WO2002081512A1 - Dust mite der p1 cd8+ t-cell epitopes and uses thereof - Google Patents

Abstract

Epitopes of the Der p1 antigen of the house dust mite Dermatophagoides pteronyssinus, a major cause of atopic allergic reactions, that are human CD8 cell epitopes and useful in treatment and prevention of allergies to the major house dust mite antigen, and of monitoring disease activity in atopic patients. Preferred embodiments include peptides of amino acid sequences: Y L A Y R N Q S L, M M I E E Y P Y V, S L D L A E Q E L and K I V L A I A S L.

Description

DUST MITE DER P1 CD8+ T-CELL EPITOPES AND USES

THEREOF

The present invention relates to antigens, and epitopes thereof, specifically the Der pi antigen of the house dust mite Dermatophagoides pteronyssinus, a major cause of atopic allergic reactions. The present inventors have identified epitopes within Der pi that are human CD8 cell epitopes. Peptides comprising the epitopes have medical indications, especially in treatment and prevention of allergies to the major house dust mite antigen, and of monitoring disease activity in atopic patients.

Atopy (Greek: atopos, meaning out of place) defines a general predisposition to develop allergic reactions to<- therwise innocuous substances. Persons with atopy produce IgE antibodies against common environmental allergens, and thus may develop allergic diseases such as atopic dermatitis, allergic rhinitis, and atopic asthma. However, there are some allergic diseases such as contact dermatitis and hypersensitivity pneumonitis, which develop through IgE independent mechanisms and in this sense can be considered non-atopic allergic conditions.

At present allergic rhinitis, atopic asthma and atopic eczema are amongst the leading causes of chronic ill health. In recent years there has been a marked increase in prevalence in these conditions, especially in Western Europe, the USA and Australasia. For example in Sweden over a 12 year period the number of children suffering with these conditions has doubled¹. Further treating these conditions adds considerably to the burden of healthcare costs. This is borne out by the fact that the annual cost of treating asthma in the USA is about 6 billion dollars². Understanding the immune and genetic pathogenesis of atopic allergic conditions provides insights into more targeted and specific therapy for them³.

One antigen that plays a major role in allergic disorders is Dermatophagoides pteronyssinus Der pi. Dermatophagoides pteronnysinus is the most abundant mite species world-wide²⁷'

²⁸ , with two other species namely Dermatophagoides farinae and Euroglyphus maynei also present in high numbers depending upon the region and climate²⁹.

In households, house dust mites occur in mattresses, pillows, bedclothes, carpets rugs and any other area which can provide appropriate ecologic factors (i.e. a sufficient supply of food and humidity) for survival of the mite³¹. Since they feed primarily on human skin dander, they have a close association with humans²⁷. Household dust provides an excellent resource for these mites as humans shed up to 1.5 grams of skin scales per week³³.

There are a great deal of data on the identification, purification and characterisation of mite allergens in the genus Dermatophagoides. The allergens produced by these mites fall into two main immunologically important groups; Der 1 (Der pi and Der fl) and II (Der piI and Der f II) . Group I allergens are 24000 mw glycoproteins found primarily in mite faeces³⁴. They are structurally homologous with very similar N terminal amino acid sequences³³. The Der pi allergen is a cysteine protease secreted by mites during digestion and is released in faecal pellets that are 10 - 40 micrometers in size³⁵. It is found at a concentration of 10 mg/ml (0.2 ng per faecal pellet) in mite faeces and elutes rapidly from faecal pellets³⁶. International standards for both Der p and Der f have been established for allergen quantification using highly sensitive enzyme linked immunosorbent assays (ELISA)³⁷.

Although the critical role of the Th2 CD4+ T cell is fairly well defined in allergen sensitization and pathogenesis, the role of the CD8+ T cell in this process is poorly defined. Surprisingly the inventors have been able to identify CD8+ T cells specific for Der pi in atopic individuals and furthermore have been able to specifically map the epitopes to nonamer peptides. These specific epitopes lie within regions of high immunogenicity which, on the basis of the inventors' methodology, are likely to be rich in CD8+ T cell epitopes that may be presented through a wide range of HLA types. The present invention in various aspects provides peptides comprising or consisting of such epitopes, uses of such peptides in compositions and methods for immunising and/or therapeutic treatment against, diagnosing or monitoring Der pi allergic reaction, especially atopic reaction.

Brief Description of the Figures

Figure 1 shows IFN gamma ELISpot results expressed as spot forming cells per million PBMC's for the different peptides tested (peptide 1-7 of the Der pi protein) in HLA A*0201 non- atopic, atopic asymptomatic and atopic symptomatic subjects.

Figure 2 shows IL4 ELISpot results expressed as spot forming cells per million PBMC's for the different peptides tested

(peptide 1-7 of the Der pi protein) in HLA A*0201 non-atopic, atopic asymptomatic and atopic symptomatic subjects. Figure 3 shows IL10 ELISpot results expressed as spot forming cells per million PBMC's for the different peptides tested (peptide 1-7 of the Der pi protein) in HLA A*0201 non-atopic, atopic asymptomatic and atopic symptomatic subjects.

Figure 4 shows tetramer positive cells per million CD8 T cells recognising peptides 6, 3, 1 and 5 of the Der pi protein in HLA A*0201 non-atopic, atopic asymptomatic and atopic symptomatic subjects.

Figure 5 shows the full length sequence of Der pi with predicted A*0201 epitopes highlighted either by shading or as boxes .

The present invention provides peptides that comprising CD8⁺ T- cell epitopes. Such epitopes bind to a HLA class 1 molecule that can be recognised by a T cell receptor of a specific CD8+ T cell.

A peptide epitope according to the present invention may bind or be recognised by an HLA class 1 molecule selection from any allele of HLA-A, HLA-B or HLA-C subregions . Preferred alleles include A2 , B8, B35, B7, Al and A3, especially A*0201, which is the most prevalent. Others include All, A23(9), A24(9), A25(9), A26(9), A29(9), A30, A31(19), A32(19), A33(19), A34(10), A43(10), A66 (10) , A68(28), A69(28), A74(19) and A80, for HLA-A, and for HLA-B: B13, B15, B18, B27, B37, B38(16), B39(16), B40, B41, B42, B44(12), B45(12), B46, B47, B48, B49(21), B50(21), B51(5), B52(5), B53, B54(22), B55(22),

B56(22), B57(17), B58(17), B59, B60(40), B61(40), B62(15), B63(15), B64(14), B66(14), B67, B70, B71(7), B72(70), B73, B75(15), 1376(15), B77(15), B78, B81 and B82. The serological specificities in brackets () denote the broad group within which a particular serological specificity belongs.

In an isolated form, such peptides have not previously been identified, it not having been established until the work described herein that Der pi is involved with a CD8⁺ T-cell response in humans .

According to one aspect of the present invention there is provided a peptide which is a fragment of human Der pi consisting of a CD8⁺ T-cell epitope.

In preferred embodiments a peptide according to the present invention comprises or consists of an amino acid sequence selected from the group consisting of peptide fragments with amino acid sequences found within amino acids 1-20 or 138-168 of Der pi, such peptide fragments containing a CD8⁺ T-cell ι epitope.

Further preferred embodiments are peptides comprising or consisting of an amino acid sequence selected from the group consisting of: Y L A Y R N Q S L M M I E E Y P Y V S L D L A E Q E L K I V L A I A S L

Other peptides of the invention are shown in the Sequence Listing herein, and embodiments of the invention consist of or comprise amino acid sequences within any one or more of the sequences set out in the sequence listing, which peptides contain a CD8⁺ T-cell epitope. In a further embodiment, the present invention provides a peptide that consists of or comprises amino acid sequence LAIASLLAL or YLAYRNQSL or ALAQTHSAI and contain a CD8⁺ T-cell epitope .

A peptide according to the present invention may be provided in a fusion with additional amino acids. Additional amino acids may be fused at one or both of the N-terminus and the C- terminus of the peptide. The additional amino acids may be an amino acid sequence that is not a fragment of Der pi protein, or may be an amino acid sequence that is part of that protein. As noted below, nucleic acid encoding peptides and polypeptides, including fusions, according to invention are provided as further aspects of the invention

Generally, a peptide according to the present invention is immunogenic or able to raise a T-cell immune response on administration to an individual. This may be in a therapeutic context, which may include treatment of an existing symptomatic condition or prophylaxis, immunisation or vaccination against an allergic or other reaction.

A peptide with the amino acid sequence of a said selected peptide may provided in isolated form, e.g. after its production by expression from encoding nucleic acid. As noted further below, one or more peptides in accordance with the present invention may be provided by peptide synthesis.

A plurality of peptides each with an amino acid sequence of a different selected peptide may provided in isolated form, individually or in a mixture.

Peptides and antibodies to the peptides (see below) may be used in quantifying and characterising the CD8+ T-cell response in individuals, useful in identifying or diagnosing patients who have the atopic tendency at any early stage. Through avoiding the specific allergens to which one could show T-cell reactivity, disease severity may be reduced.

Similarly, an individual's reactivity to the critical CD8+ T- cell epitopes may be monitored to predict disease exacerbations prior to clinical manifestation. For instance, prediction of acute deteriorations of asthma, which carries significant morbidity and mortality, has extensive clinical application.

Preferred peptides of the invention comprise or consist of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In one embodiment, for example, a peptide consists of a fragment of Der pi found within amino acids 1-20 or within amino acids 138-168 of Der pi and consisting of sufficient amino acids to contain a CD8⁺ T-cell epitope. Such a peptide may have up to 20 amino acids. Such a peptide may have 5, 6, 7, 8 or 9 amino acids. Nonamer peptides (with 9 amino acids) are preferred in certain embodiments of the present invention.

Peptides and polypeptides (e.g. fusion molecules including a peptide as discussed) in accordance with the present invention may be made using any of a variety of techniques at the disposal of the ordinary person skilled in the art.

Peptides may be synthesized using standard peptide chemistry such as by the common method employing Fmoc (Fluorenilmetil- ossicarbonil) t-Bu (tert-butil), as described in Atherton and Sheppard (1989) , Solid Phase Peptide Synthesis, a Practical Approach, IRL Press, Oxford. A convenient way of producing a peptide or polypeptide according to the present invention is to express nucleic acid encoding it, by use of the nucleic acid in an expression system.

Accordingly, the present invention also encompasses a method of making a peptide or polypeptide (as disclosed) , the method including expression from nucleic acid encoding the peptide or polypeptide (generally nucleic acid according to the invention) . This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide. Peptides and polypeptides may also be expressed in in vi tro systems, such as reticulocyte lysate.

Polynucleotides encoding peptides and polypeptides according to the present invention represent further aspects of the invention.

In one aspect there is provided a polynucleotide encoding a peptide as disclosed.

In a still further aspect, .a polynucleotide is provided which includes a plurality of nucleotide sequences encoding peptides or polypeptides according to the invention. This allows for production of a mixture of peptides or polypeptides in a single expression reaction.

Nucleic acid encoding a peptide or polypeptide according to the present invention may be used in nucleic acid immunisation in order to raise an immune response in a mammal, such as a human individual for a therapeutic or prophylactic purpose, or a non-human mammal for such a purpose or in order to produce antibodies for subsequent manipulation and/or use (e.g. in diagnostic or therapeutic contexts as discussed further below) .

Nucleic acid encoding a peptide or polypeptide according to the present invention may be used in a method of gene therapy, in prevention and/or treatment of allergy. This requires use of suitable regulatory elements for expression and a suitable vector for deliver of the expression unit (coding sequence and regulatory elements) to host cells. A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see e.g. US Patent No. 5,252,479 and WO 93/07282. In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses, including HSV and EBV, and retroviruses . Many gene therapy protocols in the prior art have used disabled murine retroviruses. A variety of adenovirus and adeno-associated viral vectors have been developed. Alternatives to viral vectors include transfer mediated by liposomes and direct DNA uptake and receptor- mediated DNA transfer.

Host cells containing nucleic acid encoding a peptide or polypeptide (or mixture thereof) according to the present invention may themselves be used in therapeutic or prophylactic treatment of individuals for or against allergic symptoms, (i.e. therapeutic treatment of an individual with an allergic reaction or prophylactic treatment of an individual prior to allergic reaction, especially an individual susceptible to such a reaction) .

Nucleic acid is generally provided as DNA or RNA, though may include one or more nucleotide analogues, and may be wholly or partially synthetic. Nucleic acid molecules and vectors according to the present invention may be provided in isolated and/or purified form, e.g. in substantially pure or homogeneous form. The term "isolate" may be used to reflect all these possibilities. Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.

Where it is desired to express a peptide or polypeptide from encoding nucleic acid, the nucleic acid includes appropriate regulatory control sequences. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al . eds . , John Wiley & Sons, 1992.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli .

A further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. The nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. The nucleic acid may be on an extra-chromosomal vector within the cell.

A still further aspect provides a method which includes introducing the nucleic acid into a host cell. The introduction, which may (particularly for in vi tro introduction) be generally referred to without limitation as a transformation, may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. As an alternative, direct injection of the nucleic acid could be employed. Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded peptide or polypeptide is produced. If the peptide or polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium. Following production by expression, a peptide or polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).

A peptide or polypeptide according to the present invention may be used as an immunogen.

Peptides may be used to induce tolerance in reactive T cells for allergic disease. In previous immunotherapy strategies, whole antigen has been used to tolerise, but these have been beset with risks of an IgE-mediated analphylactic reaction. Peptides may be used to circumvent the IgE response whilst still influencing the T cell response.

Antibody molecules against peptides of the invention are useful in purification and other manipulation of polypeptides and peptides.

According to a further aspect of the present invention there is provided a method of obtaining one or more antibody molecules containing a binding site able to bind a peptide of the invention, the method including bringing into contact a population of antibody molecules and a peptide according to the present invention, and selecting one or more antibody molecules of the population able to bind said peptide. The method may involve bringing the population of antibodies into contact with a plurality of peptides according to the invention.

As noted, the peptides may be provided in a fusion with additional amino acids.

Antibody molecules may routinely be obtained using technologies such as phage display, by-passing direct involvement of an animal's immune system.

Instead of or as well as immunising an animal, a method of obtaining antibodies as disclosed may involve displaying the population of antibody molecules on the surface of bacteriophage particles, each particle containing nucleic acid encoding the antibody molecule displayed on its surface. Nucleic acid may be taken from a bacteriophage particle displaying an antibody molecule able to bind a peptide or peptides of interest, for manipulation and/or use in production of the encoded antibody molecule or a derivative thereof (e.g. a fusion protein, a molecule including a constant region or other amino acids, and so on) . Instead of using bacteriophage for display (as for example in WO92/01047) , ribosomes or polysomes may be used, e.g. as disclosed in US-A-5643768, US-A-5658754 , W095/11922.

A peptide or peptides may be administered to a non-human mammal to bring them into contact with a population of antibody molecules produced by the mammal's immune system, then one or more antibody molecules able to bind the peptide or peptides may be taken from the mammal, or cells producing such antibody molecules may be taken from the mammal. The mammal may be sacrificed.

If cells are taken from the mammal, antibody molecules may be taken from said cells or descendants thereof. Such descendants in particular may include hybridoma cells.

Antibody molecules may be provided in isolated form, either individually or in a mixture. A plurality of antibody molecules may be provided in isolated form.

Preferred antibodies according to the invention are isolated, in the sense of being free from contaminants such as antibodies able to bind other polypeptides and/or free of serum components. Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies are within the scope of the present invention. Indeed, polyclonal mixtures able to bind one or more peptides or polypeptides according to the present invention are preferred in some embodiments, as discussed. Thus, the present invention in a further aspect is directed to a mixture of different antibodies able to bind one or more peptides or polypeptides according to the invention. Such a mixture may be provided in a composition including at least one additional component, such as a pharmaceutically acceptable excipient or vehicle.

The present invention also extends to methods of obtaining and/or raising antibodies to one or more peptides or polypeptides of the invention. Such methods may include administering a peptide or polypeptide or mixture of peptides or polypeptides to a mammal in order to raise an antibody response. For the production of antibodies or antibody- producing cells to be isolated and used for any of a variety of purposes, a step of sacrificing a non-human mammal may be included. Such a non-human mammal may be for example mouse, rat, rabbit, dog, cat, pig, horse, donkey, goat, sheep, camel, Old World monkey, chimpanzee or other primate. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to peptide or polypeptide of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, Nature, 357:80-82, 1992) .

Antibodies according to the present invention may be modified in a number of ways. Indeed the term "antibody" should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments and derivatives comprising an antibody antigen-binding domain enabling it to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included. As noted already, peptides, polypeptides and nucleic acid in accordance with the present invention may be formulated into compositions, and are useful in pharmaceutical contexts. These compositions may include, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes .

Compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Naked DNA may be used for immunization (see e.g. Cohen, J, 1993) , and one or more mimotope sequences may be cloned into suitable vectors (see e.g. Major et al . , 1995) . Naked DNA may be delivered using direct injection or by use of gene-guns (Yang et al . , 1990) or any other suitable technique. The peptide, polypeptide or nucleic acid according to the present invention that is to be given to an individual, administration may be in an immunogenic amount, that is sufficient to raise an immune response in the individual, or in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) . A prophylactic effect is sufficient to potentiate the immune response of an individual to a subsequent challenge with Der pi. Most preferably the effect is sufficient to prevent the individual from suffering one or more clinical symptoms as a result of subsequent Der pi. A therapeutic effect is sufficient to potentiate the immune response of an individual to pre-existing allergic reaction, preferably sufficient to antagonise the reaction, wholly or partially. Most preferably the effect is sufficient to ameliorate one or more clinical symptoms. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington=s Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

Further aspects of the invention provide methods of treatment including administration of a peptide, mixture of peptides, antibody molecule or mixture of antibody molecules, as provided, pharmaceutical compositions including such a peptide, mixture of peptides, antibody molecule or mixture of antibody molecules, and use of such a peptide, mixture of peptides, antibody molecule or mixture of antibody molecules, in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition including formulating the specific binding member with a pharmaceutically acceptable excipient.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated and the availability of alternative or additional treatments.

One aspect of the present invention provides use of a peptide as disclosed in the manufacture of a medicament for raising in a mammal an immune response against Der pi.

Another aspect provides a method of immunising a mammal against Der pi challenge, the method including administering a peptide or mixture of peptides to the mammal.

Aspects and embodiments of the present invention will now be illustrated further and experimentally exemplified with reference to various figures. Further aspects and embodiments of the present invention will be apparent to those of ordinary skill in the art.

Experimen tal

The inventors identified allergen specific CD8⁺ T cells in the blood of atopic symptomatic individuals and compared their frequency with that in atopic asymptomatic persons and non atopic individuals. In order to assess the frequency of these allergen specific CD8⁺ cells they used an ELISpot assay and peripheral blood lymphocyte staining using specific peptide HLA tetrameric complexes.

These cells were found to be present in the blood and functional characterisation of these cells was undertaken using IFNγ, IL4 and IL10 ELISpot assays.

The Der pi amino acid sequence was obtained from the protein data base SWISSPROT P08176 and also searching other published references of these sequences. The reported sequence variation between the different studies are small, and not significant in the present context.

Seven potential A2-binding nonamer peptides were identified within the Der pi protein. The peptides were tested for immunogenicity in ELISpot assays (IFNγ, IL10, IL4) and four were found to be recognized by CD8+ T cells in the peripheral blood of affected individuals. The peptides were then used to generate HLA peptide tetrameric complexes by refolding with HLA heavy chain and beta2 microglobulin. The tetrameric complexes were used to stain peripheral blood mononuclear cells from atopic individuals and confirmed the presence of the specific CD8+ T cells to the four peptides. In view of this surprising result, the Der pi sequence was then analysed for other potential CD8+ T cell epitopes relevant for other common HLA class I types (Al, A3, B7 , B8) and multiple potential peptides were identified. Two regions of Der pi (1- 20 amd 138-168) were found to be rich in predicted epitopes providing indication that the immunogenic regions of Der pi are concentrated at two critical sites. The HLA class I molecules were selected as they are common alleles. By identifying epitope enriched regions that are presented through many different class I molecules, the findings will be relevant to the vast majority of the population.

The subjects for this study consisted of three groups. Atopic symptomatic (atopic dermatitis, atopic asthma or allergic rhinitis) who were selected from the Dermatology clinic, Churchill Hospital, atopic asymptomatic and non-atopic subjects selected from laboratory personnel. Clinical details were recorded in each of these individuals and blood was taken for HLA typing, and separation of serum/PBL. An individual was considered atopic if they were skin test positive following a prick test using a panel of common allergens and/or specific allergen IgE antibody positive. HLA typing with subtyping was done on each of the subjects using a sequence specific PCR based system.

RESULTS

Seven potential HLA A2 binding CD8 T cell epitopes were identified in the Der pi allergen sequence based on the preferred A*0201-peptide binding preferences, and the peptides were synthesized. The original analysis generated 17 possible peptides, of which seven were synthesized following selection by the inventors according to criteria applied by them in calculations of possible A*0201 binding affinity, with contributions from both major anchor residues and minor anchor residues. The inventors calculations generated different results from those obtained using standard computer-based algorithms, and the inventors followed their own calculations.

IFN γ, IL4 and IL10 ELISpots were performed on the PBL obtained from both HLA A*0201 and non-HLA A*0201, non-atopic, atopic asymptomatic and atopic symptomatic individuals. The ELISpot is a simple and highly sensitive assay for analysis of cell activation at the single cell level. It is particularly useful for analysing specific immune responses to whole antigens or peptides.

The assay was performed in 96 well microtiter plates. In the first step the wells were coated with high affinity monoclonal antibodies to the cytokine to be investigated. Cells up to 250 x 10³/well were added and incubated for 6-48 hours in the presence of antigen. During this period antigen specific responding cells released the cytokine which was capture in the immediate vicinity of the cells. Cells were removed by washing and a biotinylated antibody directed to a second epitope of the cytokine added. Next streptavidin conjugated with enzyme (ALP) was added. Finally, a precipitating substrate for ALP was added and the plates incubated until spots emerged at the site of the activated cells. The spots were examined and counted in a dissection microscope or image analyser system. Comparison of the number of spots with the number of cells added to the wells gave the frequency of the responding cells.

HLA peptide tetrameric complexes were synthesised as described previously by Altman. Purified HLA heavy chain and β2 microglobulin were synthesises using a prokaryotic expression system (pET, Novagen, Milwaukee, WI, USA) . The heavy chain was modified by deletion of the transmembrane/cystolic tail and COOH terminal addition of a sequence containing the BirA enzymatic biotinylation site. Heavy chain β2 microglobulin and peptide were refolded by dilution. A*0201 - binding peptides were Der P 1 YLAYRNQSL, MMIEEYPYV, SLDLAEQEL, KIVLAIASL. In addition a B8-binding peptide was YLAYRNQSL. The 45-kd refolded product was isolated using fast protein liquid chromatography (FPLC) , biotinylated by BirA (Avidity, Denver, CO, USA) in the presence of biotin (Sigma Chemical Co., St. Louis, MO, USA), ATP (Sigma Chemical Co.) and Mg²⁺

(Sigma Chemical Co.). The biotinylated product was separated from free biotin by gel filtration and ion exchange using FPLC. Streptavidin-PE conjugate (Sigma Chemical Co.) was added in a 1:4 molar ratio and the tetrameric product was concentrated to 1 mg/ml . Analysis of cells for the expression of cell surface markers was performed using a FACScan^® (Becton Dickinson & Co., Mountain View, CA, USA) and CellQuest software (Becton Dickinson & Co.).

The non-HLA A*0201 individuals did not show reactivity in the ELIspot assays (IFNγ, IL4, IL10) using the specific A*0201 binding Der pi peptides identified. The results of the different ELISpot assays (IFNγ, IL4 and IL10) in HLA A*0201 individuals are shown in Figures 1, 2 and 3 respectively. The mean (SEM) spot forming cells per million PBMC in the IFNγ and IL10 ELISpot assays for the different peptides tested in these patients is shown in Table 1.

Comparisons were made between the different groups of patients using the unpaired t test. The difference was considered significant if the p value was less than 0.05, and highly significant if less than 0.01. In the IFNγ ELISpot assay the p values were highly significant for the peptides 1, 2, 3, 5, and 6 when comparisons were made between non-atopic and atopic asymptomatic or non-atopic and atopic symptomatic individuals. When comparisons were made between the two atopic groups, there were highly significant differences for the peptides 1, 3, 5 and 6. When the same statistical analysis was done with the IL10 ELISpot results, values for the atopic asymptomatic patients were significantly more than those for the atopic symptomatic individuals for the peptides 1, 3, 5 and 6.

HLA peptide tetrameric complexes were made using four of the seven peptides identified as reacting best in the ELISpot assays (namely peptide 1, 3, 5 and 6) . These complexes were used to stain PBMC obtained from both HLA A*0201 positive and negative individuals who were non atopic, atopic asymptomatic and atopic symptomatic. In addition the A*0201 individuals PBL were also stained using a non A2 tetramer and an irrelevant tetramer. Non HLA A*0201 individuals did not show any tetramer positive staining, when stained with these tetramers. The numbers of tetramer positive cells per million CD8 T cells in the A*0201 positive individuals is shown in the Figure 4. The mean (SEM) of the tetramer positive cells per million PBMC for the three groups of subjects using HLA tetrameric complexes with peptide 1, 3 and 6 are shown in Table 2. The data for peptide 5 are not shown in this table, as the number of subjects tested at present with this tetramer is small. However the absolute number of cells detected by this tetramer in these is shown in individuals tested is shown in Figure 4. Again comparisons between the different groups using the non paired t test, showed highly significant differences in the number of specific cells detected between the three groups of individuals. The atopic asymptomatic individuals have significantly more tetramer positive cells than atopic asymptomatic individuals, who themselves have more specific cells compared to the non atopic individuals. Using the B8-based tetramer refolded around the peptide YLAYRNQSL, Derpl-specific CD8+ T cells were identified in atopic symptomatic B8-positive individuals.

In summary, the inventors have identified CD8+ T cells that react to Der pi, and have fine mapped the epitopes to their minimal sequences with functional characterisation of the reactive CD8+ T cells. A correlation was found between the frequency of reactive cells and disease severity and there were functional differences in the CD8+ T cells between symptomatic and asymptomatic atopic individuals. These unexpected data provide indication of a role of the Der pl- specific CD8+ T cells in disease pathogenesis .

Similar predictions were done for the HLA Al, A3, B7 and B8 molecules which are common class I molecules . Although potential peptides could be found throughout the Der pi protein, they were concentrated in two regions of Der pi, namely amino acids 1-20 and 138-168, providing indication that these are highly immunogenic regions of Der pi. The first epitope cluster regions lies within the leader sequence.

DISCUSSION

Four Der pi peptides have been identified that bind HLA A*0201 and are immunogenic. One of the four peptides also binds HLA- B*0801 and was found to be immunogenic. Cells responding to the peptides have been identified within peripheral blood lymphocytes using IFNγ and IL10 ELISpot assays and HLA peptide tetrameric complex staining.

The Der pi specific CD8 T cells are found in the blood of atopic persons exposed to the HDM allergen. The frequency of these cells seen in the atopic symptomatic patients are comparable to that seen in the memory CD8 T cell population of influenza virus or hepatitis C virus. There is also evidence from these data that the number of the allergen specific cells in the blood correlates with disease activity. These cells are functional in that they produce IFNγ and IL10. They show that the CD8 T cells from atopic symptomatic patients tend to produce more IFNγ, but less IL10 than atopic asymptomatic individuals .

These data provide indication that IL10 producing Der pi specific CD8+ T-cells may have a role in disease suppression.

The IL4 production was minimal in all groups of subjects tested so far. It is not known whether it is the same population of specific CD8 cells that is producing both cytokines, or whether there are actually two populations of specific CD8 T cells secreting each cytokine separately. Further characterising the functional characteristics of these cells using other cytokine assays, studying the surface marker profiles of this antigen specific population and comparing the frequency of these cells in skin samples obtained before and after a patch test challenge using the specific allergen will provide further information. REFERENCES

1. Aberg, N., Hesselmar, B., Aberg, B. & Eriksson, B. Increase of asthma, allergic rhinitis and eczema in Swedish schoolchildren between 1979 and 1991 [see comments] . Clin Exp Allergy 25, 815-9 (1995) .

2. Smith, D.H. et al . A national estimate of the economic costs of asthma. Am J^" Respir Cri t Care Med 156, 787-93 (1997) . 3. Romagnani, S. Technological advances and new insights into pathogenesis prelude novel therapeutic strategies.

Curr Opin Immunol 7, 745-50 (1995) . 26. Kern, R.A. Dust sensitisation in bronchial asthma. Med

Clin N Amer 5, 751 (1921) . 27. Maunsell, K. , Wraith, D.G. & Cunnington, A.M. Mites and house-dust allergy in bronchial asthma. Lancet 1, 1267-70

(1968) .

28. Biliotti, G. , Passaleva, A., Romagnani, S. & Ricci, M. Mites and house dust allergy. I. Comparison between house dust and mite (Dermatophagoides pteronyssinus and D. farinae) skin reactivity. Clin Allergy 2, 109-13 (1972) .

29. Arlian, L.G. Biology and ecology of house dust mites, Dermatophagoides spp. and Euroglyphus spp. Airborne Allergens 9, 339 (1989) . 31. Wharton, G.W. House dust mites. J Med Ento ol 12, 577- 621. (1976) . 33. Leeks, H.I. The mite and house dust allergy. A review of current knowledge and its clinical significance. Clin Pediatr (Phila) 12, 514-7. (1973). 34. Tovey, E.R., Chapman, M.D. & Platts-Mills, T.A. Mite faeces are a major source of house dust allergens. Nature 289, 592-3. (1981) . 35. Stewart, G.A. , Lake, F.R. & Thompson, P.J. Faecally derived hydrolytic enzymes from Dermatophagoides pteronyssinus: physicochemical characterisation of potential allergens. Int Arch Allergy Appl Immunol 95, 248-56 (1991) . 36. Geissler, W. , Maasch, H.J., Winter, G. & Wahl , R. Kinetics of allergen release from house dust mite Dermatophagoides pteronyssinus. J Allergy Clin Immunol 77, 24-31. (1986) . 37. Platts-Mills, T.A. & Chapman, M.D. Dust mites: immunology, allergic disease, and environmental control. J Allergy Clin Immunol 80, 755-75. (1987).

TABLE 1

Mean (SEM) spot forming cells per million peripheral blood lymphocytes I the IFNγ and IL10 ELISpot assays for the different peptides in the three groups tested (NA - non- atopic, AA - atopic asymptomatic, AS - atopic symptomatic)

# mean (SEM) not calculated as only one patient's results available

* p <0.01, for comparison between non-atopic and atopic symptomatic groups

IFNgamma ELISpot assay IL10 ELISpot assay

PEPTIDE NO NA AA AS NA AA AS

1 9(1.0) 113.3(12.6) 179(13.5)* # 165.5(7.2) 22(3.0) 2 3.5(0.5) 54(5.7) 38.4(8.1)* # 38.5(3.5) 6.5(1.5) 3 14.5 (0.5) 161.7(14.9) 269.8 (22.3) * # 200.5 (11.0) 31.5 (8.5) 4 6.5(1.5) 12.8(2.2) 6.8(0.9) # 6.2(1.2) 1(1.0) 5 7(2.0) 133.3(21.5) 194.2(6.2)* # 192.5(21.9) 14(1.0) 6 12(3.0) 137.5(13.8)285.2(25.9)* # 213.3(12.7)38.5(6.5) 7 5.5(0.5) 12.7(1.6) 6.4(0.8) # 6(0.5) 3(0.2)

PEPTIDE SEQUENCES

1 KIVLAIASL 2 LAIASLLAL 3 MMIEEYPYV 4 ALAQTHSAI SLDLAEQEL YLAYRNQSL TIPRQIEYI

TABLE 2

Mean (SEM) tetramer positive cells per million CD8 T cells for different peptides in the three groups of patients (NA - non-atopic, AA - atopic asymptomatic, AS - atopic symptomatic)

PEPTIDE NO NA AA AS

50 (22.4) 183.3 (30.7) 412.2 (35.1)

16.7(16.7) 166.7(21.1) 600 (46.3)

50 (22.4) 316.7 (30.7) 737.5(62.5)

SEQUENCE LISTING

A2 nonoπvers

2 K I V L A I A S L

145 Y L A Y R N Q S L

5 L A I A S L L A L

152 S L D L A E Q E L

228 A L A Q T H S A I

237 A V I I G I K D L

309 M M I E E Y P Y V

173 T I P R G I E Y I

62 G A I N H L S D L

224 K I R E A L A Q T

234 S A I A V I I G I 310 M I E E Y P Y V V

10 L L A L S A V Y A

6 A I A S L L A L S

8 A S L L A L S A V

59 S N G G A I N H L

138 V A A T E S A Y L

Al nono ers

140 A T E S A Y L A Y

172 D __ I P R G I E Y

21 S S I K T F E E Y

275 Y S N A Q G V D Y

291 D T N W G D N G Y

9 S L L A L S A V Y

29 Y K K A F N K S Y

206 A Q R F G I S N Y

243 K D L D A F R H Y

48 K __ F L E S V K Y

183 H N G V V Q E S Y

184 N G V V Q E S Y Y

259 R D N G Y Q P N Y

267 Y H A V N I V G Y

A3 nonomers

216 Q I Y P P N V N K 9 S L L A L S A V Y 15 A V Y A R P S S I 159 E L V D C A S Q H

137 G V A A T E S A Y

235 A I A V I I G I K

3 I V L A I A S L L

47 R K N F L E S V K

194 Y V A R E Q S C R

224 K I R E A L A Q T

22 S I K T F E E Y K

269 A V N I V G Y S N

B7 nonomers

203 R P N A Q R F G I

264 Q P N Y H A V N I

147 A Y R N Q S L D L

19 R P S S I K T F E 108 A P A E I D L R Q

174 I P R G I E Y I Q

312 E E Y P Y V V I L

5 L A I A S L L A L

43 E E A A R K N F L 59 S N G G A I N H L

64 I N H L S D L S L

221 N V N K I R E A L

B8 nonomers

145 Y L A Y R N Q S L 72 L D E F K N R F L E E A A R K N F L E F K N R F L M S S L D L A E Q E L V N K I R E A L A E A A R K N F L E . H L K T Q F D L N K I V L A I A S L S I K T F E E Y K S L D E F K N R F A Y R N Q S L D L L A I A S L L A L K A F N K S Y A T

Claims

CLAIMS :

1. An isolated peptide which is a fragment of human Der pi consisting of a CD8⁺ T-cell epitope.

2. An isolated peptide according to claim 1 which consists of an amino acid sequence selected from the group consisting of peptide fragments with amino acid sequences found within amino acids 1-20 or 138-168 of human Der pi, such peptide fragments containing a CD8⁺ T-cell epitope.

3. An isolated peptide according to claim 2 which consists of an amino acid sequence selected from the group consisting of: Y L A Y R N Q S L M M I E E Y P Y V S L D L A E Q E L and K I V L A I A S L.

4. An isolated peptide according to claim 1 which consists of an amino acid sequence selected from the group consisting of

L A I A S L L A L Y L A Y R N Q S L and A L A Q T H S A I.

5. An isolated peptide of 20 amino acids or less comprising a peptide according to any one of claims 1 to 4.

6. An isolated peptide according to any one of claims 1 to 5 fused to additional amino acids, which additional amino acids do not provide an amino acid sequence that is a fragment of human Der pi protein.

7. A peptide according to any one of claims 1 to 6 which consists of 20 amino acids or less.

8. A peptide according to claim 7 wherein the peptide consists of 5, 6, 7, 8 or 9 amino acids.

9. A peptide according to claim 8 which consists of 9 amino acids .

10. A plurality of peptides each with an amino acid sequence of a different peptide according to any one of claims 1 to 9 provided in isolated form, individually or in a mixture.

11. A method of producing a peptide or plurality of peptides according to any one of claims 1 to 10, the method comprising chemically synthesizing the peptide or peptides.

12. A method of producing a peptide or plurality of peptides according to any one of claims 1 to 10, the method comprising providing nucleic acid encoding the peptide or plurality of peptides in an expression system under conditions for producing the peptide or plurality of peptides.

13. A method according to claim 12 comprising growing host cells in culture, which host cells contain one or more expression vectors comprising nucleic acid encoding the peptide or plurality of peptides, under appropriate conditions for producing the peptide or plurality of peptides.

14. A method according to any one of claims 11 to 13 further comprising isolating the peptide or plurality of peptides.

15. A method according to any one of claims 11 to 14 further comprising formulating the peptide or plurality of peptides into a composition comprising at least one additional component .

16. An isolated polynucleotide encoding a peptide or plurality of peptides according to any one of claims 1 to 10.

17. An expression vector comprising a polynucleotide according to claim 16.

18. A host cell transformed with an expression vector according to claim 17.

19. An isolated antibody molecule that specifically binds a peptide according to claim 3 or claim 4.

20. A method of obtaining or producing one or more antibody molecules able to bind a peptide or peptides according to any one of claims 1 to 9, the method comprising bringing into contact a population of antibody molecules and said peptide or peptides, and selecting one or more antibody molecules of the population able to bind said peptide or peptides.

21. A method according to claim 20 comprising bringing the population of antibodies into contact with a plurality of said peptides .

22. A method according to claim 20 or claim 21 comprising bringing into contact with the peptide or peptides a population of antibody molecules displayed on bacteriophage particles or ribosomes, each particle or ribosome containing nucleic acid encoding the antibody molecule it displays.

23. A method according to claim 20 or claim 21 comprising administering the peptide or peptides to a mammal.

24 . A method according to claim 23 wherein the mammfcHti/tts non -human .

25. A method according to claim 24 wherein the mammal is sacrificed.

26. A method according to claim 24 or claim 25 wherein one or more antibody molecules able to bind the peptide or peptides are taken from the mammal, or cells producing such antibody molecules are taken from the mammal .

27. A method according to claim 26 further comprising culturing cells taken from the mammal, or descendants of said cells, to produce antibody molecules.

28. A method according to claim 22 further comprising obtaining nucleic acid from a bacteriophage particle or ribosome displaying an antibody molecule able to bind said peptide or peptides and producing antibody molecules encoded by said nucleic acid.

29. A method according to any one of claims 20 to 28 further comprising isolating the antibody molecule or antibody molecules .

30. A method according to any one of claims 20 to 29 further comprising formulating the antibody molecule or antibody molecules into a composition comprising at least one additional component. s

38

31. A peptide or plurality of peptides according to any one of claims 1 to 10 for use in a method of treatment of the human or animal body.

5 32. A peptide or plurality of peptides according to claim 31 for use in a method of raising an immune response to DerPl.

33. A peptide or plurality of peptides according to claim 32 for use in a method of treatment of a patient.

10

34. Use of a peptide or plurality of peptides according to any one of claims 1 to 10 in the manufacture of a medicament for treating a patient.

15 35. A method of raising an immune response to DerPl, the method comprising administering a peptide or plurality of peptides according to any one of claims 1 to 10 to the mammal .