USE OF PLATELET GLYCOPEPTIDE HIA EPITOPES IN THE TREATMENT OF IMMUNE THROMBOCYTOPENIC PURPURA

[001] The present invention relates to a composition for treating diseases associated with autoantibodies specific for platelet proteins, in particular autoimmune thrombocytopenic purpura .

[002] Chronic autoimmune thrombocytopenic purpura (AITP) is a bleeding disorder characterized by the production of autoantibodies that mediate platelet destruction. The clinical signs include petechial hemorrhages, hemorrhagic bullae on mucous membranes, gingival or gastrointestinal bleeding, menorrhagia, retinal hemorrhages, and, most seriously, intracranial hemorrhage. Current therapeutic strategies for AITP rely on non-specific immunosuppressive agents, or intravenous immunoglobulin or anti-D, with refractory cases undergoing splenectomy to remove a major site of autoantibody production and platelet destruction. Unfortunately, the results of these approaches are frequently unsatisfactory. A fuller understanding of the pathogenesis of AITP is therefore required, in order to develop safe, effective treatments that specifically inhibit the disease process .

[003] A major focus of research into the pathogenesis of

AITP has been the characterization of the autoantibody response. Platelet membrane glycoprotein Ilb/IIIa

(GPIIb/IIIa) has emerged as the major autoantigen that is bound by pathogenic autoantibodies from most patients. Other platelet antigens that can be targeted, but less frequently, include glycoproteins GPIb/IX, GPIa/IIa, and GPV. Although this progress in determining the specificities of the autoantibodies has led to novel diagnostic assays for AITP, the mechanisms underlying the loss of self-tolerance remain to be elucidated.

[004] The vast majority of IgG responses are driven by CD4 ⁺ helper T (Th) cells, including the production of pathogenic antibodies in murine models of autoimmune blood cell destruction. Human AITP is no exception, since the disease is associated with loss of peripheral T cell tolerance and the development of recall helper responses to platelet autoantigens . Peripheral blood Th cells from AITP patients, in comparison with those from healthy controls, exhibit accelerated proliferation when stimulated in vitro with fragments of purified or recombinant GPIIb/IIIa, indicative of prior activation in vivo. These memory Th cells are capable of driving anti-GPIIb/IIIa IgG synthesis by peripheral blood B cells from patients in vitro, with the spleen as the primary site for the autoreactive B cells to receive such help in vivo. T cells in AITP may, in addition to providing help for the autoantibody response, also contribute directly to platelet destruction. In response to the accumulating evidence that Th cells represent potential therapeutic targets, a small number of AITP patients has been treated with a humanized monoclonal antibody (mAb) that blocks the helper co-stimulatory molecule, CD40 ligand

(CD154) . The effects were to reduce both the frequency and in vitro collaboration of peripheral blood Th and B cells responsive to GPIIb/IIIa, and in some cases, treatment was associated with increased platelet counts. It is considered that any such immune inhibition may be only temporary, and not necessarily limited to the pathogenic response.

[005] According to the present invention there is provided a composition for treating diseases associated with autoantibodies specific for platelet proteins by tolerisation, the composition comprising an epitope of the platelet protein.

[006] Tolerisation is an effective way to treat autoimmune

diseases. CD4 ⁺ Th cells recognize short peptides that have been processed and displayed bound to MHC class II molecules by antigen presenting cells (APC) . Antigen-specific tolerance can be induced in vivo by synthetic peptides containing dominant helper epitopes, if administered appropriately in soluble form, for example via mucosal surfaces in the nose or gut.

[007] It has been shown that peptides containing dominant Th cell epitopes can prevent responses to the corresponding antigen when given in soluble form without adjuvant, or if administered by a tolerogenic route e.g. mucosal.

Importantly, induction to tolerance to only one dominant epitope, particularly if mediated by active immune regulation, can ablate responsiveness to the entire autoantigen from which it is derived, and also to other, associated antigens by a process of bystander suppression.

[008] In the present invention, peptides that contain the dominant Th epitopes from platelet autoantigens were mapped.

[009] The mapping took place by screening a panel of short, overlapping peptides spanning the entire sequence of platelet glycoprotein for the ability to stimulate recall responses by peripheral blood Th cells.

[0010] Conveniently the platelet protein is a membrane component .

[0011] Membrane proteins are highly involved in stimulating autoimmune responses.

[0012] Conveniently the platelet protein is GPIIIa.

[0013] GPIIIa is a major autoantigenic molecule, which is

- A - known to contain important B and T cell determinants. The present invention identifies GPIIIa peptides that contain epitopes recognized by autoreactive Th cells from AITP patients, and which are candidate tolerogens for specific immunotherapy of the disease and describes seven dominant sequences .

[0014] Autoreactive Th cells specific for platelet glycoprotein are known to be activated in AITP, but this is the first time that peptides driving the response have been mapped. The results not only provide further insight into the mechanisms of disease, but open the way for novel forms of peptide immunotherapy for AITP that selectively target the pathogenic Th cells.

[0015] Conveniently the disease is autoimmune thrombocytopenic purpura.

[0016] The need for specific, effective and safe treatment for patients with chronic AITP may be met by the development of peptide immunotherapy to re-induce Th tolerance to the platelet glycoproteins.

[0017] The composition can be formulated for various types of administration including through oral, rectal, nasal, buccal, dermal, topical, parenteral, intraarticular, inhalation, intrathecal and vaginal forms of administration.

[0018] Conveniently the composition is formulated for delivery through non-invasive routes. The composition may be formulated for delivery through mucosal tissue.

[0019] Alternatively, the composition is formulated for delivery through invasive routes such as injection, for example intraperitoneal, intramuscular, or depot injections,

or via implants.

[0020] Any of the aforementioned routes can lead to effective tolerisation.

[0021] Conveniently the epitope is immunodominant.

[0022] Immunodominance ensures that effective tolerisation takes place.

[0023] Conveniently the epitope is promiscuous.

[0024] Promiscuous epitopes elicit responses regardless of the HLA type of the patient. Accordingly, the epitopes can be used over a wider range of the population.

[0025] Conveniently the epitope contains between substantially 10 and 20 amino acids. The epitope may contain substantially 15 amino acids.

[0026] Such lengths of epitope result in effective tolerisation.

[0027] Conveniently the epitope is selected from SEQ ID No: 2, 44, 47, 53, 70, 77 and 82.

[0028] In the example of the present invention PBMC from almost all patients with AITP proliferated against members of a peptide panel spanning the sequence of GPIIIa, and such responses are strongly associated with the disease since they were rarely exhibited by samples from healthy or disease control donors. The culture conditions were biased in favor of supporting accelerated recall responses by Th cells that have previously been activated in vivo as part of the disease process, and not by naive Th cells. The vast majority of

the patients with AITP had both anti-GPIIb/IIIa antibodies and PBMC that mount recall proliferation to GPIIIa peptides, strengthening the view that the pathogenic B cell response is dependent on T cell help specific for the same autoantigenic complex. The small number of AITP patients with PBMC responsive to GPIIIa peptides, but no detectable anti-GPIIb/IIIa antibodies, may reflect the limited serological assays that could be performed in these cases. As with other autoantigens, the relatively rare and weak responses to GPIIIa peptides observed in control donors could well represent cross-reactivity with environmental antigens, particularly given the limited sequence homology between different peptides necessary for T cell cross-reactivity. It was confirmed by flow cytometric analysis that the cells from patients with AITP that responded in vitro to immunodominant GPIIIa peptides were of the CD3 ⁺ CD4 ⁺ Th phenotype, and the ability of anti-HLA antibodies consistently to block the proliferation verified that they were MHC class II restricted cells. DR appears to be the principal restricting locus, but the effects of the blocking antibodies suggest that DP and DQ molecules may also compete for presentation of particular GPIIIa peptides.

[0029] It has been shown in the present invention that multiple peptides from GPIIIa stimulated proliferation by Th cells from most AITP patients. This suggests that the diversity may follow the phenomenon of epitope spreading.

This occurs when the autoimmune helper response initially targets very few, or only one, self-determinant (s) , but further Th clones with new specificities for the same, or associated, autoantigens are recruited over time as pathology develops .

[0030] The related feature of GPIIIa recognition that resembles other autoaggressive responses is the variation,

seen in individual AITP patients over time, in the peptides that induce proliferation by peripheral blood Th cells in vitro. Such gain or loss of stimulation by peptides can reflect changes in the frequency of the corresponding Th cells in the circulation, attributable to the respective effects of epitope spreading and clonal exhaustion.

[0031] It has also been shown that despite the variation between cases in the patterns of stimulatory GPIIIa peptides, particular sequences are dominant, and stimulate responses in many patients. Seven such peptides distributed throughout GPIIIa, were identified; numbers 2 (aa6-20), 44 (aa331-345), 47 (aa361-375), 53 (aa421-435) 70 (aa591-605), 77 (aaββl- 675) , 82 (aa711-725) .

[0032] The question arises as to why these peptides should contain dominant epitopes. When considering conventional immune responses to foreign antigens, the dominant Th epitopes can often be predicted due to their ability to bind well to the restricting MHC molecules. However, the same is not true of many autoimmune diseases, where lack of tolerance in the helper compartment, whether mediated by deletion, anergy or regulation, is a prime factor in the selection of dominant helper epitopes, rather than high affinity for the restricting class II molecules. Indeed, there are well- characterized examples where inefficient presentation of self-peptides contributes crucially to the failure to tolerize the corresponding repertoire, and allows the persistence of potentially autoaggressive Th cells that can be activated to drive disease. AITP fits with this pattern, since many of the dominant peptides fail to exhibit high predicted affinity for any HLA-DR molecules from an extensive panel. Furthermore, with the exception of peptide 82 (aa711- 725), for any of the dominant sequences that do carry an HLA-DR binding motif, there is no correlation in different

patients between the expression of the respective class II molecule, and the stimulation of responses. The likely low affinity of most of the dominant GPIIIa peptides for their restricting MHC molecules would lead to poor presentation and could account for the escape of the corresponding Th cells from mechanisms that purge the immune repertoire of potentially autoaggressive lymphocytes. These Th cells would then be available to be activated in disease by events such as stimulation with higher avidity cross-reactive microbial antigens, or increased production and display of the dominant GPIIIa peptides following changes in antigen presentation in vivo. Th cells that recognize peptide 82 (aa711-725), may survive, not because of poor binding and display of the sequence by restricting MHC molecules, but because of "destructive processing" by enzymes that cleave the sequence within APC, although it should be noted that this peptide may be of less pathogenic relevance since it is the only dominant sequence to induce proliferation by Th cells from control donors .

[0033] Conveniently, a patient for which the composition is directed to is from Northern European Caucasian population. In this connection, ethnic origin can influence Th responses.

[0034] The invention will now be described, by way of illustration only, with reference to the following examples and the accompanying figures.

[0035] Figure 1 shows that PBMC from patients with AITP proliferate in response to peptides from the sequence of

GPIIIa. PBMC were isolated from representative patients

AITPl (A) , AITP8 (B) , AITPlO (C) , and AITP20 (D) tested for the ability to proliferate against the panel of 86 peptides spanning the GPIIIa molecule. The line denotes the level of proliferation taken as representing a significant

positive response (SI>3).

[0036] Figure 2 shows that PBMC from healthy control donors rarely proliferate when stimulated with peptides from the GPIIIa sequence. Shown here are proliferative responses of PBMC from representative healthy control donors C5 (A) , C6 (B), C8 (C) and C17 (D) against the panel of 86 peptides spanning the GPIIIa molecule. The line denotes the level of proliferation taken as representing a significant positive response (SI>3).

[0037] Figure 3 shows that PBMC from disease control donors rarely proliferate when stimulated with peptides from the GPIIIa sequence. Shown here are proliferative responses of PBMC from representative patients with aplastic anemia C26 (A), C27 (B), C28 (C) and C29 (D) against the panel of 86 peptides spanning the GPIIIa molecule. The line denotes the level of proliferation taken as representing a significant positive response (SI>3) .

[0038] Figure 4 shows that particular dominant peptides from GPIIIa stimulate T cells from many patients with AITP to proliferate. Shown here are the proportions of patients with AITP (solid) and healthy control donors (clear) whose PBMC proliferated in response to each of the 86 peptides from the panel spanning GPIIIa. PBMC from all individuals in both groups responded to stimulation with the control recall antigen mycobacterial PPD (hatched) .

[0039] Figure 5 shows that the pattern of GPIIIa peptides that stimulate PBMC from AITP patients to proliferate can evolve over time. Proliferative responses of PBMC from a representative patient (AITP22) against the panel of 86 peptides spanning the GPIIIa molecule were compared on three different occasions, at presentation (A; platelet count

76xlO9/L), then after 44 weeks (B; platelet count 54xlO9/L) and 56 weeks (C; platelet count 84xlO9/L) . The line denotes the level of proliferation taken as representing a significant positive response (SI>3).

[0040] Figure 6 shows that PBMC from patients with AITP that respond to GPIIIa peptides are predominantly of the helper phenotype. PBMC from patients AITPlO (A) and AITP18 (B) were either left unstimulated in culture, or incubated with GPIIIa peptides 2 or 53 that induce proliferative responses in these patients, before being stained for CD4 expression and the activation marker CD71. Results are shown gated on the CD3+ population.

[0041] Figure 7 shows that the proliferation of T cells from AITP patients against GPIIIa peptides is dependent on HLA- class II molecules. Cultures of PBMC from a representative patient (AITP9) were stimulated with dominant GPIIIa peptides 2, 47, 53 or 82, and class II restricted responses were blocked by addition of antibody specific for HLA-DR, -DQ or

-DP. For each peptide stimulus, the line denotes the level of inhibition taken as significant (>50%). Similar results were obtained with PBMC from another three patients.

Example 1

Patients and control subjects

[0042] Approval for the study was received from the Grampian Local and Regional Ethics Committee (number 00/0052). Informed written consent was obtained from all patients and healthy controls. Samples of whole blood were obtained from 31 patients (21 female and 10 male) with AITP, who attended the outpatient hematology clinic at Aberdeen Royal Infirmary. The details of the patients, who are all North European Caucasian, are summarized in Table 1. The diagnosis of AITP was made by exclusion of other causes of thrombocytopenia and

in compliance with the British Committee for Standards in Haematology Guideline. The majority (29/31) of the patients were being treated with immunosuppressive drugs at the time of sampling, and eight had undergone splenectomy.

[0043] Samples of whole blood for PBMC isolation were also taken from 25 healthy control blood donors (18 female and seven male) . None was on any medication. PBMC samples from a further group, of five patients with aplastic anaemia (four male and one female), were included as disease controls, since this condition responds to immunosuppression and is considered to have an autoimmune basis, and patients also have low platelet counts.

Platelet recovery and preparation of eluates

[0044] Platelets from AITP patients and controls were isolated by differential centrifugation of anti-coagulated

(citrate-phosphate-dextrose) blood. Antibody was eluted from the surface of platelets as described by Hϋrlimann-Forster et al and stored at -80 ⁰ C until used.

Detection of antiplatelet autoantibodies against GPIIb/lIIa from serum and platelet eluates of AITP patients and controls [0045] Anti-GPIIb/IIIa autoantibody concentrations in sera and eluates were measured by ELISA using published methods of Bessos H et al in Vox Sang 1999; 77 : 103-106 and Sukati H et al in Transfusion 2005 ; 45 : 1165-1177. Briefly, samples were screened in duplicate wells of microtitre plates coated with purified GPIIb/IIIa. Background binding was determined by incubating each sample in uncoated wells, and control samples positive and negative for antibody were also included. Absorbance was read at 540nm using a multiscan plate reader (Labsystems, Finland) . Specific optical densities (OD) > 0.1 and > 0.05 were interpreted as positive

results for serum and eluate samples respectively (determined from the mean of healthy control samples + 2 SD) .

HLA Class II DNA typing using PCR-SSP [0046] Genomic DNA preparation from the whole blood of AITP patients and controls and HLA class II typing was carried using PCR-SSP as reported in Sukati H et al in Transfusion 2005 ; 45 : 1165-1177. Visual interpretation of positive bands after gel electrophoresis were confirmed using HELMBERG SCORETM software v3.000T (provided by Dr. W. Helmberg, Institute for Transfusion Medicine, University of Graz, Austria, www.genoversion.com).

Preparation of antigens and mitogens [0047] The human platelet membrane GPIIIa amino acid sequence (Genebank Accession no: M35999) was synthesized

(Pepceuticals, Nottingham, UK) as a complete panel of 86 15- mer peptides, overlapping by 5-10 amino acids (Table 3) .

Peptide purity was monitored by amino acid analysis and mass spectrometry as reported previously by Barker RN et al in Blood 1997;90:2701-2715, Scott L et al in Blood 2000 ; 96 : 4011- 4019 and Cairns LS et al in J Am Soc Nephrol 2003; 14 : 2801- 2812. The peptides were used for stimulation of T cells at the previously determined optimum concentration of 20 mg/mL in culture.

[0048] The antigen mycobacterial purified protein derivative (PPD) (Statens Serumintitut , Denmark) was added to cultures at 20mg/mL to stimulate positive control recall T cell responses. Concanavalin A (Con A) (Sigma, Poole, Dorset, UK) was used at 20mg/mL as a positive control T cell mitogen.

Isolation of peripheral blood mononuclear cells (PBMC) [0049] Mononuclear cells were recovered from anti-coagulated

samples of peripheral blood from AITP patients and control donors by density gradient centrifugation (Lymphoprep; Nycomed, Denmark) . Cell viability determined by trypan blue exclusion was greater than 90% in all samples.

T cell proliferation assay

[0050] Assays of T cell proliferation were carried out, as described by Scott L et al in Blood 2000 ; 96 : 4011-4019, Cairns LS et al in J Am Soc Nephrol 2003; 14 : 2801-2812 and Sukati H et al in Transfusion 2005; 45 : 1165-1177 , under culture conditions designed to favor responses by previously activated T cells, rather than primary responses. Briefly, PBMC were cultured at 1.25x106 cells per mL in Alpha Modification of Eagle's Medium (Sigma, Poole, Dorset, UK) supplemented with 5% autologous serum. Synthetic GPIIIa peptides or control stimuli were added to cultures, which were incubated at 37 ⁰ C in a humidified atmosphere of 95% air and 5% CO ₂ . T cell proliferation was estimated from the incorporation of 3H-thymidine in triplicate 10OmL samples withdrawn from the cultures 5 days after stimulation, when recall responses peak. Results are presented either as the mean counts per minute (CPM) + SD of the triplicate samples, or as a stimulation index (SI) expressing the ratio of mean CPM in stimulated versus unstimulated control cultures. An SI >3 is interpreted as a positive response.

Flow cytometric characterization of lymphocytes responding to stimulation

[0051] As previously described by Sukati H et al in Transfusion 2005; 45 : 1165-1177 , cultures of unstimulated PBMC, and those proliferating in response to peptides, were analyzed for expression of the T cell marker CD3, the T helper marker CD4, and the activation marker CD71 by 3-colour flow cytometry. All antibodies and control immunoglobulins were supplied by Beckman Coulter (Bucks, UK) . A total of

10,000 cells per sample was counted using an Epics XL cytometer (Beckman Coulter) and the results analyzed with Expo 32 software (Beckman Coulter) .

HLA restriction of PBMC proliferating in response to GPIIIa peptides

[0052] To determine the HLA Class II restriction of proliferating T cells, 2.5mg/mL anti-DR, anti-DQ or anti-DP blocking monoclonal antibodies (Pharmingen, Oxon, UK) were added to replicate cultures before stimulation.

Prediction of peptide binding motifs for HLA-DR molecules

[0053] Protein sequences were entered into ProPred predictive software (www.imtech.res.in/raghava/propred), which is based on quantitative matrices derived by Stumiolo T et al in Nat Biotechnol 1999; 17 : 555-561. An algorithm allows the sequences to be scanned for motifs predicted to have high affinity for binding to many of the commonly expressed HLA-DR molecules.

Statistical Analysis

[0054] Non-parametric Chi-square and Fisher Exact Tests were used for statistical analysis, with p<0.05 considered to represent significance.

Results

Mapping peptides derived from the GPIIIa sequence that stimulate proliferation by PBMC from AITP patients or healthy controls

[0055] The prime aim was to identify the peptide sequences from GPIIIa that contain Th epitopes. PBMC were obtained from the group of 31 patients with AITP (clinical details summarized in Table 1) and from 25 healthy control blood

donors. A panel of 86 synthetic overlapping 15-mer peptides, spanning the entire sequence of the platelet GPIIIa (Table 2) was screened for the ability to stimulate the proliferation of PBMC from each of the patients and controls. The platelet glycoprotein-responsive Th cells that are associated with AITP have previously been shown to be activated in vivo, as would be expected for autoaggressive lymphocytes of pathogenic relevance. Therefore, to map the epitopes recognized by these cells, the culture conditions were based on those previously designed to favor fast developing recall, rather than slower primary, responses.

[0056] Representative results from four AITP patients, demonstrating GPIIIa peptides that elicit PBMC proliferation, are illustrated in Figure 1, and the stimulatory peptides for each of the 31 patients are listed in Table 3. It can be seen that PBMC from all but three patients responded to at least one member of the peptide panel, and that, typically, multiple sequences induced proliferation.

[0057] The presence of anti-platelet antibodies reactive with GPIIb/IIIa was confirmed in 27 of the 31 AITP patients

(Table 3) . The individuals generating anti-GPIIb/IIIa included 21 patients with serum antibodies, 14 of whom also had platelet-bound antibodies demonstrated after elution, plus a further six with no detectable serum antibodies but positive eluates. All eight patients who had undergone splenectomy had persisting antibodies. Comparison with the results of PBMC stimulation reveals that the vast majority of the AITP patients (25/31) had both anti-GPIIb/IIIa antibody and proliferative responses against GPIIIa peptides. This association between detectable anti-GPIIb/IIIa and peptide responsiveness is not absolute since, for example, three of the four antibody-negative patients did show PBMC proliferation to peptides. However, in these cases it was

possible to screen only sera for anti-GPIIb/IIIa, and the testing of platelet eluates was often necessary to detect the antibody. Table 3 also illustrates that there is no simple relationship between the number, or the identities, of the stimulatory peptides and the platelet count of the AITP patients at the time of sampling.

[0058] In contrast to the results obtained in AITP patients, responses were rarely seen when the peptide panel was used to stimulate PBMC from healthy control donors. Examples of results from the control group are depicted in Figure 2, with the data summarized in Table 4A. No anti-GPIIb/IIIa antibodies were detected in serum or platelet eluate samples from this group. PBMC from only nine of the control donors demonstrated proliferation to any of the peptide panel, and, in each of these cases, responsiveness was limited to one or two sequences. It should be noted that PBMC from all patients and control donors proliferated normally when stimulated with the control recall antigen mycobacterial PPD (Figure 4) or the mitogen Con A (results not shown), indicating that any lack of response to the GPIIIa peptide panel is specific, and not attributable to a general loss of immune function or lymphocyte viability. The background levels of proliferation in the absence of antigen or mitogen are generally higher in the control donors than in the AITP patients, reflecting the effects of disease state and immunosuppressive treatment. The difference in the total number of peptide responses in the patient and control groups was highly significant (a total of 178 responses to peptides in 31 patients versus 12 in 25 healthy donors, χ2 = 115.967; p=<0.001), consistent with the view that recall Th responses specific for platelet glycoprotein are associated with AITP. To confirm that responsiveness to GPIIIa epitopes is not a feature of immune mediated disease in general, or of low platelet counts, PBMC from a group of five patients with

aplastic anemia were also stimulated with the peptide panel (results summarized in Table 4B, with representative examples illustrated in Figure 3) . It can be seen that, as in the healthy donors, responses to GPIIIa peptides in this disease 5 control group are very infrequent.

Distribution of stimulatory peptides on the platelet GPIIIa

[0059] Despite variation between AITP patients in the

10 profile of GPIIIa peptides that elicited PBMC proliferation

(Table 3), particular peptides were identified as dominant, since they stimulated responses in a high proportion of cases. These dominant sequences are shown in Figure 4, which summarizes the number of patients in which each peptide

15 induced proliferation. The four most dominant peptides are

2 (aaβ-20), 47 (aa361-375), 53 (aa421-435) and 82 (aa711-

725), with 24 patients (77%) showing PBMC responses to at least one of these sequences, and 13 (42%) to three or more.

A further three peptides, 44 (aa331-345), 70 (aa591-605) and

20 77 (aa661-675), exhibited a lower level of dominance, with each stimulating proliferation by PBMC from at least 5 (15%) patients .

[0060] Analysis of the GPIIIa peptides eliciting the

25 relatively rare responses by control donor PBMC (Table 4,

Figure 4) reveals that they include only one of the seven sequences identified as dominant in AITP patients. This peptide, 82 (aa711-725), was the only member of the entire panel to stimulate PBMC from more than one control donor.

30 Thus, compared to those from AITP patients, the responses of healthy control PBMC to GPIIIa sequences are not only infrequent, but generally target sporadic peptides that differ from those commonly recognized in AITP.

35

Variation over time in the pattern of GPIIIa peptides that stimulate responses

[0061] Longitudinal studies of patients with chronic autoimmune diseases other than AITP demonstrate changes over time in the identities of autoantigen-derived peptides recognized by autoaggressive Th cells. To establish whether the same is true for AITP, serial PBMC samples taken over periods of weeks or months from patients (n=10) were screened for responsiveness to the GPIIIa peptide panel. Figure 5 depicts a typical set of results, where the GPIIIa peptides were tested against PBMC taken from patient AITP22 on three different occasions over 56 weeks. The dominant peptide 2

(aa6-20) elicited proliferation from all samples, whilst responsiveness to dominant sequences 47 (aa361-375), 53 (aa421-435) and 82 (aa711-725) was initially absent but appeared at later time points, and proliferation to the lower ranking dominant peptide 77 (aa661-675) was seen only in the second sample. It should be noted that these differences are consistent across all replicate cultures set up from each sample, and therefore do not represent chance inter-well variation. These results from patient AITP-22, and the other examples, illustrate a complex, dynamic pattern of responsiveness, with some peptides persistently stimulating PBMC proliferation, and others eliciting responses that fluctuate over time. Such evolution of the fine specificity of the immune response does not directly correlate with the clinical course of disease, since there is no relationship between the changes over time in the identities of the stimulatory peptides, and the platelet count of the AITP patients (Figure 5) .

Characterization of the phenotype of PBMC that proliferate in response to GPIIIa peptides

[0062] In order to confirm that the PBMC proliferating against GPIIIa peptides were of the CD3 ⁺ CD4 ⁺ Th phenotype,

selected cultures were analyzed by multi-color flow cytometry. Responding cells were labeled with antibody to the activation marker CD71, and the Th subset was identified by counter-staining with anti-CD3 and anti-CD4. Representative results (n=6) from two AITP patients are shown in Figure 6. It can be seen that, as expected, the background level of CD71 expression in control, resting cultures was very low, and there was a small increase (1.5- 3.8%) in numbers of activated CD71 ⁺ cells after stimulation with dominant peptides 2 (aa6-20) or 53 (aa421-435) . The size of this expansion is typical of the responses to antigen made by specific lymphocytes within a polyclonal population, and the vast majority (88-100%) of the cells that upregulated CD71 as a result of the peptide stimulation were CD3 ⁺ CD4 ⁺ .

Role of HLA Class II in responses of PBMC from AITP patients and control donors

[0063] In order to demonstrate functionally that the lymphocytes responding to GPIIIa peptides came from the Th subset, which is restricted by MHC Class II molecules, blocking antibodies specific for anti-HLA-DP, -DQ and -DR were tested for the ability to inhibit the responses. Dominant peptides 2(aa6-20), 47 (aa361-375), 53 (aa421-435) and 82 (aa711-725) were selected for these experiments and used to stimulate PBMC from four AITP patients, in the presence or absence of anti-DP, -DQ or DR. Representative results from one patient are illustrated in Figure 7. Each example of peptide-induced proliferation was blocked by at least one of the antibodies, of which anti-DR was consistently the most potent, inhibiting 15 out of the 16 responses tested.

[0064] HLA type is one of the factors that can influence predisposition to particular immune-mediated diseases. The panels of AITP patients and healthy controls were typed for

HLA-DR and HLA-DQ polymorphic beta chain genes (Tables 3 and 4A) , and the results compared with published data from the general UK population. The commonest alleles at each locus amongst patients were, respectively, DRBl*03 and DRB1*15, and DQB1*O3 and DQB1*O6, but there were no significant positive or negative associations with the disease or the ability of particular sequences to stimulate proliferation.

[0065] Table 5 demonstrates that the dominant peptides are located throughout different domains of GPIIIa, including the transmembrane/cytoplasmic area, reflecting the fact that T cells, unlike pathogenic antibody, are not limited to the recognition of epitopes accessible on the intact cell. The selection of dominant helper epitopes in autoimmune disease may be also determined by different criteria from those that shape the fine specificity of conventional responses by CD4 ⁺ T cells to foreign antigens. In particular, the major self- epitopes may be dominant due to a lack of tolerance in the corresponding Th cell repertoire, rather than because they are contained in the most efficiently presented peptides that exhibit high affinity for their restricting elements. To test whether this is true for AITP, a web-based algorithm

(www.imtech.res.in/raghava/propred) was used to predict the motifs within the sequence of GPIIIa that have high affinity for a comprehensive panel of HLA-DR molecules, including all those expressed by the AITP patients. The results in Table 5 reveal that three of the seven dominant GPIIIa peptides were predicted not to have high affinity for any of the class II molecules evaluated. Of the four dominant peptides computed to be displayed at high levels by particular HLA-DR molecules, only peptide 82 (aa711-725) showed a correlation

(χ2 = 10; p<0.05) between the ability to stimulate Th responses and the expression of the relevant class II type by AITP patients. Thus, with the exception of peptide 82 (aa711-725), the vast majority of interactions between the

dominant GPIIIa peptides and their restricting MHC molecules in AITP patients are predicted to be of low affinity.

Table 1. Clinical details of patients with AITP PDL = Prednisolone, AZP = Azathioprine; DAP = Dapsone; IVIg = Intravenous Immunoglobulin; MYC = Mycophenlate Mofetil

Table 2. Amino acid sequences of the panel of overlapping, synthetic GPIIIa peptides spanning the entire length of the GPIIIa molecule

Table 3. Summary of GPIIIa peptides eliciting PBMC proliferation from AITP patients in vitro

AITP21 7 0103/15 Pos 1, 2, 29, 34, 44, 47, 49, 81, 82

AITP22 76 15/15 Pos 2, 47, 53, 77, 82 AITP23 5 07/15 Pos 5, 30, 31, 36,

47, 54, 60,

61, 86

AITP24 163 01/07 wPos 2, 3, 44, 47,

53, 56, 70, 82

AITP25 76 NT Neg 82 AITP26 NT NT Pos 2, 47, 53, 62,

63

AITP27 95 NT Neg None AITP28 260 NT Neg 4, 7, 9, 11, 21, 23, 24, 43, 57, 63, 69, 70

AITP29 217 NT Pos 5, 9, 17, 32, 33, 36, 38, 40, 52, 53, 57,70

AITP30 121 NT Neg 2, 20, 41, 44, 47, 53, 76 AITP31 327 NT Neg 2, 37, 47, 65, 82

NT = Not Tested; Pos = Positive Reaction; wPos = Weak Positive Reaction; Neg = Negative Reaction; SI = Stimulation Index

NT = Not Tested; Neg = Negative Reaction; SI = Stimulation Index

TABLE 4B. Summary of GPIIIa peptides eliciting PBMC proliferation from disease control donors in vitro

Table 5. Summary of predicted motifs in dominant GPIIIa peptides for binding to HLA-DR molecules and responsiveness of PBMC from AITP patients

^e From structural analysis of b3 integrin

Predicted using the Propred algorithm (www. imtech. res . in/raghava/propred)

*Significant association between response to peptide 82 and expression of HLA-DR molecules to which peptide predicted to bind with high affinity (χ ² = 10; p < 0.05)