8), which is typically present on the surface of an antigen presenting cell (AFC). 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 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 knovm 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, er are freshly isolated T cells (such as in the form of freshly isolated MCs or PBMCs) which are used directly ex vivo, ie. they are not cultured before being used in me method. Thus the T cells have not been restunulated in en antigen specific manner in vitro. However me 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 fhe agent(s) are typicaDy present on fhe 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, sach as short term ceH fines (for example as described in Ota et al (1990) Nature 346, p!83-187).
The APC tirat is typically present in the memodmay be from the same individual as me T cell or from a different host The APC may be a naturally occurring APC or an artificial APC. The APC is a ceH mat is capable of presenting the peptide to aToelL ft is typically a B cell, dendritic cefl or macrophage. ftis typically separated from the same sample as the T cell and ife typically co-purified with fee TceE Thus fee APC may be present in MCs or PBMCs: The APC is . typically a freshly isolated ex vivo cell or a catasred'cell. It may be in the form of a ceH line, such as a short term or immortalised cell toe. The APC may express empty MHC class n 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 is intended totest orthe T cells can be divided and placed into separate assays each of which contain one or more of the agents.
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).
hi one embodiment agent perse is added directly to an assay comprising T cells and APCs. As discussed 0bove 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 pi) bound to a MHC molecule are an example of such an agent
hi one embodiment the agent is provided to the APC in the absence of the T cell The-APC is-feen provided to fee T ceB, typically after being allowed to present the agent on its surface. The peptide may have been taken up inside fee 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 fte T cells wffl vary depending on the method used for determining recognition of the peptide. Typically 10s to 107, preferably 5xl05 to 106 PBMCs are added to each assay, m fte case where agent is added directly to the assay its concentration is from 10"1 to 10 ug/ml, preferably 0.5 to SOug/ml or 1 to lOug/rnL
Typically me lenglh 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 ec WHO PBMCs ft has been found mat 03x10* PBMCs can be incubated in lOug/ml of peptide for 12 hours at37°C. •"
The de*ermmation of the recognition of the agent by the T cells may be done by measuring 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 wfll be deemed to occur if the frequency of cells sorted using €be 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.
The detennination 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 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. Typically from 0.001 to 1000 ug, for example from 0.01 to 100 fig or 0.1 to 10 ug of agent is administered.
In one embodiment a product can be administered which is capable of providing the agent in vivo. Thus a polyrmcleotide capable of expressing the agent can be administered, typically in any of the ways described above for me administration of the agent The polynucleotide typically has any of the characteristics of me polynucleotide provided by Hie invention which is discussed below. The agent is caressed from the polynucleotide in vivo. Typically from 0.001 to 1000 ug, for example from 0.01 to 100 ug or 0.1 to 10 ug of polynucleotide is administered.
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 mat brads the . agent is typically earned out by contacting a sample from the individual (such as any of the samples mentioned here, optionally processed in any manner mentioned herein) with the agent and determining whether an antibody hi the sample binds the agent, such a binding indicating mat the individual has, or is susceptible to coeliac disease. Any suitable format of binding assay may be used, such as any such format mentioned herein.
Therapy
The identification of the inununodominant epitope and other epilopes 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 Ihe 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 tolerisation of the T celi that carries the TCR, Such agents, optionally in association with a carrier, can Iherefore 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 Ihe immune system in a • 'tolerising' context
epitope by the immune system. In the case of a T cell epitope this can be caused by the deletion oranergising 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 admmisljered.
Methods of presenting antigens -to the immune system in such a contract are known and are described for example in Yoshida et aL Clin. ImmunoL ImmunopamoL 82, 207-215 (1997), Thurau et aL Clin. Exp. ImmunoL 109, 370-6 (1997), and Werner et al. Res. tamunoL 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 mat favour a Tb2 response (e.g. EL-4, TGF-jJ or DL/-10). Products or agent may be administered at a dose mat causes tolerisation.
The invention provides a protein that comprises a sequence able la act as an antagonist of Hie 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 me antagonist may bind ELA-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 (he ait la one embodiment the antagonist is a. homologue of the epitopes mentioned above and may have any of the sequence, binding or other properties of me 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 me art The mutations are typically at the amino acid positions mat contact the TCR.
The antagonist may differ Jrom the epitope by a substitution within the sequence mat is equivalent to me sequence represented by ammo 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,67Mor65T.
Since me 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.
lie 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 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 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 man one substance.
Testing whether a composition is capable of causing coeliac disease
As mentioned above me 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. transglutammase to as sequence comprising the agent or epitope of the invention (such transgjntaminase activity may be a human intestinal tcansghrtmninaRe activity). Typically this is performed by using a binding assay in which a moiety which binds to me sequence in a specific manner is contacted with me composition and the fonnation of sequence/moiety complex is detected and used to ascertain the presence of the agent Such a moiety may be airy suitable substance (or type of substance) mentioned herein, and is typically a specific antibody. Any suitable format of 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 man one substance,
The composition typically comprises material from a plant mat expresses a gliadin which is capable of causing coeliac disease (for example any of the gliadins
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 giiadin. The processing of food material and testing in suitable binding assays is routine, for example as mentioned in Kriclca LJ, J. Biolumin. Chemilumin. 13,189-93(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 mat 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 me progress of me 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 giiadin protons
The invention provides a fdiadin protein in which an epitope sequence of the invention, or sequence which can be modified by a transgiutanrinase to provide such a sequence has been mutated so that it no longer causes, or is recognised by, a T cell response mat recognises me epitope. In mis context the term lecognition 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 wfld type of the mutated giiadin is one which causes coeliac disease. Such a giiadin may have horoology with SEQ ID NO:3, for example to the degree mentioned above (in relation to the analogue) across all of SEQ ID NO3 or across 15,30,60,100 or200 contiguous 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 mat giiadin. The sequences of ofter natural gh'adin proteins are known in the art
The mutated gliadin will not cause coeliac disease or wul 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 abijity to bind to or to be recognised (i.e. cause antigen-specific functional activity) by T cells that recognise tiie 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 onetjr 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 NO2 or across any of SEQIDNOS: 18-22,31-36,39-44, and 46; or across 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-gtiadin (or in an equivalent position in other gtiadins). Typically the naturally occurring glutamine at this position is substituted to any of the amino acids shown in Table 3, preferably to bistidine, tyrosine, tryptophan, tysine, proline, orargirune.
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 flie 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 feat comprises a sequence which is able to bind
which sequence is able to cause antagonism of a T cell mat 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 gHadfris or with non-gh'adin proteins.
The equivalent wild type protein to the mutated gliadin protein is typically from a graminaceous monocotyledon, such as a plant of genus Triticum, e.g wheat, rye, barley, oats or triticale. The protein is typically an a, ap, p, y or e> gliadin. The gliadin may be an A-gliadin.
Kits
The invention also provides a kit for carrying out fhe method comprising one . or more agents and optionally a means to detect the recognition of the agent by (he T cell Typically fhe 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 IFN-y. The moiety is typically immobilised on a solid support This means mat after binding the moiety the substance win 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 me substance. Quantifying me spots, and typically comparing agamet a control, allows determination of recognition of the agent
The kit may also comprise a means to detect the sabstance/moiety complex. A detectable change may occur in the moiety itself after binding me 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

the T cells directly in the sample without requiring any separation of the components of thesamp]e.
The kit may comprise an instrument which allows administration of the agent, such as inlradermal or epidermal administration. Typically such an instrument comprises plaster, dressing or one or more needles. The instalment may allow ballistic delivery of the agent The agent in the kit may be in me 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 me above methods. Typically in fee kits designed to determine recognition in vitro me 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
f ,
mononuclear cells or T cells from a sample from the host
PolynncleotideSj-cells, transgenic mammals and antibodies
The invention also provides a polyuucleotide which is capable of expression to provide the agent or mutant gliadin proteins. Typically the polynncleotide is DNA or EN A, and is single or double stranded. The potynucleotide win preferably comprise at least SO bases or base pans, for example 50 to 100,100 to 500,500 to 1000 or 1000 to 2000 or more bases or base paiis. The polynucleotide therefore comprises a sequence which encodes the sequence of SEQ ID NO: 1 or2oranyof the other agents mentioned herein. To the 5' and 31 of mis coding sequence the polynucleotide of the invention has sequence or codons which ate different from the sequence or codons 51 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 51 and/or 3' to the coding sequence may comprise sequences which aid expression, such as transcription and/or translation, of the sequence encoding me agent The polynucleotide may be capable of expressing the agent piokaryotic or eukaryotic cell In one embodiment me polynucleotide is
capable of expressing (he 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 tiiat encodes SEQ ID NO:3 at a level significantly above background. Selective hybridisation is typically achieved using conditions of medium to high stringency (for example Q.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 me art (see Sambrook et al (1989), Molecular Cloning: A Laboratory Manual).:For example, if high stringency is requited, suitable 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 polynncleotides described herein.
The polynucleotide may form or be incorporated into a replicable vector. 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 me invention is opeiably linked to a control sequence which is capable of providing for the expression of me polynucleotide. The vector may contain a selectable marker, such as the ampicfllin resistance gene. *
The polynucleotide or vector may be present in a cell. Such a cell may have been transformed by me polynucleotide or vector. The cell may express the agent The cell wfll be chosen to be compatible wife the said vector and may for example be a prokaryotie (bacterial), yeast, insect or mammalian cell The polynucleotide or vector may be introduced into host cells using conventional techniques including calcium phosphate precipitation, DEAE-dextran transfection, or electroporation.
The invention provides processes for the production of the proteins of the invention by lecombinant means. This may comprise (a) cultivating a transformed cell as defined above under conditions mat allow me expression of the protein; and preferably (b) recovering the expressed polypeptide. Optionally, the polypeptide 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
present in the any form mentioned hereb (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 Ihe 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 wefl as single-chain antibodies.
A method for producing a polyclonal antibody comprises nrnnrmismg a suitable host animal, for example an experimental m^mal, wifc the immunogen and isolating imnxunoglobulins from the serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from me animal and me 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 wife tumour cells (Kohler and Mflstein (1975) Nature 256,495-497).
An immortalized ceH 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 nnmunocomprornised host Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus. •
For ihe production of bom monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, me 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 earner. The earner molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.
The polynucleotide, agent, protein or antibody of the invention, may cany a detectable label. Detectable labels which allow detection of the secreted substance by visual inspection, optionally with fee 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 peroxidasej 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 me invention may be in substantially purified form. They may be in substantially isolated form, in which case they wfll generally comprise at least 80% e.g. at least 90,95,97 or 99% of ihe polynucleotide, peptide, antibody,-cells or dry mass in the preparation. The polyrincleotide, agent, protein or antibody is typically substantially free of other cellular components. The polynncleotide, 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 maybe 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 marnmal may also express BLA-DQ2 or -DQ8 or HLA-DR3-DQ2 and/or may be given a diet comprising a gKadfrn which cause coeliac disease (e.g. any of ihe 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 fee 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 prophykctic) agents or diagnostic substances (the agents, proteins and poryaucleotides of foe invention).
These substances are formulated for clinical administration by mixing them with a pbannaceulically acceptable carrier or diluent For example they can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular, intradermai, 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; me age, weight and condition of the patient to be treated; the mode of administration used; the severity of the condition to be treated; and me required clinical regimen. As a guide, the amount of substance administered by injection is suitably from 0.01 mg/kg to 30 mg/fcg, preferably from 0.1 ing/kg to 10 mg/kg.
The routes of administration and dosages described are intended only as a guide since a slcHled practitioner wffl be able to determine readily me 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 me 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 (he 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.
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 coeiiac 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 coeiiac 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 me expression of such a gliadin or to change the amino acid sequence of fee gliadin so mat it no longer causes coeiiac 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 me 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 coeiiac disease)).
In the case of plants or plant cells that express a protein (hat 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 coeiiac disease). Preferably though the presence of the antagonist sequence will cause reduced coeiiac disease symptoms (such as no symptoms) in an individual who ingests a food comprising protein from the plant or plant cefl.
The polymicleotide 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 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 promoters include those described by Dalta et al (Biotechnology Ami. Rev. (1997), 3, pp.269-296). Particular examples of seed-specific promoters are napin promoters (EP-A-Q 255,378), phaseolin promoters, glutenine promoters., helianthenme promoters (WO92/17580), albumin promoters (WO98/45460), oleosin promoters (WO98/45461) and ATS I and ATS3 promoters (PCT/US98/0679.8).
The cell may be in any form. For example, ft 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 Thecenmaybeofanytype(e.g.ofany ' type of plant part). For example, an ^differentiated cell, such as a callus cell; or a differentiated cell, such as a cell of a type found in embryos, pollen, loots, shoots or 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 polymicleotide or vector of me invention to give a transgenic plant cell Any suitable transformation method may be used (in the case of wheat the techniques disclosed in Vasfl 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
•*
The cell of the invention or thus obtained cell may bfe 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 aud/or division of the transgenic cell. Similarly, techniques such as somatic embryogenesis and meristem culture may be used. Regeneration techniques are well knovra 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, WO91/02071 and WO 95/06128.
In many such techniques,-one step is the formation of a callus, Le. 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 example, the invention provides transgenic plant tissues and parts, including embryos, meristems, seeds, shoots, roots, stems, leaves and flower parts. These may be chimeric in me sense mat some of their cells are ceDs of the invention and some are not Transgemc plant parts and tissues, plants and seeds of the invention may be of any of the plant species mentioned herein.
Regeneration procedures wfll 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 memods of obtaining ttansgenic plants of former generations from this first generation plant These are known as progeny transgenic plants. Progeny plants of second, third, fourm, fifth, sixfli and former generations may be obtained from (he 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 me 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 Jransgenic seed; and/or
propagating clonally a tansgenic plant of Jhe invention belonging to a previous generation to give a transgenic progeny plant of tbe invention belonging to a new generation; and/or
crossing a first-generation transgenic plant of me invention belonging to a previous generation with another compatible plant to give a transgenic progeny plant of file 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 mat 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, farmer 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 me'polynucleotides of me 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 mose coding for other herbicide resistance traits, e.g. tolerance to: Glyphosate (e.g. using an EPS? synrhase gene (e.g. EP-A-0 293,358) or a gryphosate oxidoreductase (WO 92/000377) gene); or tolerance to fosametin; a dihalobenzonitrile; glufosinate, e.g. using a phosphinothrycin aceryl transferase (PAT) or glutamine synrhase gene (cf. EP-A-0 242,236); asulam, e.g. using a dflrydropteroate synmase gene (EP-A-0 369367); or a sulphonyiurea, 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 imide such as chlorophmalim; 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 WO91/02701 orWO95/06128.
Typically, a protein of the invention is expressed in a plant of the invention. Depending on fee 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 fhe 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 axe 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 ftiat might mimic those produced hi 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 autoirnmune diseases.
Clinical and A-gliadin specific T-cell responses with 3 and 10 day bread challenge In a pflot 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 dafly in addition to iheir 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 mOd nausea at one week. TheEMA became positive in Subject 2 one week after fte bread challenge, indicating foe bread used bad caused a relapse of Coeliac disease. But in Subject 1, EMA remained negative up to two mon&s 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 breiad challenge. But from me day after bread withdrawal (Day-4) in Subject 1 a single pool of 5 overlapping peptides spanning A-gliadin 51-85 (Pool 3) treated wim tTG showed potent IFNy responses (see Figure la), In Subject 1, me PBMC SFNy 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 o-chymotrypsin digested gUadtn. PBMC IFNy responses to tTG^breated Pool 3 were consistently 5 to 12-fold greater than Pool 3 not treated with tTG, and responses to a-cbymotrypsb digested gliadin were 3 to 10-fold greater if treated wim tTG. In Subject 2, Pool 3 treated with tTG was also the only immunogenic set of A-gliadin peptides on Day 8, but mis response was weaker than Subject 1, was not seen on Day 4 and by Day 11 me response to Pool 3 had dimmished and other tTG-4reated pools of A-gliadin peptides elicited stronger IFNa
responses (see Figure Ib).
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 ten days instead of three, T cell responses to olher A-gliadin peptides appear, consistent wilh'epitope spreading.
Coeliac disease-specific IFN-g induction by tTG-treated A-gliadin peptides
In five out of six further Coeliac disease subjects on gluten free diet (see Table 1), bread challenge for three days identified tTG-lreated peptides in Pool 3, and in particular, peptides conesponding to 56-70 (12) and 60-75 (13) as fee sob A-gliadin components eliciting IFNy from PBMC (see Figure 2). IL-10 ELISPOT assays run in parallel to IFNy' ELISPOT showed no H/-10 response to tTG-feeated peptides 12 or 13. fa one subject there were no IFNy responses to any A-gliadin peptide or a-chymotrypsin digested gtiadin 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 bealfey, EMA-aegative subjects wife (he HIA-DQ afldes al*0501, pi *0201 (ages 28-52,2 females) who had been challenged for three days with bread after following a gluten free diet for one month, showed no IFNy responses above the negative control to any of file A-gh'adin peptides wife or without tTG treatment Thus, induction of IFNy in PBMC to fKMreatedPool 3 and A- • gjiadia peptides 56-70 (12) and 60-75 (13) were CoeBac disease specific (7/8 vs. 0/4,. pO.Ol by On-squared analysis).
Fine mapping offfie minimal A-gliadin Tcell epitope
tTG-treated peptides representing truncations of A-gh'adin 56-75 revealed that fee same core peptide sequence QPQLP (SBQ ID NO:9) was essential for antigenicity in all of fee five Coeliac disease subjects assessed (see Figure 3). PBMC IFNy responses to fTG4reated peptides spanning tins core sequence beginning with the 7-*ner PQPQLPY (SEQ ID NO:4) and increasing in length, indicated feat fee tTG-*ceated 17-mer QLQPFPQPQLPYPQPQS (SEQ ID NO:10) (A-gliadia 57-73)
possessed optimal activity in the IFNy ELISPOT (see Figure 4).
Deamidation ofQ65 by tTG generates tlie immunodomvnant Tcell 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 tiiat out of the BIX ghrtamine (Q) residues contained in A-' gliadin 56-75, Q65 was preferentially deamidated by tTG (see Figure 5). Bioaetivity 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 TOth Q65 after tTG4reatment (see Figure 4a). Replacement of Q57 and Q72 by E together or alone, with E65 did not enhance antigenicify of the 17-roer in the three Coeliac disease subjects studied (see Figure 6). Q57 and Q72 were investigated because glutamine residues followed by proline in gHpd?n peptides are not deamidated by £TG in vitro (W. Vader et al, Proceedings 8ih International .Symposium CoeHac Disease). Therefore, the fanmunodominant T cell epitope was defined as QLQPFPQPELPYPQPQS (SBQ ID NO:2).
Immimodominant Tcell epitope response is DQ2-restricted and CD4 dependent
In two Coeliac disease subjects homozygous for HLA-DQ al*0501, Pl*0201, anti-DQ monoclonal antibody blocked the EUSPOT IFNy 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 &om two Coeliac disease subjects indicated the IFNY response to tTG-treated A-^iadin 56-75 is CD4 Tcell- • 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 bloodof 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 al*0501, pl*020I. Tissue transglutaminase action in vitro selectively deamidates Q65. Elicited peripheral blood IFNg responses to synthetic A-gliadin pepiides 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 pepiides.
We havfc 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 mat may occur with the use of T cell clones hi 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-gliadm'peptides; A-gliadin 57-73 modified by fTG 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 gh'adms of equal or greater importance in the pamogenesis of Coeliac disease. Indeed, me peptide sequence at the core of the epitope in A-gliadin that we have identified PQPQLPY (S6Q ID KO:4) is shared by several other ghadins (SwissProt and Trembl accession numbers: P02863, Q41528, Q41531, Q41533, Q9ZP09, P04722, P04724, P18573). However, A-gliadin peptides feat have previously been shown to possess bioactivity in biopsy challenge and in vivo studies (for example: 31-43,44-55, and 206-217)4>s 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 Tcett recognition of substitutions in the immanodominant epitope
The effect of substituting the gluramate 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 jig/ml). The responses were measured hi 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 ghitamate to histidine,
tyrosine, tryptopaan, lysine, proline or argrnine stimulated a response whose magnitude was less than 10% of the magnitude of the response to the immunodominanl epitope. Tbus mutation of A-gliadin at mis position could be used to produce a mutant gliadin with reduce or absent imimraoreactivity.
Example 3
Testing the immunoreactivity of equivalent peptides from otiier naturally occurring gliadins
The immunoreaotivity of equivalent peptides form other naturally occurring wheat gliadins was assessed using synthetic peptides corresponding to me naturally occurring sequences which were men treated with transglutaminase. 'These peptides were tested in an ELJSPOT in the same manner and with PBMCs from the same subjects as described in Example 2. At least five of the peptides show irmnunoreactiviry comparable to me A-gliadin 57-73 E65 peptide (after transglutaminase treatment) indicating mat other gh'adin proteins in wheat are also likely to induce mis Coeliac disease-specific immune response (Table 4 and Figure
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 Wool HLA-DQA and DQB genotypmg was performed by PCR using sequence-specific primer mixes6*.
Anti-endomysial antibody assay: BMA were detected by indirect immunofluorescence using patient serum diluted 1:5 with monkey oesophagus, followed by FlTC-conjugated goat anti-human IgA, IgA was quantitated prior to EMA, none of the subjects were IgA deficient
Antigen Ciiallenge: Coeliac disease subjects following a gluten free diet, consumed 4 slices of gluten-containing bread (SOg/slice, Sainsbuxy'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 andJL-10 EUSPOT: PBMC were prepared from 50-100 ml of venous blood by Ficbll-Hypaque density centri&gation. After three" washes, PBMC were vesuspended in complete KPMI containing 10% heat inactivated human AB serum. ELJSPOT assays for single cell secretion of IFNy and IL-10 were performed using commercial kits (Mabtech; Stockholm, Sweden) wim 96-welI plates (MAIP-S-45; MiUipore, Bedford, MA) according to the manufacturers instructions (as described elsewhere9) with 2-SxlO5 (IFNy) or 0.4-lxlO5 (JDL-10) PBMC in each well Peptides were assessed in duplicate wells, and Mycobacterium tuberculosis purified protein derivative (PPD RT49) (Serum Institute; Copenhagen, Denmark) (20 ug/ml) was included as a positive control in all assays.
Peptid&s: Synthetic peptides were purchased from Research Genetics (HuntsvQle, Alabama) Mass-spectroscopy and HPLC verified peptides' authenticity and >70% purity. Digestion of gliadin (Sigma; G-3375) (100 nag/ml) with a-chymotrypsin (Sigma; C-3142) 200:1 (w/w) was performed at room temperature in 0.1 M NH«HCO3 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-Chymotrypsm-digested gliadin (640 jig/ml) and synthetic gliadin peptides (15-mers: 160 ugftnl, other peptides: 0.1 mM) were individually treated with tTG (Sigcna; T-5398) (50 |ig/ml) in PBS+CaCU 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 ofpeptides: 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 494A, Applied Biosystems, Foster City, California). The sequence of unmodified G56-75 was confirmed as: LQLQPFPQPQLPYPQPQSFP (SEQ ED 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 percentof the combined amount of glutamine and glutamate recovered in cycles 2,4, 8,10,15 and 17 of fee amino acid sequencing. Deamidation attributable to tTG was defined as (% deamidation of glutamine in fee tTG treated peptide - % deamidation in the untreated peptide) / (100 - % deamidation in fee untreated peptide).
CD4/CD8 and HLA doss n Restriction: AntKJM or anti-CDB coated magnetic beads (Dynal, Oslo, Norway) were washed four times with RPMI fiien incubated wife PBMC in complete RPMI containing 10% heat inactivated human AB serom (5x10 cells/ml) for 30 minutes on ice. Beads were removed using a magnet and celts remaining counted. In vivo HLA-classH restriction of me immune response to tTG-heated A-gUadin 56-75 was established by incubating PBMC (5x10* cells/ml) with anti-HLA-DR (L243), -DQ (L2), and -DP (B7.21) monoclonal antibodies (10 ug/tnl) at room temperature for one hour prior to fhe addition of peptide.
Example 4
Mucosal integral expression bygiadin -specie peripheral blood lymphocytes
Interaction between endothelial and lymphocyte adressins facilitates homing of organ-specific lymphocytes. Many adressins are known. The heterodimer 04*7 is specific for lamina propria gut and other mucosal lymphocytes, and a% is specific and mtra-epimelial lymphocytes in the gut and slon. Approximately 30% of peripheral blood CD4 T cells express a$j and are presumed to be in transit to a mucosal site, while 5% of peripheral blood T cells express o6^. Jmmunomagnetic beads coated with antibody specific for aE or p 7 deplete PBMC of cells expressing a% or aEpV and o^p?, respectively. In combination with HUSpot assay,

immunomagnetic bead depletion allows determination of gliadin-specific T cell addressin expression feat may identify these cells as homing to a mucosal surfSace. Interestingly, gluten challenge in vivo is associated wife rapid influx of CD4 T cells to fiie small intestinal lamina propria (not intra-epithelial sites), where over 90% lymphocytes express 0$?.
Irxnmmomagnetic beads were prepared and used to deplete PBMC from coeliac subjects on day 6 or 7 after commencing 3 day gluten challenge. FACS analysis demonstrated a E beads depleted approximately 50% of positive CD4 T cells, while p 7 beads depleted alrp7 positive CD4 T cells. Depletion of PBMC using CD4- or P 7-beads, but not CDS- or aE -beads, abolished responses in the interferon • gamma ELESpot tTG gliadin and PPD responses were abolished by CD4 depletion, but consistently affected by integral-specific bead depletion.
Thus A-guadin 57-73 QE65-Bpepific T cells induced after gluten challenge in coeliac disease express the integrin, tt$j, 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 ammo acids in length spanning
the
core of the dominant T cell epitope in A-gliadin indicated mat the 17mer, A-ghadin 57-73 QE65 (SEQ ID NO2) induced T»ayimal responses in fee interferon gamma Elispot using peripheral blood mononuclear cells (PBMQ from coeliac volunteers 6 days after commencing a 3-day gluten challenge.
Peptides representing expansions form me core sequence of the dominant T cell epitope in A-gliadin were assessed in the JFN gamma ELISPOT using peripheral blood mononuclear cells (PBMQ from coeliac volunteers in 6 days after commencing a 3-day gluten challenge (o=4). Peptide 13: A-gtiadin 59-71 QE65 (13mer), peptide 15:58-72 QE65 (ISmer), _, peptide 27:52-78 SE65 (27mer).
As shown hi Figure 11 expansion of fiie A-gliadin 57-73 QE65 sequence does not substantially enhance response in the BFNgamma Elispot. Subsequent Examples
characterise the agonist and antagonist activity of A-gUadin 57-73 QE65 using 17mer peptides.
Example 6 5 Comparison ofA-gliadin 57-73 'QE65 with other DQ2-restricted Tcell epUopes in
coeliac disease
Dose response studies were performed 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. D Responses ID peptides were expressed as percent of response to A-gliadin 57-73
QE65.' All subjects were BDLA-DQ2-}- (none were DQ8+).
The studies indicate that A-gliadin 57-73 QE65 is the most potent gKadin
peptide for induction of interferon gamma in me ELXSpot assay using coeliac PBMC
after gluten challenge (see Figure 12a-h, and Tables 5 and 6). The second and third 5 epitopes are suboptimal fragments of larger peptides Le. A-gliadin 57-73 QE65 and
GDA4_WEOEATP04724-84-100QE92. Tne epitope is only modes%bioactive
(approximately 1/20* as active as A-gliadin 57-73 QE55 after blank is subtracted). A-gliadin 57-73 QE65 is more potent man other known T cell epitopes in
coeliac disease. There are 16 polymorphisms of A-gliadin 57-73 (including the sequence PQLPY (SEQ ID NO.12)) amongst sequenced gjiadin genes, their
bioactivity is assessed next.
Example 7
Comparison of gKadin- and A-gliadin 57-73 QE65-specific responses inperipheml
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 o.9-gliadin generates the peptide QLQPFPQPELPY (SEQ ID NO: 13), mat is a truncation of me optimal epitope
sequence QLQPFPQPELPYPQPQS (SEQ ID N02) (see above). Transglulaminase-treatmen.t substantially increases the potency of cbymotrypsin-digested gliadin in proliferation assays of gliadin-specific T cell clones and lines. Hence, transglutaminase-treated chymotrypsin-digested gHadin (tTG gliadin) may not be an ideal antigen, but responses against this mixture inay approximate the "total" number of peripheral blood lymphocyte specific for gliadin. Comparison of responses against A-gliadin 57-73 QE65 and tTG gliadin in me ELISpot assay gives an indication of the contribution of this dominant epitppe 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 cbymotrypsin-digested gliadin +/- tTG treatment and compared with ELISpot responses to an optimal concentration of A-gliadin 57-73 QB65 (25mcg/nu). TTG treatment of gliadin enhanced PBMC responses in the EOSpot approximately 10-fold (tTG was comparable to blank when assessed alone) (see Figure 13a-c). fa fee four coeliac subjects studied, A-gliadm 57-73 QE65 (25 meg/ml) elicited responses between 14 and 115% those of fTG gliadin (500 meg/ml), and me greater the response to A-gliadin 57-73 QE65 the greater proportion it represented of me 1TG gliadin response.
Relatively limited data suggest feat A-gliadin 57-73 QE65 responses are comparable to tTG gliadin in some subjects. Epitope spreading associated with more evolved anti-gnadin T cell responses may account for fee smafier contribution of A-' gKadin 57-73 QE65 to "total" gliadin responses in peripheral blood in some individuals. Epitope spreading may he inqii?fomgd in indlvidflalg wifti H^SP sfrfcfly gluten free diets.
Example 8
Dilution of gliadin peptides bioacKve in coeliac disease: polymorphisms ofA-
gliadin 57-73
Overlapping ISmer peptides spanning the complete sequence of A-gliadin were assessed in order to identify the immunodonrinant 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-Prol 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/ml), unmodified peptide and transglutaminase-treated peptides were assessed at 25 meg/ml only. Bioactivity was expressed as % of response associated with A-gliadin 57-73 QE65 25 meg/ml in individual subjects (n=4). (See Fig 14).
Bioaetivity of "wild-type" peptides was substantially increased (>5-fold) by treatment with transglutaminase. Transglutaminase treatment of wild-^type peptides resulted in bioactivity similar to (hat of the same peptides substituted wife glutamate at position 10. Bioactivities of five gjLutamate-substitnted peptides (B, C, K, L» M), were >70% lhat of A-gliadin 57-73 QE65 (A), but none was significantly more bioactive than A-gUadin 57-73 QE65. PBMC responses to glutamate-substituted peptides at concentrations of 2.5 and 250 meg/ml were comparable to those at 25 mcgfaoL Six glutamate-substituted gliadin peptides (H, I, J, N, O, P) were At least six gtiadin-derived peptides are .equivalent in potency to A-gliadin 57-73 QE65 after modification by transglutamrnase:' Relatively non-bioactive polymorphisms of A-guadin 57-73 also exist. These data indicate mat transglutaminase modification of peptides from several gliadins of Tr&cum aesttvum, T. uartu and T. speJta 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 gUadin genes. Generation of wheat containing gliadins or other proteins or peptides incorporating sequences defining altered peptide ligand antagonists of A-gliadm 57-73 is an alternative strategy to
generate genetically modified wheat that is therapeutic rather than "non-toxic" in coeliac disease.
Example 9
Definition of Core Bpitope Sequence:
Comparison of peptides corresponding to truncations of A-gliadia 56-75 from the N- and C-tenninal indicated that fee 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-gliadia feat are substituted at residues that substantially contribute to its bioactiviry.
Peptides corresponding to A-gJiadin 57-73 QE65 wife alanine (Figure 15) or lysine (Figure 16) substituted for residues 57 to 73 were compared in fee IFN gamma ELISPOT using peripheral blood mononnclear cells (PBMC) from coeliac volunteers 6 days after commencing a 3-day gluten challenge (n=8). (BL is blank, E is A-gliadm 57-73 QE65: QLQPFPQPELPYPQPQS (SEQ ID NO2)).
It was found feat residues corresponding to A-gliadin 60-70 QE65 (PFPQPELPYPQ (SEQ ID N0:14)) contribute substantially to fee bioactivity in A-gliadm 57-73 QE65. Variants of A-gliadin 57-73 QE65 substituted at positions 60-70 are assessed in a 2-step procedme. Initially, A-gliadm 57-73 QE65 substituted at positions 60-70 using 10 different ammo acids wife contrasting properties are assessed. A second group of A-gliadin 57-73 QE65 variants (substituted wife all ofeer naturally occurring amino acids except cysteine at positions feat prove are sensitive to modification) are assessed in a second round
Example 10
Agonist activity of substituted variants of A-gliadin 57-73 QJE65
A-gliadin 60-70 QE65 is fee core sequence of fee dominant T cell epitope in A-gliadin. Antagonist and non-agonist peptide variants of feis epitope are most likely generated by modification of feis core sequence. Initially, A-gliadin 57-73 QE65 substituted at positions 60-70 using 10 different amino acids wife contrasting properties will be assessed in fee IFNgamma ELtSPOT using PBMC from coeliac subjects 6 days after starting 3 day gluten challenge. A second group of A-gliadin
57-73 QE65 variants (substituted with all oilier Baturally occurring amino acids except cysteine) at positions 61-70 were also assessed. Both groups of peptides (all at 50 meg/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 meg/ml.
Results are expressed as mean response in spot forming cells (95% confidence interval) as % A-G 57-73 QB65 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 ihan A-G 57-73 QE65 at a level of significance of p0.01) from blank (buffer without peptide). Peptides wifli agonist activity 30% or less that of A-gliadin 57-73 QE65 were considered "suitable" partial or non-agonists to assess for antagonistic activity (see Table 8 and Figures 17-27).
The lENgamma 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 Kahancing binding to HLA-DQ2 since the motif for flns HLA molecule indicates a preference for bulky hydrophobia resides at positions 1 and 9. Eighteen non-agonist peptides were identified. Bioactivities of me variants (50 meg/ml): P65, K64, K65 and Y65 (bioactivity 7-8%) were comparable to blank (7%). In total, 57 mutated variants of A-gliadin 57-73 QB65 were 30% or less bioactive than A-gliadin 57-73 QE65.
The molecular specificity of the peripheral blood lymphocyte (PBL) T cell response to me dominant epitope, A-gUadin 57-73 QE65, is consistently reproducible amongst BLA-DQ24- coeliac subjects, and is highly specific to a restricted number of ammo acids in the core 7 amino acids. Certain single-amino acid variants of A-gliadin 57-73 QE65 are consistently non-agonist? in all HLA-DQ2+ coeliac subjects.
Example 33
Antagonist activity of substituted variants
The homogeneity of the PEL 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 PEL 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 weaMybioactive naturally occurring polvmorphisms of A-gliadin 57-73 QE65 have also been identified (see below) and may be "naturally occurring" APL antagonists. It has also been suggested (hat competition for binding MHC may also antagonise antigen^specific T cell immune. Hence, non-gliadin peptides that do not induce IFNgamma responses in coeliac PEMC 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 flat bind avidly to HLA-DQ2 are HLA class 1 o 46-60 (HLA la).(PRAPWBBQEGPEYW(SEQ ID N0:15)) and thyroid peroxidase (to) 632-645Y (TOVWI/3GIiAENFLPY Simultaneous addition of peptide (50pg/ml) or buffer and A-gliadin 57-73 QE65 (lOug/ml) 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 ammo acid-substituted variants of A-gliadin 57-73 QS65 reduce me interferon gamma PBMC ELISPOT response to A-gtiadin 57-73 QE65 (pO.Ol) by between 25% and 28%, 13 other peptide variants reduce the ELISPOT response by between 18% and 24% (p QE90 (PQPQPFPPELPYPQPQS (SEQ ED NO: 17)) reduces responses to A-gliadin 57-73 QE65 by 19% (p Interferon gamma responses of PBMC to A-gliadin 57-73 QE65 in EL1SPOT assays are reduced by co-administration of certain single-ammo acid A-ghadin 57-73 QE65 variantSj-a polymorphism of A-gliadin 57-73 QE65, and an unrelated peptide known to bind'HLA-DQ2 in five-fold excess. These finding suggest mat 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 PEL T cell responses to A-gliadin 57-73~QE65.
Tbese findings support two strategies to interrupt the T cell response to the dominant A-gliadin epitope in HLA-DQ2-f 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 gliadjn genes in •wheat
2. Use of high affinity HLA-DQ2 binding peptides to competitively inhibit
presentation of A-gliadin 57-73 QE65 in association wife HLA-DQ2.
These two approaches may be mutually compatible. Super-agonists were generated by replacing F61 and Q70 wfih tyroane residues. It is likely mese super-agonists resulted from improved binding to HLA-DQ2 rather than enhanced contact with flie T ceu 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 ofinteiferon gamma ELESpot 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
fcgMC measuredln^ielnterferon gamma EOSpot follows gluten challenge in almost all DQ2+ coeliac subjects following a long term strict gluten fiee diet (GFD) but aot in healmy DQ2+ subjects after 4 weeks following a strict GED. 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+eoeliac subjects were recruited from fhe 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'challengers 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-gJiadin 57-73 QE65 (A), P04724 84-100 QE92 (B) (alone and combined) and A-gliadin 57-73 QP65 (P65) (non-bioactive variant, see above) (afl 25 meg/ml) were assessed.
All bat one newly diagnosed coeliac patient was DQ2+ (one was DQ8+) (n-11). PBMC from newly diagnosed coeliacs mat were untreated, or after lor 2 weeks following GFD did not show responses to A-gliadin 57-73 QE65 and P04724 84-100 QE92 (alone or combined) that were not significantly different from blank or A-$iadin 57-73 QP65 (n=£) (see Figure 28). Gluten challenge in coeliacs who had followed GFD for only one week did not substantially enhance responses to A-giadin 57-73 QE65 orP04724 84-100 QE92 (alone or combined). But gluten challenge 2 weeks after commencing GFD did induce responses to A-ghadin 57-73 QB65 and P04724 84-100 QE92 (alone or combmed) 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 fee 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 thai "immune unresponsiveness" to tMs dominant T cell epitope prevails in untreated coeliac disease and for at least one week alter starting GFD. The optimal timing of a 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 tiae peripheral blood in unteeated coeliacs, and can only be induced by gluten challenge after at least 2 weeks GFD (antigen exclusion). Therefore, timing of a diagnostic test using mis methodology is crucial and further studies are needed for its optimization. These finding ate consistent with functional energy 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 human disease, and supports the possibility that T cell anergy may be inducible with peptide therapy in coeliac disease.
Example 13
Compreliensive Mapping of Wheat Gliadin T CeH Epitopes
Antigen chaUeogemduces antigen-specific T cells in peripheral blood, hi 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 me molecular specificity of gluten T cell epitopes. As described above, we have identified a single dominant T cell epitope in a model gluten protein, A-gliadin (57-73 deamidated at Q65). In this Example, 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 andHLA-DQ8 associated coeliac disease. Seven of the 9 coeliac subjects with me classical HLA-DQ2 complex (ELA-DQAl*05,.HLA-DQB1*02) present in over 90% of coeliacs had an inducible A-gfiadin 57-73 QE65-and gh'adin-specific T ceD response in peripheral blood. A-gh'adin 57-73 was fte
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, tiiere were as many as 5 families of structurally related peptides that were between 10 and 70% as potent as A-gliadin 57-73 in the interferon-? ELISpot assay. These new T cell epitopes were derived from 7- and o-gliadins and included common sequences that were structurally very similar, but not identical to me core sequence of A-gliadin 57-73 (core sequence: FPQPQLPYP (SEQ 3D NO:18));for example: FPQPQQPFP (SEQ JD NO:19) and PQQPQQPFP (SEQ ID NO:20). Although no homologues of A-gliadin 57-73 have been found hrrye or barley, fee other two cereals toxic in coeliac disease, the newly defined T cell epitopes in y- and c^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 me 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 HLA-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 ceH epitope was identified with the core sequence LQPQNPSQQQPQ(SEQIDNO:21). The tonsglutainniase-dearmdated version of this peptide was more potent than the non-deamidated peptide. Previous studies suggest feat the transglutammase-deamidated peptide would have the sequence LQPENPSQBQPE (SEQ ID NO22); but former studies are required to confirm this sequence. Amongst flje healthy HLA-DQ2 (10) and HLA-DQ8 (1) subjects who followed a gluten free diet tor a month, gjiadin peptide-specafic 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 ELA-DQ2-associated coeliac disease, of which A-gliadin 57-73 is the most potent HLA-DQ2- and HLA-DQ8-associated coeliac disease do not share the same T cell specificity.
We have shown ihat shorMenn gluten challenge of individuals wim coeliac disease following a gluten free diet induces gliadin-specific T cells in peripheral blood. The frequency of mese 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 0,4(37 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 15mer 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 epilope 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 o/p1-gliadin fraction of wheat gluten is consistently toxic in coeliac disease, and most studies have focused on these proteins. The gene cluster coding for a/pv-gliadins is located on wheat chromosome 6C. There are no homologues of o/JJ-gliadins in rye or barley. However, all three of the wheat gtiadin subtypes (fl/P,y, and ca) are toxic hi coeliac disease. The y- and ffi~gliadin genes ate 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 o-gliadin sequence. Anderson et al (1997) have estimated that mere are a total of about 150 distinct a-gliadin genes in T. aestivum, but many ate 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-gliadm peptides almost identical to A-gUadin 57-73 as potent T cefl epitopes specific to coeliac disease. Over 90% of coeliac patients are HLA-DQ24-, and BO 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 Hie only T-cell epitopes recognized by T cells induced by gluten challenge in bom HLA-DQ2+ and HLA-DQ8+ coeliac disease. If mis were the case, design of peptide therapeutics for coeliac disease might only require one peptide.
Homologues ofA~gliadm 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 ) only revealed o/p-gliadins. However, our fine-mapping studies of the A-gliadin 57-73 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 X]OXXXXPQ[ILMP]fPSTJXXXXXX (SEQ H> N0:23). Homologues were identified amongst y-gliadins, gjutenins, hordeins and secalins (see Table 12). A further homologue was identified in o-giiadin by visual search of the three o>-gliadin entries in Genbank.
These homologues of A-gliadin 57-73 were assessed after deamidation by tTG (or synthesis of file glutamate(QE)-substituted variant in four close homologues) using the XFNy ELISpot assay with peripheral blood mononuclear cells after gluten challenge in coeliac subjects. The In order to identify all possible T cell epitopes coded by fiie known wheat (Triticum aestivum) gliadin genes or gene fragments (61 o/pS 47 y-, and 3 o-fdiadin 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 mappuig
Healthy controls (HLA-DQ2+ n=10. and HLA-DQ8+ n=l) 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-DQ8AX) (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 300ml 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 (DFNy) EUSpot assays.
Peptides were synthesized in batches of 96 as Pepsets (Mimotopes Inc., Melbourne Australia). Approximately 0.6 micromole of each of 652 20mets-was provided. Two maricer 20mer peptides were included in each set of 96 (VLQQHNIAHGSSQVLQESTY-peptide 161 (SEQ ID NO34), and IKDfHVYFKESRDALWKGPG (SEQ 3D NO:25)) and were characterized by reverse phase-HPLC and amino acid sequence analysis. Average purities of these .madaerpeptides were 50% and 19%, respectively. Peptides were initially dissolved inacetonitrile (10%) and Hepes lOOmM to lOmg/mL
The final concentration of individual peptides in pools (or alone) incubated with PBMC for the ffNy ELISpot assays was 20 ug/ml. Fiv&4nnes concentrated solutions of peptides and pools in PBS with calcium chloride LmM were afiquotied and stored in 96-well plates according to fee template later used in ELISpot assays. Deanridated peptides and pools of peptides were prepared by incubation wiih guinea pigtissue tTG (Sigma T5398) in fee ratio 10032 vg/ml for two hours at 37°C. Peptides solutions were stored at —20°C and freshly thawed prior to use.
Gliadin (Sigma G3375) (100 mg/rnl) in endotoxin-fiee water and 2M urea was boiled for 10 minutes, cooled to room temperature and incubated wife filter (0.2 Hm)-sterilised pepsin (Sigma P6887) (2 mg/ml) in HCl 0.02M or chymotrypsin (C3142) (4mg/ml) in ammonium bicarbonate (02M). After incubation for 4 hours, pepsin-digested gliacfin was neutralized with sodium hydroxide, and men bom pepsin- and chymotrypsin-digested gliadin were boiled for 15 minutes. Identical incubations with protease in which gliadin was omitted were also performed.
Samples were centrifuged at 1 5 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 ia the ratio 2500:64
IFNy ELISpot assays (Mabtech, Sweden) were performed in 96-well plates (MAIP S-45, Millipore) in which each well contained 25ji! of peptide solution and l00µl of PBMC (2-8xl05/well) in. RPMJ 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 bom day 0 and day 6. All wells in the plate containing deamidated peptides included tTG (64 ug/ml). 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) (QLQPFPQPELPYPQPQP (SEQ ID NO27)), P02863 77-93 (QLQPFPQPQLPYSQPQP (SEQ IDNO:28)), P02863 77-93 QE85 (QLQPFPQPELP YSQPQP (SEQ ID NO29)), and chymotrypsm-digested gliadin (500 ug/ml), pepsin-digested gliadin (500 ug/ml), chymotrypsin (20 fig/ml) alone, pepsin (10 fig/ml) alone, and blank (PBS-h/-tTG) (in triplicate).
After development and drying, IFNy ELISpot plates were assessed using the MAIP automated ELISpot.plate counter. In HLA-DQ2 healthy and coeliac subjects, induction of spot forming cells (sic) by peptide pools in the IFNy ELISpot assay was tested using a one-tailed WHcoxon Matched-Pans Sigried-Ranks test (using SPSS software) applied to spot forming cells (sib) per million PBMC minus blank on day 6 versus day 0 ("net response"). Significant induction of an IFNy response to peptide pools in PBMC by in vivo gluten challenge was denned as a median "net response" of at least 10 sfc/mfllion PBMC and pO.05 level of significance. Significant response to a particular pool of peptides on day 6 was followed by assessment of individual peptides wiihin each pool using PBMC drawn the same day or on day 7.
For IFNy ELISpot assays of individual peptides, bioactivity was expressed as & percent of response to P04722 77-93 QE8S 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 wifh mean bioactivity greater thanlO% that of P04722 QE85
were analyzed for common structural motifs.
Results
Hedthy HLA-DQ2 subjects
None of the healthy KLA-DQ2+ subjects following a gluten fiee diet for a month had IFNy ELISpot responses to homologues of A-gliadin 57-73 before or afler gluten challenge. However, in 9/10 healthy subjects, gluten challenge was assocaated -with a significant increase in IFNy responses to both peptic- and chymotryptic-digests of gliadin, from a median of 0-4 Etc/million on day 0 to a median of 16-29 sfc/nrillion (see Table 14). Gliadin responses in healthy subjects were unaffected by deamidation (see Table 15). Amongst healthy subjects, mere was no consistent induction of IFNy responses to specific gliadin peptide 'pools with gluten challenge (see Figure 30, and Table 16). EPNy ELISpot responses were occasionally found, but these were weak, and not altered by deamidation. Many of me strongest responses to pools were also present on day 0 (see Table 17, subjects H2,B8 and H9). Four heaKhy subjects did show definite responses to pool 50,and the two with strongest responses on day 6 also had responses on day 0. in bom subjects,-1he post-challenge' responses to pool 50 responses were due to peptide 390 (QQTYPQEPQQPFPQTQQPQQ (SEQ ID NO30)). 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 sfctoMon (see Table 4). One of the six coeliac subjects (CQ6) did not respond to P04722 77-93 QE85 (2 sfc/mflJion) and had only weak responses to gliadin peptide pools (maximum: Pool 50-tfTG 27 sfc/milUon). Consistent with earlier work, bioactivity of wild-type P04722 increased 6.5 times with deamidation by 1TG (see Table 15). Interfcron-gamma responses to gliadin-digests were present at baseline, but were substantially increased by gluten challenge from a median of 20 up to 92 sfb/mfllion for chymotryptic-gtiadin, and from 44 up to 176 sic/million for peptide-gh'adin.
Deamidation of gliadin increased bioactivity by a median of 3.2 times for chymotryptic-gliadin and 1.9 limes for peptjc-gliadin (see Table 15). (Note feat ilie acidity required for, digestion by pepsin is likely to result in partial deamidation of gliadin.)
In contrast to healthy subjects, gluten challenge induced JFNy ELISpot responses to 22 of the 83 tTG-treated pools including peptides from a-, y- and One hundred and seventy individual tTG-deanridated peptides fiom 21 of fee most bioactive pools were separately assessed. Seventy-two deamidated peptides were greater man 10% as bioactive as P04722 77-93 QE85 at an equivalent concentration (20 ng/inl) (see Table 19). The five most patent peptides (85-94% bioactivity of P04722 QE85) were previously identified a-gliadm homologues A-gliadin 57-73. Fifty of the bioactive peptides were not homologues of A-gJiadin 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: PCK3PQQPOOPPPOPOOPFPW (SEQ ID NO31) (peptide 626, median 72% bioactivity of P04722 QE85),
(Sequence Removed)
(core sequences are underlined). All of these sequences include gmtamine residues, predicted to be susceptible to deamidation by bansgluiaminase (e.g. QXP, QXPF (SEQ ID N037), QXJqFn (SEQ ID N0:38)) (see Vader et al 2002). Some bioactive peptides contain two core sequences from different families.
Consistent with the possibility tbat 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 63L(bomologue of A-gliadin 57-73) 61%, 636 (bomologue of 626) 51%, and 635 19%, 629 16%, and 634 13% (all homplogues of 396).
Although likely to be an oversimplification, the contribution of each "peptide
family" to the summed IFNy ELISpot response to gliadin peptides was compared in
the HLA-DQ2-f coeliac subjects (see Figure 32). Accordingly, fee contribution of
P04722 77-73 E85 to the summed response to gliadin peptides is between 1/5 and
2/3. ' •
Using fee peptide homology search programme, WWW PepPepSearch, which can be accessed through fee world wide web of fee internet at, for example, "cbrg-inf ethzjcb/subsection3_l_5 JrimL", and by direct comparison wife Genbank sequences for rye secalins, exact matches werefoand for fee core sequences QQPFPQPQQPFP (SEQ ID NO39) in barley horfeins (HORS) and rye secalins (A23277, CAA26449, AAG35598), QQPFPQQPQQPFP (SEQ ID NO:40) inbadey hoideins (HOG1 and HORS), and for PIQPQQPFPQQP (SEQ ID NO:43) also in barley hordeins (HORS).
HLA-DQ8-assotiated coeliac disease
' Seven HLA-DQ8+coehac subjects were studied before and after gluten challenge. Five of feese HLA-DQ8+ (HLA-DQAO*0301-3, HLA~DQBO*03D2) subjects also earned one or bom of fee coeliac disease-associated HLA-DQ2 complex (DQAO*05, DQBO*02). Two of fee ftree subjects wife bofe coeliac-associated HLA-DQ complexes had potent responses to gliadin peptide pools (and individual peptides including P04722 77-93 B85) feat were qualitatively and quantitatively identical to HLA-DQ2 coeliac subjects (see Figures 33 and 34, and Table 18). Deamidated peptide pool 74 was biqactive in bofe HLA-DQJ2/8 subjects, but only in one of flie 6 HLA-DQ2/X subjects. Ptetteatment of pool 74 wife flTG enhances bioactivity between 3.8 and 22-times, and bioactivity of tTG-treated pool 74 in fee three lesponders is equivalent to between 78% and 350% fee bioactivity of
P04722 77-93 E85. Currently, it is not known which peptides are bioactive in Poo! 74 in subject C02, C07, and COS.
Two of the four HLA-DQ8 coeliac subjects that laclced both or one of the HLA-DQ2 alleles associated with coeliac disease showed very weak IFNy 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 tQPQNPSQQQPjQ (SEQ ID NO:42) (see Table 20). Previous work (by us) has demonstrated that three glutainine residues in this sequence are susceptible to fTG-mediated deamidation (underlined). Homology searches using WWW PepPepSearch have identified close matches to LQPQNPSQQQPQ (SEQ ID NO:43) only in wheat a-gliadms.
The fourfli HLA-DQ8 subject (Cl 1) had inducible IFNy ELISpot responses to fTG"-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 fTG (underlined Gin are deamidated and convey bioactiviry) (van der Wai et al 1998). Cuttentty, 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 qjamring all known 12 ammo acid sequences in wheat gliadin has thus identified at least 72 peptides at 10% as bioactive as fee known a-gjiadin epitope, A-gHadin 57-73 E65. However, these bioactive peptides can be reduced to a set of perhaps as few as 5 distinct bat closely related femflies of peptides. Almost all mese peptides are rich inproline, glutamine, phenylalanine, and/or tyrosine and include the sequence PQ(QL)P(FY)P (SEQ ID NO:45). This sequence fecflitates deamidation of Q inposMon 2 by tTG. By analogy with deamidation of A-gliadin 57-68 (Aientz-Hansen 2000), the enhanced bioactivity of mese,peptides generally found with deamidatioaby tTG may be due to increased affinity of binding for HLA-DQ2.
Cross-reactivity amongst T cells in vivo recognizing more than one of mese bioactive gfiadin 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 IFNy in response to gliadin after gluten challenge.
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-^Jiadin.57-73 E65 homologues. Two different epitopes were dominantin two EDLA-DQ84- coeliacs. TTtie dominant epitope in one of these HLA-DQ8-J- individuals has not been identified previously (LQPQNPSQQQPQ (SEQ ID NO:46)).
Given the teaching herein, design of an imnlunotherapy for coeliac disease utilizing all the commonly recognised T cell epitopes is practical and may include fewer than six distinct peprides. Epitopes in wheat y- and co-gliadins are also present in barley hordems and rye secauns..
Example 14
Several EOSpot assays were performed as previously described and yielded the following results and/or conclusions: Examination of multiple a-gliadin polymorphisms wflft PQLPY
(Sequence Removed)
. Dashes indicate
identity with the GO 1 sequence in the particular position.
Gluten challenge induces A-gliadin 57-73 QE65 T cells only after two weeks of gluten-free diet in newly diagnosed coeliac disease
Additional analyses indicated flat tTG-deamidated gliadin responses change after two weeks of gluten-free diet in newly diagnosed coeliac disease. Omer
analyses indicated that deamidated gliadiu-specific T cells are OX'o^fb HLA-DQ2 restricted.
Optimal epitope (clones versus gluten challenge)
A "dominant" epjtope is defined by ylFN ELISpot after gluten challenge. QLQPFPQPELPYPQPQS (100% ELISpot response). Epitopes defibaed by intestinal T cell clones (Sequence Removed)

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. Dcamidation generates almost all gliadin epitopes. HLA-DQ2, DQ8, andDR4 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-hased therapeutics. •
Other analyses indicated me following: HIA-DR3-DQ2 (85-95%) and HLA-DR4-DQ8 (5-15%).
Oilier analyses indicated the following:
BQLA-DQ HLA-DQA1 HLA-DQB1 Duodenal Gluten EMAon
(T able Removed)
Another analysis was earned out-to determine the bioactrvity of individual fTG-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%X Sequence 19 (3%), Sequence 20 (0%), Sequence 21 (0%). The predicted deamidated sequence is LQPENPSQEQPE.
Individual ELlSpot responses by PBMC (Spot forming cells determined by ELXSpot
Reader)
Peptide (see Table 23) (T able Removed) 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 wife another stractaany related peptide, expression of a particular peptide response can be as a percentage of a "dominant" peptide response. Alternately, the expression can be a "reIatednessM as correlation- coefficients between peptide responses, or via bioinformatics.
Additional epUopes
A representative result is as follows:
Combination of peptides with P04722E (an 20mcgtofl) (n=4)

(Sequence Removed)
Irnmunomagnetic depletion of PBMC by beads coated with anti-CD4 and by anti-integrin pv depleted IFNy ELISpot responses, while imraunoroagnetic depletion of PBMC by beads coated with anti-CD8 or anti-alphaE integrin. Thus, flie PBMC secreting IFNy are CD4+ and 04^7+, associated with homing to the lamina propria in the gut
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.
Tcell epitopes fn coetiac disease
Other investigators have characterized certain intestinal T cell clone epitopes. See, e.g; Vader et aL, Gastroenterology 2002,122:1729-37; AtentzrBansen et aL, Gastroenterology 2002,123:803-809. These axe examples of epitopes whose relevance is at best unclear because of the in vitro techniques used to clone T cells.
intestinal versus peripheral blood clones
Intestinal: 1) intestinal biopsies, 2) T cell clones raised against peptic-tryptic digest
of gluten, 3) all HLA-DQ2 restricted, 4) clones respond to gliadin deamidated by
transghitaminase.
PeiqjbjBial blood: 1) T.cell clones raised against gluten are HLA-DR, DQ and DP
lestricted. Result: Intestinal T cell clones can be exclusively used to map coeliac
disease associated epitopes
GDA_9Wheat 307 aa Etefinition Alpha/Beta-Gliadin MM1 Precursor (Prolamin) Accession P1S573 — Genbank (which is incorporated herein by reference in its entirety)
Intestinal Tcell clone epitopes
A definition of intestinal T cell clone epitopes can be found in, for example, Arente-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-gUadins include PQPQLPY singly or duplicated. A majority of T cell clones respond to eithei/or DQ2-aI: QLQPFPQPELPY DQ2-aII: PQPELPYPQPELPY
Dominant gliadin T cell epitopes-
AU deamidated by tansglutaininase.
Peripheral blood day 6 after gluie.n challenge: A-gliadin 57-73:
QLQPFPQPELPYPQPQS
Intestinal T cell clones: DQ2-oI: QLQPFPQPELPY DQ2-oIfc PQPELPYPQPELPY
Intestinal T-cell Clone Epitope Mapping
(Sequence Removed)
Gluten exposure anduiduction of 'IFNy'-secreting A-Gliadin 57-73QE65-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 QB65 (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 PPBMQ
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/mfflion PPBMQ
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
Characterization of anti-gliadin T cell response was carried oul in peripheral blood on day 6-8 after 3-day gluten challenge.
Result 1: PBMC Day 6 Long-term gluten free diet (preincubation with anti-HLA-DR and -DQ antibody) (expressed as % inhibition)
DR-: tTG-gliadin 100 meg/ml (105), A-gliadin 57-73 QE65 50 meg/ml (90), PPD5mcg/mT(30)
DQ-: tTG-gliadin 100 meg/ml (5), A-glladin 57-73 QE65 50 meg/ml (22), PPD 5 meg/ml (78).
Result 2: PBMC Day 6 Long-term gluten free diet (expressed as % CD8-depleted PBMC response)
B7 depletion: tTG-gliadin n=6 (7), A-^liadin 57-73 n=9 (6), PPD n=8 (62)
AE depletion: tTG-fUadinn=6 (120), A-gliadin 57-73 n=9 (80), PPD n=8 (110).
CD4 depletion: tTG-gliadin n=6 (10), A-^iadin 57-73 n=9 (9), PPD n=8 (10).
Therapeutic peptides include, but are not limited to (Sequence Removed)

Briefly after oral antigen challenge, specificities of peripheral blood T cells reflect those of intestinal T cell clones. In peripheral blood, epitopes of intestinal T cell clones are sub-optimal compared to A-gliadin 57-73 QE65, which is an optimal a-gliadin epitope.
Example 15
ELISpot assays were also carried out for mapping purposes as follows.
Fine-mapping the dominant DQ-8 associated epitope
(Sequence Removed)

Fine-mapping dominant epitope (2) Pool 33
Example 16 "
Bioactivity of gliadin epitopes in IFNy-EUSpot (25 meg/ml, n~6) (expressed as %A-
gliadin 57-73 QE65 response)
(Sequence Removed)
Dose response ofA-Gliadin 57-73 QE65 (G01E) (n*=8) (expressed as %GOIE maximal response)
0.025 meg/ml (1), 0.05 meg/ml (8), 0.1 mcgfeil (10), 0.25 meg/ml (22), 0.5 meg/mi (38), 1 mcg/nd (43), 2.5 meg/ml (52), 5 mog/ml (70), 10 meg/ml (81), 25 meg/ml (95), 50 meg/ml (90), 100 meg/ml (85).
IFNy ELlSpot response to gliadin qtitopes alone or mixed with A-gliadin 57-75 (G01E) (aD 50 meg/ml, tTG-fJkdin 100 meg/ml, PPD 5 mcgfeal; n=5) (expressed as % GO IE response)
Alone: DQ2-A1 (20), DQ2-A2 (55), Omega Gl (50), fTG Gliadin (80), PPD (220), DQ2 binder (0)
G01E+: DQ2-A1 (90), DQ2-A2.(95), Omega Gl (100), tTG GKadm (120), PPD (280), DQ2 binder (80)
Effect ofalmane and lysine substitution of A-gliadin 57-73 QE65 on IFNy EHSpot responses in individual coeliac subjects (n—8) Epitope sequence: QLQPFPQPELPYPQPQS
Alanine substitution at positions 57-59 and 72-73 showed litfle to no . decrease in % A-gliadin 57-73 QE65 response. Alanine substitution at positions 60-62 and 68-71 showed moderate decrease in % A-^iadin 57-73 QE65 response.
Alauine substitution at positions 63-67 showed most decrease in % A-gliadin 57-73 QE65 response.
Effect of lysine substitution of A-gliadin 57-73 QE65 on DRNy 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 69-70 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. Mofcerg O, et aL Nature Med. 4,713-717 (1998).
2. Quanten E, et aL Eur. J. ImnnmoL 29,2506-2514 (1999).
3. GreenbergCSetal FASEB 5,3071-3077 (1991).
4. MantzarisG, Jewell D. Scand. J. Gastroenterol. 26,392-398 (1991).
5. Maori L, et at Scand. J. GasttoenteroL 31,247-253 (1996).
6. Bancel&etaL Tissue Antigens 46,355-367 (1995).
7. Olerup O, et al. Tissue antigens 41,119-134 (1993).
8. MullighanCG,etal. Tissue-Antigens. 50,688-92 (1997).
9. PlebansMMetal Eur. J. JmmunoL 28,4345^4355 (1998).

10. Anderson DO, Greene £C. The alpha-gliadin gene family. H.DNA and protein
sequence variation, subfamily structure, and origins of pseudogenes. Theor Appl
Penet (1997) 95:59-65.
11. Arentz-Hansen H, Komer R, Molberg O, Quarsten EL, Van der Wai Y, Kooy
YMC, Lundin KEA, Koning F, Roepstorff P, Soffid IM, McAdam SN. The
mtestinal T cell response to alpha-gliadin in adult ceh'ac disease is focused on a
single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 2000; 193:603-12.
12. Vader LW, de Ru A, van der Wai, Kooy YMC, Benckhuijsen W, Mearin ML,
Drijfkoul 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 aer'Wal Y, Kooy Y, van Veelan P, Pena S, Mearin L, Papadopoulos G,
Koning F. Selective deamidation by tissue transglutaminase strongly enhances
glkdin-specific T cell reactivity. JImmunoL 1998; 161:1585-8.
14. van der Wai Y, Kooy Y, van Veelan P, Pena S, Mearin L, Molberg 0, Lundin
KEA, SoDid L, Mutis T, Bencldiuijsen WE, Drijfliout JW, Koning F. Proc Nati
Acad Sci USA 1998; 95:10050-10054.
15. Vader W, Kooy Y, Van Veelen Petal The gluten tesponse in children
with celiac disease is directed toward multiple gjiadin and glutenin
peptides. Gastroenterolc»gy2002,122:1729-37
16. Arenrz-HansenH, Me Adam SN, Molberg O.etaL Celiac lesion T cells
recognize epitopes that cluster in regions of gliadin rich in proliae
residues. Gastroerrferology 2002,123:803-809.
Each of fte PCT publications, U.S. patents, otherpatenis, journal references, and any other publications cited or referred to herein is incorporated herein by reference in their entirety.
Table 1. A-Gliadin protein sequence (based on amino acid sequencing)(T able Removed)








119
WE CLAIM:
1. An agent typically in the form of a peptide for the preparation of a medicament for treating or preventing coeliac disease, wherein the agent comprises:
(a) a peptide comprising at least one epitope comprising a sequence selected from the group consisting of transglutaminase-deamidated SEQ ID NOs: 19-20, 31-35 and 39-41, and equivalents thereof; and
(b) an analogue of (a) which is capable of being recognised by a T cell receptor that recognises the epitope 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.
2. An agent as claimed in claim 1 wherein the agent is HLA-DQ2-restricted.
3. An agent as claimed in claim 1 wherein the agent is HLA-DQ8-restricted.
4. An agent as claimed in claim 1 wherein the agent has one or more epitopes selected from group consisting wheat epitope, barley epitope and rye epitope.
5. A protein typically gluten comprising a sequence which is able to bind to a T cell receptor, which T cell receptor recognises an agent as claimed in claim 1, and which sequence is able to cause antagonism of a T cell that carries such a T cell receptor.
6. A mutant gliadin protein whose wild-type sequence can be modified by a transglutaminase to a sequence which is an agent as claimed in claim 1, which mutant gliadin protein comprises a mutation preferably at position 65 in A-giiadin or in an equivalent position in other gliadins which prevents its modification by a transglutaminase to a sequence which is an agent as defined in claim 1; or a fragment of such a mutant gliadin protein which is at least 15 amino acids long and which comprises the mutation.
120
7. A polynucleotide preferably having at least 50 bases or base pairs that has a coding sequence that encodes a protein or fragment as defined in claim 5 and a regulatory sequences operably linked to the coding sequence, which regulatory sequences are capable of securing the expression of the coding sequence in a cell.
8. A polynucleotide as claimed in claim 7 wherein the regulatory sequence(s) allow expression of the coding sequence in a prokaryotic cell.
9. A polynucleotide as claimed in claim 7 which is a vector.
10. A kit for carrying out a method of diagnosing coeliac disease having an agent as claimed in claim 1 and a means to detect the recognition of the peptide by the T cell.
11. A kit as claimed in claim 10 wherein the means to detect recognition comprises an antibody to IFN-Y.
12. A kit as claimed in claim 11 wherein the antibody is immobilised on a solid support and optionally the kit also has a means to detect the antibody/IFN-Y complex.

Documents:

32-delnp-2005-abstract.pdf

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32-delnp-2005-claims.pdf

32-delnp-2005-complete specification (granted).pdf

32-DELNP-2005-Correspondence Others-(14-07-2011).pdf

32-DELNP-2005-Correspondence-Others-(08-07-2010).pdf

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32-DELNP-2005-Form-1-(22-02-2011).pdf

32-delnp-2005-form-1.pdf

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32-delnp-2005-form-2.pdf

32-DELNP-2005-Form-3-(08-07-2010).pdf

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32-delnp-2005-pct-210.pdf

32-delnp-2005-pct-304.pdf

32-delnp-2005-pct-409.pdf

32-delnp-2005-petition-137.pdf

32-delnp-2005-petition-138.pdf


Patent Number 248510
Indian Patent Application Number 32/DELNP/2005
PG Journal Number 29/2011
Publication Date 22-Jul-2011
Grant Date 21-Jul-2011
Date of Filing 05-Jan-2005
Name of Patentee ISIS INNOVATION LIMITED
Applicant Address EWERT HOUSE,EWERT PLACE, SUMMERTOWN OXFORD OX2 7SG, UNITED KINGDOM
Inventors:
# Inventor's Name Inventor's Address
1 ANDERSON, ROBERT PAUL AUTOIMMUNITY AND TRANSPLANTATION DIVISION, WALTER AND ELIZA HALL INSTITUTE,C/O ROYAL MELBOURNE HOSPITAL PO, GRATTAN STREET, PARKVILLE, VICTORIA 3050, AUSTRALIA.
2 HILL ADRIAN VIVIAN SINTON WELCOME TRUST CENTRE FOR HUMAN GENETICS,UNIVERSITY OF OXFORD, ROOSEVELT DRIVE, OXFORD OX3 7BN, UNITED KINGDOM
3 JEWELL, DEREK PARRY GASTROENTEROLOGY UNIT, GIBSON BUILDING, RADCLIFFE INFIRMARY, WOODSTOCK ROAD, OXFORD OX2 6HE, UNITED KINGDOM
PCT International Classification Number C07K 14/415
PCT International Application Number PCT/GB03/02450
PCT International Filing date 2003-06-05
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0212885.8 2002-06-05 U.K.