Diagnosis of coeliac disease using a gliadin epitope Number:7,144,569 from the United States Patent and Trademark Office (PTO) owispatent 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 or 2, or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO: 3, (ii) an epitope comprising sequence comprising: SEQ ID NO: 1, or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO: 3, which epitope is an isolated oligopeptide derived from a gliadin protein, (ii) an analogue of (i) or (ii) which is capable of being recognised by a T cell receptor that recognises (i) or (ii), which in the case of a peptide analogue is not more than 50 amino acids in length, or (iv) a product comprising two or more agents as defined in (i), (ii) or (iii), and (b) determining in vitro whether T cells in the sample recognises the agent; recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease. Therapeutic compositions which comprise the epitope and gliadin proteins which do not cause coeliac disease are also provided.<H Diagnosis, epitope, Diagnosis, of, co, 7144569.html, Patent, pto, 7144569

Title: Diagnosis of coeliac disease using a gliadin epitope

Abstract: 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 or 2, or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO: 3, (ii) an epitope comprising sequence comprising: SEQ ID NO: 1, or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO: 3, which epitope is an isolated oligopeptide derived from a gliadin protein, (ii) an analogue of (i) or (ii) which is capable of being recognised by a T cell receptor that recognises (i) or (ii), which in the case of a peptide analogue is not more than 50 amino acids in length, or (iv) a product comprising two or more agents as defined in (i), (ii) or (iii), and (b) determining in vitro whether T cells in the sample recognises the agent; recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease. Therapeutic compositions which comprise the epitope and gliadin proteins which do not cause coeliac disease are also provided.

Patent Number: 7,144,569 Issued on 12/05/2006 to Anderson, et al.

Inventors: Anderson; Robert Paul (Headington, GB), Hill; Adrian Vivian Sinton (Oxford, GB), Jewell; Derek Parry (Oxford, GB)
Assignee: ISIS Innovation Limited (Oxford, GB)

Appl. No.: 10/089,700

Filed: October 2, 2000

PCT Filed: October 02, 2000

PCT No.: PCT/GB00/03760

371(c)(1),(2),(4) Date: January 09, 2003

PCT Pub. No.: WO01/25793

PCT Pub. Date: April 12, 2001

Foreign Application Priority Data


Oct 01, 1999 [GB]			9923306.6

Current U.S. Class: 424/9.81 ; 424/185.1; 435/7.24; 435/7.94; 435/7.95; 435/975; 514/12; 514/13; 514/14; 514/15; 514/16; 514/2; 530/324; 530/325; 530/326; 530/327; 530/328; 530/329; 530/372; 530/374; 530/402

Current International Class: A61K 39/35 (20060101); C07K 7/04 (20060101); G01N 33/53 (20060101)

Field of Search: 424/9.81,185.1 435/7.24,7.94,7.95,975 514/5,12-16 530/324-329,372,374,402

References Cited [Referenced By]

U.S. Patent Documents


4459355	July 1984	Cello et al.
4536475	August 1985	Anderson
4945050	July 1990	Sanford et al.
5036006	July 1991	Sanford et al.
5100792	March 1992	Sanford et al.
5177010	January 1993	Goldman et al.
5179022	January 1993	Sanford et al.
5187073	February 1993	Goldman et al.
5204253	April 1993	Sanford et al.
5371014	December 1994	Matsuyama et al.
5405765	April 1995	Vasil et al.
5464763	November 1995	Schilperoort et al.
5478744	December 1995	Sanford et al.
5484956	January 1996	Lundquist et al.
5489520	February 1996	Adams et al.
5508468	April 1996	Lundquist et al.
5510318	April 1996	Patel et al.
5538877	July 1996	Lundquist et al.
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Foreign Patent Documents


0242236	Oct., 1987	EP
0255378	Feb., 1988	EP
0293358	Nov., 1988	EP
442174	Aug., 1991	EP
486233	May., 1992	EP
486234	May., 1992	EP
604662	Jul., 1994	EP
672752	Sep., 1995	EP
0693119	Jan., 1997	EP
0 905 518	Mar., 1999	EP
539563	Oct., 2001	EP
WO 91/02071	Feb., 1991	WO
WO 92/000377	Jan., 1992	WO
WO 92/17580	Oct., 1992	WO
WO 92/20809	Nov., 1992	WO
WO 94/13863	Jun., 1994	WO
WO 95/06128	Mar., 1995	WO
WO 98/23960	Jun., 1998	WO
WO 98/45460	Oct., 1998	WO
WO 98/45461	Oct., 1998	WO
WO 99/20775	Apr., 1999	WO
WO 02/083722	Oct., 2002	WO
WO 03/066079	Aug., 2003	WO
WO 03/096984	Nov., 2003	WO
WO 03/104273	Dec., 2003	WO

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Primary Examiner: Saunders; David
Attorney, Agent or Firm: O'Connor, P.C.; Cozen

Claims

The invention claimed is:

1. An isolated peptide comprising the amino acid sequence SEQ ID NO:1.

2. The peptide of claim 1 wherein the peptide is 7 to 50 amino acids in length.

3. The peptide of claim 1 comprising the amino acid sequence SEQ ID NO:2.

4. The peptide of claim 3 wherein the peptide is up to 50 amino acids in length.

5. A fusion protein comprising SEQ ID NO:1 and a non-gliadin sequence.

6. A kit comprising a peptide of claim 1 or 5 and a means to detect the recognition of the peptide by a T cell.

7. The kit of claim 6 wherein the means to detect recognition comprises an antibody to IFN-.gamma..

8. The kit of claim 7 wherein the antibody is immobilised on a solid support and, optionally, comprises a means to detect any complexes formed between the antibody and IFN-.gamma..

9. A method of diagnosing coeliac disease or susceptibility to coeliac disease in an individual comprising: a) contacting the individual or a sample from the individual with a peptide of claim 1 or 5; and b) determining whether a T cell in the sample recognises the peptide, wherein recognition by the T cell indicates that the individual has or is susceptible to coeliac disease.

10. The method of claim 9 wherein a) comprises administering the peptide to the skin of the individual, and b) comprises detecting the presence of inflammation at the site of administration, wherein detection of inflammation indicates that the T cell of the individual recognises the peptide.

11. The method of claim 9 wherein the sample is a blood sample.

12. The method of 9 wherein the T cell is not re-stimulated in an antigen specific manner in vitro before determining whether the T cell in the sample recognises the peptide.

13. The method of claim 9 wherein the recognition of the peptide by the T cell is determined by detecting the secretion of a cytokine from the T cell.

14. The method of claim 13 wherein the cytokine is IFN-.gamma..

15. The method of claim 13 wherein the cytokine is detected by allowing the cytokine to bind to an immobilised antibody specific to the cytokine and then detecting the presence of any complex formed between the antibody and cytokine.

16. The method of claim 9 wherein b) comprises measuring whether the peptide binds a T cell receptor.

17. An isolated product comprising two or more of: a peptide comprising the amino acid sequence SEQ ID NO:1; a peptide 7 to 50 amino acids in length comprising the amino acid sequence SEQ ID NO:1; a peptide comprising the amino acid sequence SEQ ID NO:2; a peptide up to 50 amino acids in length comprising the amino acid sequence SEQ ID NO:2; and a fusion protein comprising SEQ ID NO:1 and a non-gliadin sequence.

18. A composition comprising a peptide or a fusion protein, and a pharmaceutically acceptable carrier or diluent, wherein the peptide is: a peptide comprising the amino acid sequence SEQ ID NO:1; a peptide 7 to 50 amino acids in length comprising the amino acid sequence SEQ ID NO:1; a peptide comprising the amino acid sequence SEQ ID NO:2; or a peptide up to 50 amino acids in length comprising the amino acid sequence SEQ ID NO:2; and wherein the fusion protein comprises SEQ ID NO:1 and a non-gliadin sequence.

19. A method of diagnosing coeliac disease or susceptibility to coeliac disease in an individual comprising detecting the presence of an antibody that binds to a peptide comprising SEQ ID NO:1 in a sample from the individual, wherein the presence of the antibody indicates that the individual has or is susceptible to coeliac disease.

Description

The invention relates to the diagnosis and therapy of coeliac disease, and to a gliadin protein which does not cause coeliac disease.

An immune reaction to gliadin (a component of gluten) in the diet causes coeliac disease. It is known that immune responses in the intestinal tissue preferentially respond to gliadin which has been modified by an intestinal transglutaminase. Coeliac disease is diagnosed by detection of anti-endomysial antibodies, but this requires confirmation by the finding of a lymphocytic inflammation in intestinal biopsies. The taking of such a biopsy is inconvenient for the patient.

Investigators have previously assumed that only intestinal T cell responses provide an accurate indication of the immune response against gliadins. Therefore they have concentrated on the investigation of T cell responses in intestinal tissue. Gliadin epitopes which require transglutaminase modification (before they are recognised by the immune system) are known.sup.2.

The inventors have found the immunodominant T cell epitope recognised by the immune system in coeliac disease, and have shown that this is recognised by T cells in the peripheral blood of individuals with coeliac disease. Such T cells were found to be present at high enough frequencies to be detectable without restimulation (i.e. a `fresh response` detection system could be used). The epitope was identified using a non-T cell cloning based method which provided a more accurate reflection of the epitopes being recognised. The immunodominant epitope requires transglutaminase modification (causing substitution of a particular glutamine to glutamate) before immune system recognition.

Based on this work the inventors have developed a test which can be used to diagnose coeliac disease at an early stage. The test may be carried out on a sample from peripheral blood and therefore an intestinal biopsy is not required. The test is more sensitive than the antibody tests which are currently being used.

The invention thus provides a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising:

(a) contacting a sample from the host with an agent selected from (i) the epitope comprising sequence which is: SEQ ID NO: 1 or 2, or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO:3, (ii) an epitope comprising sequence comprising: SEQ ID NO:1, or an equivalent sequence from a naturally occurring homologue of the gliadin represented by SEQ ID NO:3, which epitope is an isolated oligopeptide derived from a gliadin protein, (iii) an analogue of (i) or (ii) which is capable of being recognised by a T cell receptor that recognises (i) or (ii), which in the case of a peptide analogue is not more than 50 amino acids in length, or (iv) a product comprising two or more agents as defined in (i), (ii) or (iii), and (b) determining in vitro whether T cells in the sample recognise the agent, recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease.

The invention also provides use of the agent for the preparation of a diagnostic means for use in a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual, said method comprising determining whether T cells of the individual recognise the agent, recognition by the T cells indicating that the individual has, or is susceptible to, coeliac disease.

The finding of an immunodominant epitope which is modified by transglutaminase also allows diagnosis of coeliac disease based on determining whether other types of immune response to this epitope are present. Thus the invention also provides a method of diagnosing coeliac disease, or susceptibility to coeliac disease, in an individual comprising determining the presence of an antibody that binds to the epitope in a sample from the individual, the presence of the antibody indicating that the individual has, or is susceptible to, coeliac disease.

The invention additionally provides the agent, optionally in association with a carrier, for use in a method of treating or preventing coeliac disease by tolerising T cells which recognise the agent. Also provided is an antagonist of a T cell which has a T cell receptor that recognises (i) or (ii), optionally in association with a carrier, for use in a method of treating or preventing coeliac disease by antagonising such T cells. Additionally provided is the agent or an analogue that binds an antibody (that binds the agent) for use in a method of treating or preventing coeliac disease in an individual by tolerising the individual to prevent the production of such an antibody.

The invention provides a method of determining whether a composition is capable of causing coeliac disease comprising determining whether a protein capable of being modified by a transglutaminase to an oligopeptide sequence as defined above is present in the composition, the presence of the protein indicating that the composition is capable of causing coeliac disease.

The invention also provides a mutant gliadin protein whose wild-type sequence can be modified by a transglutaminase to a sequence that comprises an epitope comprising sequence as defined above, but which mutant gliadin protein has been modified in such a way that it does not contain sequence which can be modified by a transglutaminase to a sequence that comprises such an epitope comprising sequence; or a fragment of such a mutant gliadin protein which is at least 15 amino acids long and which comprises sequence which has been modified in said way.

The invention also provides a protein that comprises a sequence which is able to bind to a T cell receptor, which T cell receptor recognises the agent, and which sequence is able to cause antagonism of a T cell that carries such a T cell receptor.

Additionally the invention provides a food that comprises the proteins defined above.

The invention is illustrated by the accompanying drawings in which:

FIG. 1 shows freshly isolated PBMC (peripheral blood mononuclear cell) IFN.gamma. ELISPOT responses (vertical axis shows spot forming cells per 10.sup.6 PBMC) to transglutaminase (tTG)-treated and untreated peptide pool 3 (each peptide 10 .mu.g/ml) including five overlapping 15mers spanning A-gliadin 51 85 (see Table 1) and a-chymotrypsin-digested gliadin (40 .mu.g/ml) in coeliac disease Subject 1, initially in remission following a gluten free diet then challenged with 200 g bread daily for three days from day 1 (a). PBMC IFN.gamma. ELISPOT responses by Subject 2 to tTG-treated A-gliadin peptide pools 1 10 spanning the complete A-gliadin protein during ten day bread challenge (b). The horizontal axis shows days after commencing bread.

FIG. 2 shows PBMC IFN.gamma. ELISPOT responses to tTG-treated peptide pool 3 (spanning A-gliadin 51 85) in 7 individual coeliac disease subjects (vertical axis shows spot forming cells per 10.sup.6 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 IFNg Elispot responses to tTG-treated overlapping 15mer peptides included in pool 3; bars represent the mean (.+-. SEM) response to individual peptides (10 .mu.g/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.)

FIG. 3 shows PBMC IFN.gamma. ELISPOT responses to tTG-treated truncations of A-gliadin 56 75 (0.1 .mu.M). Bars represent the mean (.+-.SEM in 5 Coeliac disease subjects. (In individual subjects, responses were calculated as the % of the maximal response elicited by any of the peptides tested.)

FIG. 4 shows how the minimal structure of the dominant A-gliadin epitope was mapped using tTG-treated 7 17mer A-gliadin peptides (0.1 .mu.M) including the sequence, PQPQLPY (A-gliadin 62 68) (a), and the same peptides without tTG treatment but with the substitution Q.fwdarw.E65 (b). Each line represents PBMC IFNg ELISPOT responses in each of three Coeliac disease subjects on day 6 or 7 after bread was ingested on days 1 3. (In individual subjects, ELISPOT responses were calculated as a % of the response elicited by the 17mer, A-gliadin 57 73.)

FIG. 5 shows the amino acids which were deamidated by tTG. A-gliadin 56 75 (LQLQPFPQPQLPYPQPQSFP) (0.1 .mu.M) was incubated with tTG (50 .mu.g/ml) at 37.degree. C. for 2 hours. A single product was identified and purified by reverse phase HPLC. Amino acid analysis allowed % deamidation (Q.fwdarw.E) of each Gln residue in A-gliadin 56 75 attributable to tTG to be calculated (vertical axis).

FIG. 6 shows the effect of substituting Q.fwdarw.E in A-gliadin 57 73 at other positions in addition to Q65 using the 17mers: QLQPFPQPELPYPQPEIS (E57,65), QLQPFPQPELPYPQPES (E65,72), ELQPFPQPELPYPQPES (E57, 65, 72), and QLQPFPQPELPYPQPQS (E65) in three Coeliac disease subjects on day 6 or 7 after bread was ingested on days 1 3. Vertical axis shows % of the E65 response.

FIG. 7 shows that tTG treated A-gliadin 56 75 (0.1 .mu.M) elicited IFN-g ELISPOT responses in (a) CD4 and CD8 magnetic bead depleted PBMC. (Bars represent CD4 depleted PBMC responses as a % of CD8 depleted PBMC responses; spot forming cells per million CD8 depleted PBMC were: Subject 4: 29, and Subject 6: 535). (b) PBMC IFN.gamma. ELISPOT responses (spot forming cells/million PBMC) after incubation with monoclonal antibodies to HLA-DR (L243), -DQ (L2) and -DP (B7.21) (10 .mu.g/ml) 1 h prior to tTG-treated 56 75 (0.1 .mu.M) in two coeliac disease subjects homozygous for HLA-DQ a1*0501, b1*0201.

FIG. 8 shows the effect of substituting Glu at position 65 for other amino acids in the immunodominant epitope. The vertical axis shows the % response in the 3 subjects in relation to the immunodominant epitope.

FIG. 9 shows the immunoreactivity of naturally occurring gliadin peptides (measuring responses from 3 subjects) which contain the sequence PQLPY with (shaded) and without (clear) transglutaminase treatment.

FIG. 10 shows CD8, CD4, .beta..sub.7, and .alpha..sup.E-specific immunomagnetic bead depletion of peripheral blood mononuclear cells from two coeliac subjects 6 days after commencing gluten challenge followed by interferon gamma ELISpot. A-gliadin 57 73 QE65 (25 mcg/ml), tTG-treated chymotrypsin-digested gliadin (100 mcg/ml) or PPD (10 mcg/ml) were used as antigen.

FIG. 11 shows the optimal T cell epitope length.

FIG. 12 shows a comparison of A-gliadin 57 73 QE65 with other peptides in a dose response study.

FIG. 13 shows a comparison of gliadin and A-gliadin 57 73 QE65 specific responses.

FIG. 14 shows the bioactivity of gliadin polymorphisms in coeliac subjects.

FIGS. 15 and 16 show the defining of the core epitope sequence.

FIGS. 17 to 27 show the agonist activity of A-gliadin 57 73 QE65 variants.

FIG. 28 shows responses in different patient groups.

DETAILED DESCRIPTION OF THE INVENTION

The term `coeliac disease` encompasses a spectrum of conditions caused by varying degrees of gluten sensitivity, including a severe form characterised by a flat small intestinal mucosa (hyperplastic villous atrophy) and other forms characterised by milder symptoms.

The individual mentioned above (in the context of diagnosis or therapy) is human. They may have coeliac disease (symptomatic or asymptomatic) or be suspected of having it. They may be on a gluten free diet. They may be in an acute phase response (for example they may have coeliac disease, but have only ingested gluten in the last 24 hours before which they had been on a gluten free diet for 14 to 28 days).

The individual may be susceptible to coeliac disease, such as a genetic susceptibility (determined for example by the individual having relatives with coeliac disease or possessing genes which cause predisposition to coeliac disease).

The Agent

The agent is typically a peptide, for example of length 7 to 50 amino acids, such as 10 to 40, or 15 to 30 amino acids in length.

SEQ ID NO:1 is PQPELPY. SEQ ID NO:2 is QLQPFPQPELPYPQPQS. SEQ ID NO:3 is shown in Table 1 and is the sequence of a whole A-gliadin. The glutamate at position 4 of SEQ ID NO:1 (equivalent to position 9 of SEQ ID NO:2) is generated by transglutaminase treatment of A-gliadin.

The agent may be the peptide represented by SEQ ID NO:1 or 2 or an epitope comprising sequence that comprises SEQ ID NO:1 which is an isolated oligopeptide derived from a gliadin protein; or an equivalent of these sequences from a naturally occurring gliadin protein which is a homologue of SEQ ID NO:3. Thus the epitope may be a derivative of the protein represented by SEQ ID NO:3. Such a derivative is typically a fragment of the gliadin, or a mutated derivative of the whole protein or fragment. Therefore the epitope of the invention does not include this naturally occurring whole gliadin protein, and does not include other whole naturally occurring gliadins.

The epitope may thus be a fragment of A-gliadin (e.g. SEQ ID NO:3), which comprises the sequence of SEQ ID NO:1, obtainable by treating (fully or partially) with transglutaminase, i.e. with 1, 2, 3 or more glutamines substituted to glutamates (including the substitution within SEQ ID NO:1).

Such fragments may be or may include the sequences represented by positions 55 to 70, 58 to 73, 61 to 77 of SEQ ID NO:3 shown in Table 1. Typically such fragments will be recognised by T cells to at least the same extent that the peptides represented by SEQ ID NO:1 or 2 are recognised in any of the assays described herein using samples from coeliac disease patients.

In the case where the epitope comprises a sequence equivalent to the above epitopes (including fragments) from another gliadin protein (e.g. any of the gliadin proteins mentioned herein or any gliadins which cause coeliac disease), such equivalent sequences will correspond to a fragment of a gliadin protein typically treated (partially or fully) with transglutaminase. Such equivalent peptides can be determined by aligning the sequences of other gliadin proteins with 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 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 the presence of an antigen presenting cell (APC) (such as any of the APCs mentioned herein), the analogue inhibits the recognition of (i) or (ii), i.e. the analogue is able to compete with (i) or (ii) in such a system.

The analogue may be one which is capable of binding the TCR which recognises (i) or (ii). Such binding can be tested by standard techniques. Such TCRs can be isolated from T cells which have been shown to recognise (i) or (ii) (e.g. using the method of the invention). Demonstration of the binding of the analogue to the TCRs can then shown by determining whether the TCRs inhibit the binding of the analogue to a substance that binds the analogue, e.g. an antibody to the analogue. Typically the analogue is bound to a class II MHC molecule (e.g. HA-DQ2) in such an inhibition of binding assay.

Typically the analogue inhibits the binding of (i) or (ii) to a TCR. In this case the amount of (i) or (ii) which can bind the TCR in the presence of the analogue is decreased. This is because the analogue is able to bind the TCR and therefore competes with (i) or (ii) for binding to the TCR.

T cells for use in the above binding experiments can be isolated from patients with coeliac disease, for example with the aid of the method of the invention. Other binding characteristics of the analogue may also be the same as (i) or (ii), and thus typically the analogue binds to the same MHC class II molecule to which the peptide binds (HLA-DQ2). The analogue typically binds to antibodies specific for (i) or (ii), and thus inhibits binding of (i) or (ii) to such antibodies.

The analogue is typically a peptide. It may have homology with (i) or (ii), typically at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99% homology with (i) or (ii), for example over a region of at least 15 more (such as the entire length of the analogue and/or (i) or (ii), or across the region which contacts the TCR or binds the MHC molecule) contiguous amino acids. Methods of measuring protein homology are well known in the art and it will be understood by those of skill 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 their 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 the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. 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. Natl. Acad. Sci. USA 90: 5873 5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

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 mutation may be measured across any of the regions mentioned above in relation to calculating homology. The substitutions are preferably `conservative`. These are defined according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

TABLE-US-00001 ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R AROMATIC H F W Y

Typically the amino acids in the analogue at the equivalent positions to amino acids in (i) or (ii) which contribute to binding the MHC molecule or are responsible for the recognition by the TCR, are the same or are conserved.

Typically the analogue peptide comprises one or more modifications, which may be natural post-translation modifications or artificial modifications. The modification may provide a chemical moiety (typically by substitution of a hydrogen, e.g. of a C--H bond), such as an amino, acetyl, hydroxy or halogen (e.g. fluorine) group or carbohydrate group. Typically the modification is present on the N or C terminus.

The analogue may comprise one or more non-natural amino acids, for example amino acids with a side chain different from natural amino acids. 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 which 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 the sequence of SEQ ID NO:1 or 2, or any of the other peptides mentioned herein; and non-gliadin sequence.

In one embodiment the analogue is or mimics (i) or (ii) bound to a MHC class II molecule. 2, 3, 4 or more of such complexes may be associated or bound to each other, for example using a biotin/streptavidin based system, in which typically 2, 3 or 4 biotin labelled MHC molecules bind to a streptavidin moiety. This analogue typically inhibits the binding of the (i) or (ii)/MHC Class II complex to a TCR or antibody which is specific for the complex.

The analogue is typically an antibody or a fragment of an antibody, such as a Fab or (Fab).sub.2 fragment. The analogue may be immobilised on a solid support, particularly an analogue which mimics peptide bound to a MHC molecule.

The analogue is typically designed by computational means and then synthesised using methods known in the art. Alternatively the analogue can be selected from a library of compounds. The library may be a combinatorial library or a display library, such as a phage display library. The library of compounds may be expressed in the display library in the form of being bound to a MHC class II molecule, such as HLA-DQ2. Analogues are generally selected from the library based on their ability to mimic the binding characteristics (i) or (ii). Thus they may be selected based on ability to bind a TCR or antibody which recognises (i) or (ii).

Typically analogues will be recognised by T cells to at least the same extent as any of the agents (i) or (ii), for example at least to the same extent as the equivalent epitope and preferably to the same extent as the peptide represented by SEQ ID NO:2, is recognised in any of the assays described herein, typically using T cells from coeliac disease patients. Analogues may be recognised to these extents in vivo and thus may be able to induce coeliac disease symptoms to at least the same extent as any of the agents mentioned herein (e.g. in a human patient or animal model).

Analogues may be identified in a method comprising determining whether a candidate substance is recognised by a T cell receptor that recognises an epitope of the invention, recognition of the substance indicating that the substance is an analogue. Such TCRs may be any of the TCRs mentioned herein, and may be present on T cells. Any suitable assay mentioned herein can be used to identify the analogue. In one embodiment this method is carried out in vivo. As mentioned above preferred analogues are recognised to at least the same extent as the peptide SEQ ID NO:2, and so the method may be used to identify analogues which are recognised to this extent.

In one embodiment the method comprises determining whether a candidate substance is able to inhibit the recognition of an epitope of the invention, inhibition of recognition indicating that the substance is an analogue.

The agent may be a product comprising at least 2, 5, 10 or 20 agents as defined by (i), (ii) or (iii). Typically the composition comprises epitopes of the invention (or equivalent analogues) from different gliadins, such as any of the species or variety of or types of gliadin mentioned herein. Preferred compositions comprise at least one epitope of the invention, or equivalent analogue, from all of the gliadins present in any of the species or variety mentioned herein, or from 2, 3, 4 or more of the species mentioned herein (such as from the panel of species consisting of wheat, rye, barley, oats and triticale).

Diagnosis

As mentioned above the method of diagnosis of the invention may be based on the detection of T cells which bind the agent or on the detection of antibodies that recognise the agent.

The T cells which recognise the agent in the method (which includes the use mentioned above) are generally T cells which 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 in vitro or in vivo. Thus the invention provides the agent for use in a method of diagnosis practiced on the human body. Different agents are provided for simultaneous, separate or sequential use in such a method.

The in vitro method is typically carried out in aqueous solution into which the agent is added. The solution will also comprise the T cells (and in certain embodiments the APCs discussed below). The term `contacting` as used herein includes adding the particular substance to the solution.

Determination of whether the T cells recognise the agent is generally done by detecting a change in the state of the T cells in the presence of the agent or determining whether the T cells bind the agent. The change in state is generally caused by antigen specific functional activity of the T cell after the TCR binds the agent The change of state may be measured inside (e.g. change in intracellular expression of proteins) or outside (e.g. detection of secreted substances) the T cells.

The change in state of the T cell may be the start of or increase in secretion of a substance from the T cell, such as a cytokine, especially IFN-.gamma., IL-2 or TNF-.alpha.. Determination of IFN-.gamma. secretion is particularly preferred. The substance can typically be detected by allowing it to bind to a specific binding agent and then measuring the presence of the specific binding agent/substance complex. The specific binding agent is typically an antibody, such as polyclonal or monoclonal antibodies. Antibodies to cytokines are commercially available, or can be made using standard techniques.

Typically the specific binding agent is immobilised on a solid support. After the substance is allowed to bind the solid support can optionally be washed to remove material which is not specifically bound to the agent. The agent/substance complex may be detected by using a second binding agent which will bind the complex. Typically the second agent binds the substance at a site which is different from the site which binds the first agent. The second agent is preferably an antibody and is labelled directly or indirectly by a detectable label.

Thus the second agent may be detected by a third agent which is typically labelled directly or indirectly by a detectable label. For example the second agent may comprise a biotin moiety, allowing detection by a third agent which comprises a streptavidin moiety and typically alkaline phosphatase as a detectable label.

In one embodiment the detection system which is used is the ex-vivo ELISPOT assay described in WO 98/23960. In that assay IFN-.gamma. secreted from the T cell is bound by a first IFN-.gamma. specific antibody which immobilised on a solid support. The bound IFN-.gamma. is then detected using a second IFN-.gamma. 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 which can be measured may be the increase in the uptake of substances by the T cell, such as the uptake of thymidine. The change in state may be an increase in the size of the T cells, or proliferation of the T cells, or a change in cell surface markers on the T cell.

In one embodiment the change of state is detected by measuring the change in the intracellular expression of proteins, for example the increase in intracellular expression of any of the cytokines mentioned above. Such intracellular changes may be detected by contacting the inside of the T cell with a moiety that binds the expressed proteins in a specific manner and which allows sorting of the T cells by flow cytometry.

In one embodiment when binding the TCR the agent is bound to an MHC class II molecule (typically HLA-DQ2), which is typically present on the surface of an antigen presenting cell (APC). However as mentioned herein other agents can bind a TCR without the need to also bind an MHC molecule.

Generally the T cells which are contacted in the method are taken from the individual in a blood sample, although other types of samples which contain T cells can be used. The sample may be added directly to the assay or may be processed first. Typically the processing may comprise diluting of the sample, for example with water or buffer. Typically the sample is diluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10 fold.

The processing may comprise separation of components of the sample. Typically mononuclear cells (MCs) are separated from the samples. The MCs will comprise the T cells and APCs. Thus in the method the APCs present in the separated MCs can present the peptide to the T cells. In another embodiment only T cells, such as only CD4 T cells, can be purified from the sample. PBMCs, MCs and T cells can be separated from the sample using techniques known in the art, such as those described in Lalvani et al (1997) J. Exp. Med. 186, p859 865.

In one embodiment the T cells used in the assay are in the form of unprocessed or diluted samples, or are freshly isolated T cells (such as in the form of freshly isolated MCs or PBMCs) which are used directly ex vivo, i.e. they are not cultured before being used in the method. Thus the T cells have not been restimulated in an antigen specific manner in vitro. However the T cells can be cultured before use, for example in the presence of one or more of the agents, and generally also exogenous growth promoting cytokines. During culturing the agent(s) are typically present on the surface of APCs, such as the APC used in the method. Pre-culturing of the T cells may lead to an increase in the sensitivity of the method. Thus the T cells can be converted into cell lines, such as short term cell lines (for example as described in Ota et al (1990) Nature 346, p183 187).

The APC which is typically present in the method may be from the same individual as the T cell or from a different host The APC may be a naturally occurring APC or an artificial APC. The APC is a cell which is capable of presenting the peptide to a T cell. It is typically a B cell, dendritic cell or macrophage. It is typically separated from the same sample as the T cell and is typically co-purified with the T cell. Thus the APC may be present in MCs or PBMCs. The APC is typically a freshly isolated ex vivo cell or a cultured cell. It may be in the form of a cell line, such as a short term or immortalised cell line. The APC may express empty MHC class II molecules on its surface.

In the method one or more (different) agents may be used. Typically the T cells derived from the sample can be placed into an assay with all the agents which it is intended to test or the T cells can be divided and placed into separate assays each of which contain one or more of the agents.

The invention also provides the agents such as two or more of any of the agents mentioned herein (e.g. the combinations of agents which are present in the composition agent discussed above) for simultaneous separate or sequential use (eg. for in vivo use).

In one embodiment agent per se is added directly to an assay comprising T cells and APCs. As discussed above the T cells and APCs in such an assay could be in the form of MCs. When agents which can be recognised by the T cell without the need for presentation by APCs are used then APCs are not required. Analogues which mimic the original (i) or (ii) bound to a MHC molecule are an example of such an agent.

In one embodiment the agent is provided to the APC in the absence of the T cell. The APC is then provided to the T cell, typically after being allowed to present the agent on its surface. The peptide may have been taken up inside the APC and presented, or simply be taken up onto the surface without entering inside the APC.

The duration for which the agent is contacted with the T cells will vary depending on the method used for determining recognition of the peptide. Typically 10.sup.5 to 10.sup.7, preferably 5.times.10.sup.5 to 10.sup.6 PBMCs are added to each assay. In the case where agent is added directly to the assay its concentration is from 10.sup.-1 to 10.sup.3 g/ml, preferably 0.5 to 50 .mu.g/ml or 1 to 10 .mu.g/ml.

Typically the length of time for which the T cells are incubated with the agent is from 4 to 24 hours, preferably 6 to 16 hours. When using ex vivo PBMCs it has been found that 0.3.times.10.sup.6 PBMCs can be incubated in 10 .mu.g/ml of peptide for 12 hours at 37.degree. C.

The determination 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 which recognise the agent will be deemed to occur if the frequency of cells sorted using the agent is above a `control` value. The frequency of antigen-experienced T cells is generally 1 in 10.sup.6 to 1 in 10.sup.3, and therefore whether or not the sorted cells are antigen-experienced T cells can be determined.

The determination of the recognition of the agent by the T cells may be measured in vivo. Typically the agent is administered to the host and then a response which indicates recognition of the agent may be measured. The agent is typically administered intradermally or epidermally. The agent is typically administered by 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 which can typically be used to administer the agent. Typically from 0.001 to 1000 .mu.g, for example from 0.01 to 100 .mu.g or 0.1 to 10 .mu.g of agent is administered.

In one embodiment a product can be administered which is capable of providing the agent in vivo. Thus a polynucleotide capable of expressing the agent can be administered, typically in any of the ways described above for the administration of the agent. The polynucleotide typically has any of the characteristics of the polynucleotide provided by the invention which is discussed below. The agent is expressed from the polynucleotide in vivo. Typically from 0.001 to 1000 .mu.g, for example from 0.01 to 100 .mu.g or 0.1 to 10 .mu.g 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 that binds the agent is typically carried 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 in the sample binds the agent, such a binding indicating that the individual has, or is susceptible to coeliac disease. Any suitable format of binding assay may be used, such as any such format mentioned herein.

Therapy

The identification of the immunodominant epitope allows the therapeutic products to be made which target the T cells which recognise this epitope (such T cells being ones which participate in the immune response against gliadin). This finding also allows the prevention or treatment of coeliac disease by suppressing (by tolerisation) an antibody or T cell response to the epitope.

Certain agents of the invention bind the TCR which recognises the epitope of the invention (as measured using any of the binding assays discussed above) and cause tolerisation of the T cell that carries the TCR Such agents, optionally in association with a carrier, can therefore be used to prevent or treat coeliac disease.

Generally tolerisation can be caused by the same peptides which can (after being recognised by the TCR) cause antigen specific functional activity of the T cell (such as any such activity mentioned herein, e.g. secretion of cytokines). Such agents cause tolerisation when they are presented to the immune system in a `tolerising` context.

Tolerisation l